182 / Caffeine / Monographs FCC 9

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182 / Caffeine / Monographs FCC 9
Sample solution: 0.2 mg/mL in Mobile phase. Shake,

Caffeine
.
and sonicate, if necessary, to dissolve.

First Published: Prior to FCC 6 Chromatographic system, Appendix IIA
Last Revision: Third Supplement, FCC 8 Mode: High-performance liquid chromatography
Detector: UV 275 nm
Column: 4.6-mm × 15-cm column packed with
1,3,7-Trimethylxanthine
octadecylsilane chemically bonded to porous silica or
ceramic microparticles1
Flow rate: 1 mL/min
Injection volume: 10 µL
Monographs
System suitability
Sample: Standard solution
C8H10N4O2 Formula wt, anhydrous 194.19 [NOTE—The relative retention times for theophylline and
C8H10N4O2 · H2O Formula wt, monohydrate 212.21 caffeine are 0.69 and 1.0, respectively.]
CAS: anhydrous [58-08-2] Suitability requirement 1: The resolution, R, between
UNII: 3G6A5W338E [caffeine] theophylline and caffeine is NLT 6.0.
Suitability requirement 2: The tailing factor for the
DESCRIPTION theophylline and caffeine peaks is NMT 2.0.
Caffeine occurs as a white powder or as white, glistening Suitability requirement 3: The relative standard
needles, usually matted together. It may be compacted or deviation of the Sample is NMT 2.0% for five
compressed into free-flowing granules or pellets. It is replicates of the Standard solution.
odorless and has a bitter taste. Caffeine is anhydrous or Analysis: Separately inject equal volumes of the Standard
contains one molecule of water of hydration. Its solutions solution and the Sample solution into the
are neutral to litmus. The hydrate is efflorescent in air, and chromatograph, record the chromatograms, and
1 g is soluble in about 50 mL of water, in 75 mL of measure the responses.
alcohol, in about 6 mL of chloroform, and in 600 mL of Calculate the percentage of caffeine (C8H10N4O2) in the
ether. portion of the sample taken:
Function: Flavoring agent
Packaging and Storage: Store hydrous caffeine in tight Result = (rU/rS) × (CS/CU) × 100
containers and anhydrous caffeine in well-closed rU = peak response of caffeine from the Sample
containers. solution
IDENTIFICATION rS = peak response of caffeine from the Standard
• A. INFRARED ABSORPTION, Spectrophotometric Identification solution
Tests, Appendix IIIC CS = concentration of USP Caffeine RS in the
Reference standard: USP Caffeine RS Standard solution (mg/mL)
Sample and standard preparation: M (previously dried CU = concentration of Caffeine in the Sample
at 80° for 4 h) solution (mg/mL)
Acceptance criteria: The spectrum of the sample Acceptance criteria: NLT 98.5% and NMT 101.0% of
exhibits maxima at the same wavelengths as those in C8H10N4O2, calculated on the anhydrous basis
the spectrum of the Reference standard. IMPURITIES
• B. PROCEDURE Inorganic Impurities
Acceptance criteria: The retention time of the major • LEAD, Lead Limit Test, Flame Atomic Absorption
peak (excluding the solvent peak) in the chromatogram Spectrophotometric Method, Appendix IIIB
of the Sample solution corresponds to that of the Sample: 3 g
Standard solution in the Assay. Acceptance criteria: NMT 1 mg/kg
ASSAY Organic Impurities
• PROCEDURE • OTHER ALKALOIDS
Buffer A: 0.01 M sodium acetate Sample solution: 20 mg/mL, made to 5 mL
Mobile phase: Acetonitrile, tetrahydrofuran, and Buffer A Analysis: Add a few drops of mercuric–potassium iodide
(25:20:955, v/v). Adjust with glacial acetic acid to a pH TS to 5 mL of the Sample solution.
of 4.5. Acceptance criteria: No precipitate forms.
System suitability stock solution: 0.02 mg/mL of USP SPECIFIC TESTS
Theophylline RS in Mobile phase. Shake, and sonicate, if • MELTING RANGE OR TEMPERATURE DETERMINATION, Appendix
necessary, to dissolve. IIB
Standard solution: Transfer 5.0 mg of USP Caffeine RS Sample: Previously dried at 80° for 4 h
to a 25-mL volumetric flask. Add 5.0 mL of the System Acceptance criteria
suitability stock solution and 10 mL of Mobile phase. Anhydrous: Between 235° and 237.5°
Shake, and sonicate, if necessary. Dilute with Mobile
phase to volume, and filter. 1 Symmetry C18, 4.6-mm × 15-cm, 5 µm, Waters Corp., or equivalent.
FCC 9 Monographs / Calcium Acid Pyrophosphate / 183
• READILY CARBONIZABLE SUBSTANCES, Appendix IIB Acceptance criteria: Any turbidity produced by the
Sample solution: Dissolve 500 mg of sample in 5 mL of Sample does not exceed that produced by the Control.
95% sulfuric acid. (NMT 0.05%)
Acceptance criteria: The color of the resulting Sample • FLUORIDE, Fluoride Limit Test, Method III, Appendix IIIB
solution is no darker than that of Matching Fluid D. Analysis: Use 10 mL of 1 N hydrochloric acid instead of
• RESIDUE ON IGNITION (SULFATED ASH), Appendix IIC water to dissolve the sample.
Sample: 2 g Acceptance criteria: NMT 0.005%
Acceptance criteria: NMT 0.1% • LEAD, Lead Limit Test, Flame Atomic Absorption
• WATER, Water Determination, Appendix IIB Spectrophotometric Method, Appendix IIIB
Acceptance criteria Sample: 10 g
Monographs
Anhydrous: NMT 0.5% Acceptance criteria: NMT 2 mg/kg
Hydrous: NMT 8.5% • SULFATE, Chloride and Sulfate Limit Tests, Sulfate Limit Test,
Appendix IIIB
OTHER REQUIREMENTS Sample: 200 mg
• LABELING: Indicate whether it is anhydrous or hydrous. Control: 200 µg sulfate (20 mL of Standard Sulfate
Solution)
Acceptance criteria: Any turbidity produced by the
Sample does not exceed that by the Control. (NMT
Calcium Acetate
.
0.1%)
First Published: Prior to FCC 6
SPECIFIC TESTS
• WATER, Water Determination, Appendix IIB
Ca(C2H3O2)2 Formula wt 158.17 Acceptance criteria: NMT 7.0%
INS: 263 CAS: [62-54-4]
UNII: Y882YXF34X [calcium acetate]
DESCRIPTION
Calcium Acid Pyrophosphate
.
Calcium Acetate occurs as a fine, white, bulky powder. It is

freely soluble in water and slightly soluble in alcohol. First Published: Prior to FCC 6
Function: Buffer; stabilizer; firming agent
Packaging and Storage: Store in well-closed containers.
IDENTIFICATION
• ACETATE, Appendix IIIA
Sample solution: 100 mg/mL
CaH2P2O7 Formula wt 216.04
Acceptance criteria: Passes tests CAS: [14866-19-4]
• CALCIUM, Appendix IIIA
UNII: A7X6BBX98K [calcium acid pyrophosphate]
Acceptance criteria: Passes tests DESCRIPTION
Calcium Acid Pyrophosphate occurs as a fine, white, acidic
ASSAY
powder. It is insoluble in water, but it is soluble in dilute
• PROCEDURE
hydrochloric and nitric acids.
Sample: 300 mg
Function: Leavening agent; nutrient
Analysis: Dissolve the Sample in 150 mL of water
containing 2 mL of 2.7 N hydrochloric acid. While
stirring, preferably with a magnetic stirrer, add about IDENTIFICATION
30 mL of 0.05 M disodium EDTA from a 50-mL buret. • A. PROCEDURE
Then add 15 mL of 1 N sodium hydroxide and 300 mg Sample: 100 mg
of hydroxy naphthol blue indicator and continue the Analysis: Dissolve the Sample by warming it in a mixture
titration to a blue endpoint. Each mL of 0.05 M of 5 mL of 2.7 N hydrochloric acid and 5 mL of water.
disodium EDTA is equivalent to 7.909 mg of Add dropwise, while shaking, 2.5 mL of 6 N
Ca(C2H3O2)2. ammonium hydroxide and then add 5 mL of
Acceptance criteria: NLT 99.0% and NMT 100.5% of ammonium oxalate TS.
Ca(C2H3O2)2, calculated on the anhydrous basis Acceptance criteria: A white precipitate forms.
• B. PRODEDURE
IMPURITIES
Sample solution: Dissolve 100 mg of sample in 100 mL
Inorganic Impurities
of 1.7 N nitric acid.
• CHLORIDE, Chloride and Sulfate Limit Tests, Chloride Limit
Analysis:
Test, Appendix IIIB
Mixture A: Add 0.5 mL of the Sample solution to 30
Sample: 40 mg
mL of quimociac TS.
Control: 20 µg chloride (2 mL of Standard Chloride
Solution)
184 / Calcium Acid Pyrophosphate / Monographs FCC 9
Mixture B: Heat the remaining portion of the Sample Acceptance criteria: NMT 10.0%
solution for 10 min at 95°, and then add 0.5 mL of the
heated solution to 30 mL of quimociac TS.
Acceptance criteria: A yellow precipitate does not
Calcium Alginate
.
form with Mixture A, but forms immediately with

Mixture B.
ASSAY
• PROCEDURE Algin
Sample solution: Dissolve 300 mg of sample in 10 mL [(C6H7O6)2Ca]n Formula wt, calculated 195.16
Monographs
of 2.7 N hydrochloric acid. Formula wt, actual (avg) 219.00

Analysis: To the Sample solution, add about 120 mL of INS: 404 CAS: [9005-35-0]
water and a few drops of methyl orange TS and boil for UNII: 8P20S56HZI [calcium alginate]
30 min. Keep the volume and pH of the solution
constant during the boiling period by adding DESCRIPTION
hydrochloric acid or water if necessary. Add 2 drops of Calcium Alginate occurs as a white to yellow, fibrous or
methyl red TS and 30 mL of ammonium oxalate TS. granular powder. It is the calcium salt of alginic acid. (See
Then, add, dropwise, with constant stirring, a mixture the monograph for Alginic Acid.) It is insoluble in water,
of equal volumes of 6 N ammonium hydroxide and but it is soluble in alkaline solutions or in solutions of
water until the pink color of the indicator just substances that combine with the calcium. It is insoluble in
disappears. Digest on a steam bath for 30 min, cool to organic solvents.
room temperature, allow the precipitate to settle, and Function: Stabilizer; thickener; emulsifier
filter the supernatant liquid through a sintered-glass Packaging and Storage: Store in well-closed containers.
filter crucible using gentle suction. Wash the precipitate
in the beaker with about 30 mL of cold (below 20°) IDENTIFICATION
wash solution, prepared by diluting 10 mL of • PROCEDURE
ammonium oxalate TS to 1000 mL with water. Allow Sample: 5 mg
the precipitate to settle and pour the supernatant liquid Analysis: Place the Sample in a test tube, add 5 mL of
through the filter. Repeat this washing by decantation water, 1 mL of a freshly prepared 1:100 solution of
three more times. Using the wash solution, transfer the naphthoresorcinol:ethanol, and 5 mL of hydrochloric
precipitate as completely as possible to the filter. acid. Heat the mixture to boiling, boil gently for about
Finally, wash the beaker and the filter with two 10-mL 3 min, and then cool to about 15°. Transfer the
portions of cold (below 20°) water. Place the sintered- contents of the test tube into a 30-mL separatory
glass filter crucible in the beaker and add 10 mL of funnel with the aid of 5 mL of water, and extract with
water and 50 mL of cold, 1:6 sulfuric acid. Add 35 mL 15 mL of isopropyl ether. Perform a blank
of 0.1 N potassium permanganate from a buret and stir determination (see General Provisions).
until the color disappears. Heat to about 70° and Acceptance criteria: The isopropyl ether extract from
complete the titration with 0.1 N potassium the Sample exhibits a deeper purple hue than that from
permanganate. Each mL of 0.1 N potassium the blank.
permanganate is equivalent to 5.40 mg of CaH2P2O7.
Acceptance criteria: NLT 95.0% and NMT 100.5% of
ASSAY
• ALGINATES ASSAY, Appendix IIIC
CaH2P2O7
Analysis: Each mL of 0.25 N sodium hydroxide
IMPURITIES consumed in the assay is equivalent to 27.38 mg of
Inorganic Impurities calcium alginate (equiv wt 219.00).
• ARSENIC, Arsenic Limit Test, Appendix IIIB Acceptance criteria: A sample yields NLT 18% and
Sample solution: 1 g in 5 mL of 2.7 N hydrochloric NMT 21% of carbon dioxide (CO2), corresponding to
acid between 89.6% and 104.5% of calcium alginate (equiv
Acceptance criteria: NMT 3 mg/kg wt 219.00), calculated on the dried basis.
• FLUORIDE, Fluoride Limit Test, Appendix IIIB
Sample: 1.0 g
IMPURITIES
Acceptance criteria: NMT 0.005%
• ARSENIC, Arsenic Limit Test, Appendix IIIB
• LEAD, Lead Limit Test, APDC Extraction Method, Appendix
Sample solution: Prepare as directed for organic
IIIB
compounds.
Acceptance criteria: NMT 2 mg/kg
SPECIFIC TESTS • LEAD, Lead Limit Test, Appendix IIIB
• LOSS ON IGNITION Sample solution: Prepare as directed for organic
Sample: 1 g compounds.
Analysis: Transfer the Sample into a suitable tared Control: 5 µg Pb (5 mL of Diluted Standard Lead
crucible, ignite at 800° ± 25° for 30 min, cool in a Solution)
desiccator, and weigh. Acceptance criteria: NMT 5 mg/kg
FCC 9 Monographs / Calcium Benzoate / 185
SPECIFIC TESTS Analysis: Add 2 drops of glacial acetic acid and 5 mL of

• LOSS ON DRYING, Appendix IIC: 105° for 4 h a 100 mg/mL calcium acetate solution to the Sample
Acceptance criteria: NMT 15.0% solution.
Acceptance criteria: The solution remains clear after
standing for 5 min.
Calcium Ascorbate
.

Calcium Benzoate
.
First Published: Second Supplement, FCC 7
Monographs
Last Revision: First Supplement, FCC 8
Monocalcium Benzoate
C12H14CaO12 · 2H2O Formula wt 426.34

INS: 302 CAS: [5743-27-1]
UNII: 183E4W213W [calcium ascorbate]
DESCRIPTION C14H10CaO4 · xH2O Formula wt, anhydrous 282.31

Calcium Ascorbate occurs as a white to slightly yellow, INS: 213 CAS: [2090-05-3]
crystalline powder. It is soluble in water, slightly soluble in UNII: 3QDE968MKD [calcium benzoate]
alcohol, and insoluble in ether. The pH of a 1:10 aqueous
solution is between 6.8 and 7.4. DESCRIPTION
Function: Antioxidant Calcium Benzoate occurs as white or colorless crystals, or as
Packaging and Storage: Store in tight containers, a white powder. It contains up to three molecules of water
preferably in a cool, dry place. of hydration. It is sparingly soluble in water.
Function: Preservative; antimicrobial agent
IDENTIFICATION Packaging and Storage: Store in well-closed containers.
• A. CALCIUM, Appendix IIIA
Sample solution: 100 mg/mL IDENTIFICATION
Acceptance criteria: Passes tests • A. PROCEDURE
• B. PROCEDURE Acceptance criteria: The retention time of the major
Sample solution: 100 mg/mL peak (excluding the solvent peak) in the chromatogram
Acceptance criteria: The Sample solution decolorizes of the Sample solution corresponds to that of the
dichlorophenol-indophenol TS. Standard solution in the Assay.
• B. INFRARED ABSORPTION, Spectrophotometric Identification
ASSAY Tests, Appendix IIIC
• PROCEDURE
Reference standard: USP Calcium Benzoate RS
Sample: 300 mg
Sample and standard preparation: K
Analysis: Dissolve the Sample in 50 mL of water in a
Acceptance criteria: The spectrum of the sample
250-mL Erlenmeyer flask and immediately titrate with
exhibits maxima at the same wavelengths as those in
0.1 N iodine to a pale yellow color that persists for at
the spectrum of the Reference standard.
least 30 s. Each mL of 0.1 N iodine is equivalent to
• C. CALCIUM, Appendix IIIA
10.66 mg of C12H14CaO12 · 2H2O.
Acceptance criteria: Passes tests
C12H14CaO12 · 2H2O ASSAY
• PROCEDURE
IMPURITIES Solution A: 20 mM potassium phosphate buffer, pH
2.5. Prepare by dissolving 2.72 g of potassium
• LEAD, Lead Limit Test, Flame Atomic Absorption
phosphate monobasic in 1000 mL of water and
Spectrophotometric Method, Appendix IIIB
adjusting with phosphoric acid to a pH of 2.5.
Sample: 10 g
Mobile phase: Solution A and acetonitrile (70:30, v/v)
Diluent: Water and acetonitrile (50:50, v/v)
SPECIFIC TESTS Standard stock solution: 1.0 mg/mL of USP Benzoic
• OPTICAL (SPECIFIC) ROTATION, Appendix IIB Acid RS in Diluent. Prepare by transferring 10 mg of
Sample solution: 50 mg/mL USP Benzoic Acid RS into a 10-mL volumetric flask,
Acceptance criteria: [α]D25 between +95° and +97° adding 5 mL of Diluent, sonicating for about 1 min, and
• OXALATE then diluting with Diluent to volume.
Sample solution: 1 g in 10 mL of water
186 / Calcium Benzoate / Monographs FCC 9
Standard solution: 0.1 mg/mL of USP Benzoic Acid RS CU = concentration of the sample in the Sample
prepared by diluting the Standard stock solution with solution (mg/mL)
Diluent Mr1 = molecular weight of calcium benzoate,
Sample stock solution: 1.0 mg/mL of sample in Diluent. 282.31
Prepare by transferring 10 mg of the sample into a 10- Mr2 = molecular weight of benzoic acid, 122.12
mL volumetric flask, adding 5 mL of Diluent, sonicating N = number of benzoic acid equivalents in
for about 1 min, and then diluting with Diluent to calcium benzoate, 2
volume. Acceptance criteria: NLT 99.0% and NMT 101.0%,
Sample solution: 0.1 mg/mL of sample prepared by calculated on the dried basis
diluting the Sample stock solution with Diluent
Monographs
Salicylic acid stock solution: 1.0 mg/mL of USP Salicylic IMPURITIES

Acid RS in Diluent. Prepare by transferring 10 mg of Inorganic Impurities
USP Salicylic Acid RS into a 10-mL volumetric flask, • FLUORIDE, Fluoride Limit Test, Method I or III, Appendix IIIB
adding 5 mL of Diluent, sonicating for about 1 min, and Analysis: Proceed as directed using a 5-g sample.
then diluting with Diluent to volume. Acceptance criteria: NMT 10 mg/kg
System suitability solution: 0.1 mg/mL each of USP • LEAD, Lead Limit Test, Flame Atomic Absorption
Salicylic Acid RS and 0.1 mg/mL of USP Benzoic Acid RS Spectrophotometric Method, Appendix IIIB
in Diluent prepared by transferring 1.0 mL each of the Sample: 10 g
Standard stock solution and Salicylic acid stock solution Acceptance criteria: NMT 2 mg/kg
into a 10-mL volumetric flask, and diluting with Diluent
SPECIFIC TESTS
to volume
• CHLORINATED COMPOUNDS
Chromatographic system, Appendix IIA
Sample: 0.25 g
Mode: High-performance liquid chromatography
Control: Mix 0.5 mL of 0.1 N silver nitrate with 20 mL
Detector: UV 230 nm
of dilute nitric acid TS containing 0.5 mL of 0.01 N
Column: 4.6- × 150-mm, packed with 5-µm reversed
hydrochloric acid.
phase C18 silica gel1
Analysis: Dissolve the Sample in 10 mL of water. Acidify
Column temperature: 25°
with nitric acid and filter off the precipitate. Mix the
Flow rate: 1.0 mL/min
precipitate with 0.5 g of calcium carbonate, dry the
mixture, and then ignite. Take up the ignition residue in
System suitability
20 mL of dilute nitric acid TS, and filter. Mix the filtrate
Sample: System suitability solution. [NOTE—The
with 0.5 mL of 0.1 N silver nitrate.
retention times for benzoic acid and salicylic acid are
approximately 5.7 min and 6.7 min, respectively.]
Sample does not exceed that produced by the Control.
Suitability requirement 1: The relative standard
(NMT 0.07% as Cl2)
deviation for the benzoic acid peak area is NMT
• LOSS ON DRYING, Appendix IIC: 105° for 4 h
0.37% for five replicate injections.
Suitability requirement 2: The peak tailing factor is
• READILY OXIDIZABLE SUBSTANCES
NMT 2.0 for the benzoic acid peak.
Sample: 1 g
Suitability requirement 3: The resolution, R, is NLT
Analysis: Add 0.1 N potassium permanganate,
1.5 between the benzoic acid and salicylic acid peaks.
dropwise, to a mixture of 100 mL of water and 1.5 mL
Suitability requirement 4: The number of theoretical
of sulfuric acid heated to boiling, until a pink color
plates, N, is NLT 5000 for the benzoic acid peak.
persists for 30 s. Dissolve the Sample in the hot
Analysis: Separately inject equal volumes of the Standard
solution. Titrate with 0.1 N potassium permanganate to
solution and Sample solution into the chromatograph,
a pink color that persists for 15 s.
and measure the responses for the major peaks on the
Acceptance criteria: The volume of 0.1 N potassium
resulting chromatograms.
permanganate consumed in the titration does not
Calculate the percentage of calcium benzoate in the
exceed 0.5 mL.
sample taken:
• WATER-INSOLUBLE MATTER
Result = (rU/rS) × (CS/CU) × (Mr1/Mr2) × N × 100 Sample: 10 g
Analysis: Dissolve the Sample in 100 mL of hot water.
rU = peak area for benzoic acid in the Sample Filter through a tared Gooch crucible, and wash any
solution residue with hot water. Dry the crucible for 2 h at 105°.
rS = peak area for benzoic acid in the Standard Cool, weigh, and calculate the percentage of water-
solution insoluble matter.
CS = concentration of USP Benzoic Acid RS in the Acceptance criteria: NMT 0.3%
Standard solution, corrected for purity
based on the label claim (mg/mL)
1 Luna C18(2) (Phenomenex, Torrance, CA), or equivalent.

FCC 9 Monographs / Calcium Carbonate / 187
Calcium Bromate Calcium Carbonate

. .
First Published: Prior to FCC 6 First Published: Prior to FCC 6
Ca(BrO3)2 · H2O Formula wt 313.90 CaCO3 Formula wt 100.09

INS: 924b CAS: [10102-75-7] INS: 170(i) CAS: [471-34-1]
UNII: QJ2S78C3RO [calcium bromate] UNII: H0G9379FGK [calcium carbonate]
DESCRIPTION DESCRIPTION
Calcium Bromate occurs as a white, crystalline powder. It is Calcium Carbonate occurs as a fine, white or colorless,
Monographs
very soluble in water. microcrystalline powder. It is stable in air, and it is
Function: Maturing agent; oxidizing agent practically insoluble in water and in alcohol. The presence
Packaging and Storage: Store in well-closed containers. of any ammonium salt or carbon dioxide increases its
solubility in water, but the presence of any alkali hydroxide
IDENTIFICATION reduces the solubility.
• A. PROCEDURE Function: pH control agent; nutrient; dough conditioner;
Sample solution: 50 mg/mL in 2.7 N hydrochloric acid firming agent; yeast nutrient
Acceptance criteria: The Sample solution imparts a Packaging and Storage: Store in well-closed containers.
transient yellow-red color to a nonluminous flame.
• B. PROCEDURE IDENTIFICATION
Sample solution: 50 mg/mL • A. PROCEDURE
Analysis: Add sulfurous acid dropwise to the Sample Analysis: Add a sample to 1 N acetic acid, 2.7 N
solution. hydrochloric acid, and 1.7 N nitric acid.
Acceptance criteria: A yellow color develops that Acceptance criteria: The sample dissolves with
disappears upon the addition of an excess of sulfurous effervescence in each case.
acid. • B. CALCIUM, Appendix IIIA
Sample solutions: The three resulting solutions from
ASSAY Identification Test A
• PROCEDURE Analysis: Boil the solutions and perform the test for
Sample: 900 mg Calcium on each.
Analysis: Dissolve the Sample in 50 mL of water in a Acceptance criteria: Each solution passes tests.
250-mL glass-stoppered Erlenmeyer flask. To the flask,
add 3 g of potassium iodide followed by 3 mL of ASSAY
hydrochloric acid. Allow the mixture to stand for 5 min • PROCEDURE
and then add 100 mL of cold water. Titrate the Sample: 200 mg, previously dried
liberated iodine with 0.1 N sodium thiosulfate, adding Analysis: Transfer the Sample into a 400-mL beaker, add
starch TS near the endpoint. Perform a blank 10 mL of water, and swirl to form a slurry. Cover the
determination (see General Provisions), and make any beaker with a watch glass and introduce 2 mL of 2 N
necessary correction. Each mL of 0.1 N sodium hydrochloric acid from a pipet inserted between the lip
thiosulfate is equivalent to 26.16 mg of Ca(BrO3)2 · H2O. of the beaker and the edge of the watch glass. Swirl
Acceptance criteria: NLT 99.8% and NMT 100.5% of the contents of the beaker to dissolve the sample. Wash
Ca(BrO3)2 · H2O down the sides of the beaker, the outer surface of the
pipet, and the watch glass, and dilute the contents to
IMPURITIES about 100 mL with water. While stirring, preferably
Inorganic Impurities with a magnetic stirrer, add about 30 mL of 0.05 M
• LEAD, Lead Limit Test, Appendix IIIB disodium EDTA from a 50-mL buret, then add 15 mL of
Sample solution: Dissolve 2 g of sample in 10 mL of 1 N sodium hydroxide and 300 mg of hydroxy
water, 10 mL of hydrochloric acid, and evaporate to naphthol blue indicator. Continue the titration to a blue
dryness on a steam bath. Dissolve the residue in 5 mL endpoint. Each mL of 0.05 M disodium EDTA is
of hydrochloric acid, again evaporate to dryness, and equivalent to 5.004 mg of CaCO3.
then dissolve the residue in 40 mL of water. Acceptance criteria: NLT 98.0% and NMT 100.5% of
Control: 4 µg Pb (4 mL of Diluted Standard Lead CaCO3, on the dried basis
Solution)
Analysis: Use 20 mL of the Sample solution. IMPURITIES
Acceptance criteria: NMT 4 mg/kg Inorganic Impurities
• ACID-INSOLUBLE SUBSTANCES
Sample: 5 g
Analysis: Suspend the Sample in 25 mL of water and
agitate the suspension while cautiously adding 25 mL
of 1:2 hydrochloric acid. Add water to make a volume
of about 200 mL. Heat the solution to boiling, cover,
digest on a steam bath for 1 h, cool, and filter. Wash
188 / Calcium Carbonate / Monographs FCC 9
the precipitate with water until the last washing shows DESCRIPTION
no chloride with silver nitrate TS, and then ignite it. Calcium Chloride occurs as white, hard fragments, granules,
Acceptance criteria: The weight of the residue does or powder. It is anhydrous or contains two molecules of
not exceed 10 mg. (NMT 0.2%) water of hydration. It is deliquescent. It is soluble in water;
• ARSENIC, Arsenic Limit Test, Appendix IIIB slightly soluble in alcohol. The pH of a 1:20 aqueous
Sample solution: 1 g in 10 mL of 2.7 N hydrochloric solution is between 4.5 and 11.0.
acid Function: Firming agent
Acceptance criteria: NMT 3 mg/kg Packaging and Storage: Store in tight containers.
• FLUORIDE, Fluoride Limit Test, Method III, Appendix IIIB
Acceptance criteria: NMT 0.005% IDENTIFICATION
Monographs
• LEAD, Lead Limit Test, Appendix IIIB • CALCIUM, Appendix IIIA

Sample solution: Cautiously dissolve 5 g of sample in Sample solution: 100 mg/mL
25 mL of 1:2 hydrochloric acid and evaporate the Acceptance criteria: Passes test
solution to dryness on a steam bath. Dissolve the • CHLORIDE, Appendix IIIA
residue in about 15 mL of water and dilute to 25 mL Sample solution: 100 mg/mL
(200 mg/mL). Acceptance criteria: Passes test
Control: 12 µg Pb (12 mL of Diluted Standard Lead
Solution)
ASSAY
• PROCEDURE
Analysis: Use 20 mL of Sample solution.
Sample: 1.5 g
[NOTE—As an alternative to the above test, determine
Analysis: Transfer the Sample into a 250-mL volumetric
as directed in the Lead Limit Test, APDC Extraction
flask, dissolve it in a mixture of 100 mL of water and 5
Method, Appendix IIIB.]
mL of 2.7 N hydrochloric acid, dilute with water to
volume, and mix. Transfer 50 mL of this solution into a
• MAGNESIUM AND ALKALI SALTS
suitable container and add 50 mL of water. While
Sample: 1 g
stirring, preferably with a magnetic stirrer, add about
Analysis: Mix the Sample with 40 mL of water, carefully
30 mL of 0.05 M disodium EDTA from a 50-mL buret.
add 5 mL of hydrochloric acid, mix, and boil for 1 min.
Then, add 15 mL of 1 N sodium hydroxide and 300 mg
Rapidly add 40 mL of oxalic acid TS and stir vigorously
of hydroxy naphthol blue indicator. Continue the
until precipitation is well established. Immediately add
titration to a blue endpoint. Each mL of 0.05 M
2 drops of methyl red TS. Then, add 6 N ammonium
disodium EDTA is equivalent to 5.55 mg of calcium
hydroxide, dropwise, until the mixture is just alkaline,
chloride (CaCl2) or 7.35 mg of calcium chloride
and cool. Transfer the mixture to a 100-mL graduated
dihydrate (CaCl2 · 2H2O).
cylinder, dilute to 100 mL with water, and let stand for
Acceptance criteria
4 h or overnight. Decant the clear, supernatant liquid
Anhydrous: NLT 93.0% and NMT 100.5% of CaCl2
through a dry filter paper and place 50 mL of the clear
Dihydrate: NLT 99.0% and NMT 107.0% of CaCl2 ·
filtrate in a platinum dish. Add 0.5 mL of sulfuric acid,
2H2O
and evaporate the mixture on a steam bath to a small
volume. Carefully evaporate the remaining liquid to IMPURITIES
dryness over a free flame and continue heating until
the ammonium salts have been completely Change to read:
decomposed and volatilized. Finally, ignite the residue Inorganic Impurities
to constant weight. • ACID-INSOLUBLE MATTER (ANHYDROUS SALT)
Acceptance criteria: The weight of the residue does Filter assembly: Place a 32-mm (od) ▲ ▲ FCC9 disk filter1
not exceed 5 mg. (NMT 1%) in a suitable filter assembly comprised of a 2.5-L screw-
cap bottle cut in half horizontally and fitted with a
SPECIFIC TESTS rubber washer (35-mm od and 25-mm id), followed by
the ▲ disk filter,▲ FCC9 a 20-mesh stainless steel screen
Acceptance criteria: NMT 2%
(35-mm od), and a bottle cap with a 25-mm hole in
the top.
Sample solution: Dissolve 1 kg sample in 3 L of water
containing 10 mL of glacial acetic acid. Allow the
Calcium Chloride
.
solution to cool.
First Published: Prior to FCC 6 Analysis: Wash the Filter assembly, with the filter at the
Last Revision: FCC 9 bottom, with 100 mL of 1:300 acetic acid, followed by
100 mL of water. Remove the disk from the assembly,
CaCl2 Formula wt, anhydrous 110.98 place it on a watch glass, dry the combination at 105°
CaCl2 · 2H2O Formula wt, dihydrate 147.01 for 2 h, let cool, and weigh. Filter the Sample solution
CAS: anhydrous [10043-52-4] through the lintine disk. Rinse the walls of the Filter
INS: 509 CAS: dihydrate [10035-04-8] assembly so that all insoluble matter is transferred to
UNII: M4I0D6VV5M [calcium chloride] 1Visible Sediment Test Card Company (www.visible-sediment.com) part #
1.25 filter disc, or equivalent.
FCC 9 Monographs / Calcium Chloride Solution / 189
the disk, and wash with 100 mL of water. Place the temperature. It is nominally available in a concentration
disk on the same watch glass mentioned above, dry at range of about 35% to 45% of CaCl2.
105° for 2 h, let cool and weigh the combination, Function: Sequestrant; firming agent
being careful at all times not to lose any particles that Packaging and Storage: Store in tight containers.
may be on the disk. The difference in the two weights
is the weight of the acid-insoluble matter. Place the IDENTIFICATION
disk under a low-power magnifier (4× to 10× • CALCIUM, Appendix IIIA
magnification). Using a millimeter rule, measure the Sample solution: 100 mg/mL (CaCl2 basis)
largest dimension of each particle (or as many as may Acceptance criteria: Passes tests
be necessary) on the disk. • CHLORIDE, Appendix IIIA
Monographs
Acceptance criteria Sample solution: 100 mg/mL (CaCl2 basis)
Anhydrous: NMT 0.02%; no particles of sample Acceptance criteria: Passes test
greater than 2 mm in any dimension are present
ASSAY
• PROCEDURE
Sample solution: 1 g in 10 mL
Sample: Quantity equivalent to 1 g of CaCl2
Analysis: Transfer the Sample into a 250-mL volumetric
• FLUORIDE, Fluoride Limit Test, Method III, Appendix IIIB
flask, add 5 mL of 2.7 N hydrochloric acid and 100 mL
of water to dissolve; dilute to volume with water, and
• LEAD, Lead Limit Test, Appendix IIIB
mix. Transfer 50.0 mL of this solution into a suitable
Sample solution: 1 g in 20 mL
container and add 50 mL of water. While stirring,
preferably with a magnetic stirrer, add about 30 mL of
Solution)
0.05 M disodium EDTA from a 50-mL buret. Then add
15 mL of 1 N sodium hydroxide and 300 mg of
hydroxy naphthol blue indicator. Continue the titration
Sample: 1 g
to a blue endpoint. Each mL of 0.05 M disodium EDTA
Analysis: Dissolve the Sample in 50 mL of water, add
is equivalent to 5.55 mg of CaCl2.
500 mg of ammonium chloride, mix, and boil for 1
Acceptance criteria: NLT 90.0% and NMT 110.0%, by
min. Rapidly add 40 mL of oxalic acid TS and stir
weight, of the labeled amount of calcium chloride,
vigorously until precipitation is well established.
expressed as CaCl2
Immediately add 2 drops of methyl red TS. Then add
6 N ammonium hydroxide, dropwise, until the mixture IMPURITIES
is just alkaline, and cool. Transfer the mixture to a 100- Inorganic Impurities
mL cylinder, dilute with water to 100 mL, and let it • FLUORIDE, Fluoride Limit Test, Method III, Appendix IIIB
stand for 4 h or overnight. Decant the clear, Sample: Quantity equivalent to 1 g of CaCl2
supernatant liquid through a dry filter paper, and Acceptance criteria: NMT 0.004%, calculated on the
transfer 50 mL of the clear filtrate to a platinum dish. amount of CaCl2 as determined in the Assay
Add 0.5 mL of sulfuric acid to the dish, and evaporate • LEAD, Lead Limit Test, Appendix IIIB
the mixture on a steam bath to a small volume. Sample solution: Quantity of sample equivalent to 1 g
Carefully evaporate the remaining liquid to dryness of CaCl2, diluted to 10 mL
over a free flame, and continue heating until the Control: 4 µg Pb (4 mL of Diluted Standard Lead
ammonium salts have been completely decomposed Solution)
and volatilized. Finally, ignite the residue to constant Acceptance criteria: NMT 4 mg/kg, calculated on the
weight. amount of CaCl2 as determined in the Assay
Acceptance criteria • MAGNESIUM AND ALKALI SALTS
Anhydrous: NMT 25 mg of residue (NMT 5.0%) Sample solution: Quantity of sample equivalent to 1 g
Dihydrate: NMT 20 mg of residue (NMT 4.0%) of CaCl2, diluted to 50 mL
Analysis: To the Sample solution, add 500 mg of
OTHER REQUIREMENTS ammonium chloride, mix, and boil for 1 min. Rapidly
• LABELING: Indicate whether the salt is anhydrous or
add 40 mL of oxalic acid TS and stir vigorously until
dihydrate.
precipitation is well established. Immediately add 2
drops of methyl red TS, then add 6 N ammonium
hydroxide, dropwise, until the mixture is just alkaline,
and cool. Transfer the mixture to a 100-mL cylinder,
Calcium Chloride Solution
.
dilute to 100 mL with water, and let it stand for 4 h or

First Published: Prior to FCC 6 overnight. Decant the clear, supernatant liquid through
a dry filter paper and transfer 50 mL of the clear filtrate
UNII: OFM21057LP [calcium chloride anhydrous] to a platinum dish. Add 0.5 mL of sulfuric acid to the
dish and evaporate the mixture on a steam bath to a
DESCRIPTION small volume. Carefully evaporate the remaining liquid
Calcium Chloride Solution occurs as a clear to slightly to dryness over a free flame, and continue heating until
turbid, colorless or slightly colored liquid at room the ammonium salts have been completely
190 / Calcium Chloride Solution / Monographs FCC 9
decomposed and volatilized. Finally, ignite the residue to about 100 mL with water. While stirring, preferably
to constant weight. with a magnetic stirrer, add about 30 mL of 0.05 M
Acceptance criteria: The weight of the residue does disodium EDTA from a 50-mL buret. Add 15 mL of 1 N
not exceed 25 mg, calculated on the amount of CaCl2 sodium hydroxide and 300 mg of hydroxy naphthol
as determined in the Assay (NMT 5.0%). blue indicator, and continue the titration to a blue
endpoint. Each mL of 0.05 M disodium EDTA is
SPECIFIC TESTS equivalent to 8.300 mg of Ca3(C6H5O7)2.
• ALKALINITY (AS CA(OH)2) Acceptance criteria: NLT 97.5% and NMT 100.5% of
Sample solution: Quantity of sample equivalent to 5 g Ca3(C6H5O7)2, on the dried basis
of CaCl2 diluted to 50 mL
Monographs
Analysis: Add phenolphthalein TS to the Sample solution IMPURITIES

and titrate with 0.1 N hydrochloric acid. Each mL of Inorganic Impurities
0.1 N hydrochloric acid is equivalent to 3.71 mg of • FLUORIDE, Fluoride Limit Test, Method III, Appendix IIIB
Ca(OH)2. Sample solution: Prepare as directed using 10 mL of
Acceptance criteria: NMT 0.3% hydrochloric acid instead of 20 mL.
Analysis: Prepare a calibration curve as directed using
1.0, 5.0, and 10.0 mL of the Sodium Fluoride Solution
(equivalent to 5.0, 25.0, and 50.0 mg/kg of fluoride,
Calcium Citrate
.
respectively).
Tricalcium Citrate Sample: 10 g
SPECIFIC TESTS
Acceptance criteria: Between 10.0% and 14.0%
Ca3(C6H5O7)2 · 4H2O Formula wt 570.50

INS: 333 CAS: [5785-44-4]
Calcium Cyclamate
.
UNII: MLM29U2X85 [calcium citrate]

First Published: Third Supplement, FCC 7
DESCRIPTION
Calcium Citrate occurs as a fine, white powder. It is very Calcium Cyclohexanesulfamate
slightly soluble in water, but it is insoluble in alcohol. Calcium Cyclohexylsulfamate
Function: Sequestrant; buffer; firming agent
IDENTIFICATION
• A. PROCEDURE
Sample: 500 mg
C12H24CaN2O6S2 · 2H20 Formula wt, anhydrous 396.53
Analysis: Dissolve the Sample in 10 mL of water and 2.5
Formula wt, dihydrate 432.57
mL of 1.7 N nitric acid. Add 1 mL of mercuric sulfate INS: 952(ii) CAS: anhydrous [139-06-0]
TS, heat to boiling, and then add potassium dihydrate [5897-16-5]
permanganate TS.
Acceptance criteria: A white precipitate forms. DESCRIPTION
• B. PROCEDURE Calcium Cyclamate occurs as colorless to white crystals or
Sample: 500 mg crystalline powder. It is soluble in water and sparingly
Analysis: Completely ignite the Sample at as low a soluble in ethanol.
temperature as possible. Cool the residue and dissolve it Function: Sweetener
in a mixture of 10 mL of water and 1 mL of glacial Packaging and Storage: Store in tight containers in a
acetic acid. Filter and add 10 mL of ammonium oxalate cool, dry place.
TS to the filtrate.
Acceptance criteria: A voluminous, white precipitate IDENTIFICATION
forms that is soluble in hydrochloric acid. • CALCIUM, Appendix IIIA
ASSAY Acceptance criteria: Passes test
• PROCEDURE • INFRARED ABSORPTION, Spectrophotometric Identification
Sample: 350 mg, previously dried Tests, Appendix IIIC
Analysis: Dissolve the Sample, in a mixture of 10 mL of Reference standard: USP Calcium Cyclamate RS
water and 2 mL of 2.7 N hydrochloric acid, and dilute Sample and standard preparation: K
FCC 9 Monographs / Calcium Disodium EDTA / 191
Acceptance criteria: The spectrum of the sample Carrier gas: Helium

exhibits maxima at the same wavelengths as those in Flow rate: 1.8 mL/min
the spectrum of the Reference standard. Temperature
Injection port: 250°
ASSAY Detector: 270°
• PROCEDURE Column: See the temperature program in the table
Sample: 0.4 g below.
Analysis: Dissolve the Sample in a mixture of 50 mL of
water and 5 mL of hydrochloric acid TS, diluted. Titrate Time Temperature
the solution with 0.1 M sodium nitrite. Add the last mL (min) (°)
Monographs
of titrant dropwise until a blue color is produced 0–1 85
immediately when a glass rod dipped into the titrated 1–9 85–150
solution is streaked on a piece of starch iodide test
9–13 150
paper. Alternatively, the endpoint may be determined
potentiometrically. When the titration is complete, the
Injection volume: 1.5 µL. Use a split vent at a flow
endpoint is reproducible after the mixture has been
rate of 20 mL/min.
allowed to stand for 1 min. Each mL of 0.1 M sodium
Analysis: Separately inject equal volumes of the
nitrite is equivalent to 19.83 mg of C12H24CaN2O6S2.
Standard solution and Sample solution into the
Acceptance criteria: 98.0%–102.0%, calculated on the
chromatograph, record the chromatograms, and
anhydrous basis
measure the responses. [NOTE—The approximate
IMPURITIES retention times (relative to cyclohexanamine, which
Inorganic Impurities has a retention time of about 2.3 min) for aniline,
• LEAD, Lead Limit Test, Flame Atomic Absorption tetradecane, and N-cyclohexylcyclohexanamine are
Spectrophotometric Method, Appendix IIIB about 1.4 min, 4.3 min, and 4.5 min, respectively.]
Sample: 5 g Acceptance criteria
Acceptance criteria: NMT 1.0 mg/kg Cyclohexanamine: NMT 10.0 mg/kg
Organic Impurities Aniline: NMT 1.0 mg/kg
• CYCLOHEXANAMINE, ANILINE, AND N-Cyclohexylcyclohexanamine: NMT 1.0 mg/kg
N-CYCLOHEXYLCYCLOHEXANAMINE
SPECIFIC TESTS
Internal standard solution: Dissolve 0.02 µL/mL of
tetradecane in methylene chloride.
Acceptance criteria: 6.0%–9.0%
Solution A: Dissolve 10 mg of cyclohexanamine, 1 mg
of N-cyclohexylcyclohexanamine, and 1 mg of aniline
in water, then dilute with the same solvent to 1000
mL. Dilute 10 mL of this solution with water to 100
Calcium Disodium EDTA
.
mL.
Solution B: 42% w/v sodium hydroxide solution First Published: Prior to FCC 6
Standard solution: To 20 mL of Solution A, add 0.5 mL
of Solution B, and extract with 30 mL of toluene. Shake Calcium Disodium Ethylenediaminetetraacetate
20 mL of the upper layer with 4 mL of a mixture of Calcium Disodium (Ethylenedinitrilo)tetraacetate
equal volumes of water and an acetic acid solution Calcium Disodium Edetate
(12% w/v). Separate the lower layer, add 0.5 mL of
Solution B and 0.5 mL of the Internal standard solution,
and shake. Use the lower layer immediately after
separation.
Sample solution: Dissolve 2 g of sample in 20 mL of
water, add 0.5 mL of Solution B, and shake with 30 mL
C10H12CaN2Na2O8 · 2H2O Formula wt 410.30
of toluene. Shake 20 mL of the upper layer with 4 mL INS: 385 CAS: [23411-34-9]
of a mixture of equal volumes of an acetic acid solution
UNII: 25IH6R4SGF [edetate calcium disodium]
(12% w/v) and water. Separate the lower layer, add
0.5 mL of Solution B and 0.5 mL of the Internal DESCRIPTION
standard solution, and shake. Use the lower layer Calcium Disodium EDTA occurs as white, crystalline granules
immediately after separation. or as a white to off-white powder. It is slightly hygroscopic
Chromatographic system, Appendix IIA and is stable in air. It is freely soluble in water.
Mode: Gas chromatography Function: Preservative; sequestrant
Detector: Flame ionization Packaging and Storage: Store in well-closed containers.
Column: 25-m × 0.32-mm (i.d.) fused-silica column
with poly(dimethyl)(diphenyl)siloxane containing IDENTIFICATION
95% of methyl groups and 5% of phenyl groups • A. CALCIUM, Appendix IIIA
(DB-5, SE52) as stationary phase (film thickness 0.51 Sample solution: 50 mg/mL
µm) Acceptance criteria: Passes oxalate test
192 / Calcium Disodium EDTA / Monographs FCC 9
• B. INFRARED ABSORPTION, Spectrophotometric Identification Cupric nitrate solution, and mix. Sonicate, if necessary,
Tests, Appendix IIIC to aid in dissolution.
Reference standard: USP Edetate Calcium Disodium RS Chromatographic system, Appendix IIA
Sample and standard preparation: M Mode: High-performance liquid chromatography
Acceptance criteria: The spectrum of the Sample Detector: UV 254 nm
exhibits maxima at the same wavelengths as those in Column: 15-cm × 4.6-mm column that contains 5- to
the spectrum of the Reference standard. 10-mm porous microparticles of silica bonded to
• C. SODIUM, Appendix IIIA octylsilane (Zorbax 8, or equivalent)
Sample solution: 50 mg/mL Flow rate: about 2 mL/min
Acceptance criteria: Passes flame test Injection volume: about 50 µL
Monographs
• D. PROCEDURE System suitability

Sample: 50 mg Sample: Standard solution
Analysis: Add 2 drops of ammonium thiocyanate TS and Suitability requirement 1: The resolution between
2 drops of ferric chloride TS to 5 mL of water contained nitrilotriacetic acid and calcium disodium EDTA is
in a test tube. Add the Sample to the deep red solution NLT 4.0.
so obtained and mix. Suitability requirement 2: The relative standard
Acceptance criteria: The deep red color disappears. deviation is NMT 2.0% for three replicate injections.
Analysis: Separately inject equal volumes of the
ASSAY Standard solution and the Sample solution into the
• PROCEDURE chromatograph, record the chromatograms, and
Sample: 1.2 g measure the responses for the major peaks. [NOTE—
Analysis: Transfer the Sample to a 250-mL beaker and The retention times are about 3.5 min for
dissolve in 75 mL of water. Add 25 mL of 1 N acetic nitrilotriacetic acid and 9 min for calcium disodium
acid and 1.0 mL of diphenylcarbazone TS and titrate EDTA.]
slowly with 0.1 M mercuric nitrate to the first Acceptance criteria: The response of the
appearance of a purple color. Each mL of 0.1 M nitrilotriacetic acid peak of the Sample solution does
mercuric nitrate is equivalent to 37.43 mg of not exceed the difference between the nitrilotriacetic
C10H12CaN2Na2O8. acid peak responses obtained from the Standard
Acceptance criteria: NLT 97.0% and NMT 102.0% of solution and the Sample solution. (NMT 0.1%)
C10H12CaN2Na2O8, calculated on the anhydrous basis
SPECIFIC TESTS
IMPURITIES • MAGNESIUM-CHELATING SUBSTANCES
Inorganic Impurities Buffer solution: Dissolve 67.5 g of ammonium chloride
• LEAD, Lead Limit Test, Appendix IIIB in 200 mL of water. Add 570 mL of ammonium
Sample solution: Prepare as directed for organic hydroxide and dilute to 1000 mL with water.
compounds, using 70% perchloric acid instead of 30% Sample solution: 1 g of sample in 5 mL of water and 5
hydrogen peroxide to decompose the sample. mL of Buffer solution
[CAUTION—Handle perchloric acid in an appropriate Analysis: Add 5 drops of eriochrome black TS to the
fume hood.] Sample solution and titrate with 0.1 M magnesium
Control: 4 µg Pb (4 mL of Diluted Standard Lead acetate to the appearance of a deep wine red color.
Solution) Acceptance criteria: NMT 2.0 mL of 0.1 M magnesium
Acceptance criteria: NMT 4 mg/kg acetate titrant is required
Organic Impurities • PH, pH Determination, Appendix IIB
• NITRILOTRIACETIC ACID Sample solution: 10 mg/mL
Mobile phase: Add 10 mL of a 1:4 solution of Acceptance criteria: Between 6.5 and 7.5
tetrabutylammonium hydroxide in methanol to 200 mL • WATER, Water Determination, Appendix IIB
of water, and adjust with 1 M phosphoric acid to a pH Acceptance criteria: NMT 13.0%
of 7.5 ± 0.1. Transfer the solution into a 1000-mL
volumetric flask, add 90 mL of methanol, dilute to
volume with water, mix, filter through a membrane
filter (0.5-µm or finer porosity), and de-gas.
Calcium Gluconate
.
Cupric nitrate solution: 10 mg/mL

Standard stock solution: Transfer 100 mg of First Published: Prior to FCC 6
nitrilotriacetic acid into a 10-mL volumetric flask; add
0.5 mL of ammonium hydroxide, and mix. Dilute to
volume with water, and mix.
Standard solution: Transfer 1.0 g of sample into a 100-
mL volumetric flask. Add 100 µL of Standard stock
solution, dilute to volume with Cupric nitrate solution,
and mix. Sonicate, if necessary, to aid in dissolution. C12H22CaO14 Formula wt, anhydrous 430.38
Sample solution: Transfer 1.0 g of sample into a 100- C12H22CaO14 · H2O Formula wt, monohydrate 448.39
mL volumetric flask, dilute to volume with INS: 578 CAS: anhydrous [299-28-5]
FCC 9 Monographs / Calcium Glycerophosphate / 193
UNII: SQE6VB453K [calcium gluconate] Acceptance criteria: NMT 2 mg/kg

Organic Impurities
DESCRIPTION • SUCROSE AND REDUCING SUGARS
Calcium Gluconate occurs as white, crystalline granules or Sample: 1.0 g
powder. It is anhydrous or contains one molecule of water Analysis: Transfer the Sample into a 250-mL conical
of hydration. It is stable in air. One g dissolves slowly in flask and add 20 mL of hot water to dissolve the
about 30 mL of water at 25° and in about 5 mL of boiling sample. Cool the flask, add 25 mL of alkaline cupric
water. It is insoluble in alcohol and in many other organic citrate TS, cover the flask, and boil gently for 5 min,
solvents. Its solutions are neutral to litmus. accurately timed. Cool the flask rapidly to room
Function: Firming agent; stabilizer; texturizer temperature, add 25 mL of 0.6 N acetic acid, 10.0 mL
Monographs
Packaging and Storage: Store in well-closed containers. of 0.1 N iodine, and 10 mL of 2.7 N hydrochloric acid.
Immediately titrate with 0.1 N sodium thiosulfate,
IDENTIFICATION using starch TS as the indicator. Perform a blank
determination (See General Provisions) and make any
Sample: 20 mg/mL
necessary correction. Each mL of 0.1 N sodium
thiosulfate consumed is equivalent to 2.7 mg of
• THIN-LAYER CHROMATOGRAPHY, Appendix IIA
reducing substances (as dextrose).
Sample solution: 10 mg/mL (Heat in a water bath at
60°, if necessary, to dissolve the sample.)
Standard solution: 10 mg/mL of USP Potassium SPECIFIC TESTS
Gluconate RS • LOSS ON DRYING, Appendix IIC: 105° for 16 h
Adsorbent: 0.25-mm layer of chromatographic silica gel Acceptance criteria:
Developing solvent system: Alcohol, water, ammonium Anhydrous: NMT 3.0%
hydroxide, and ethyl acetate [50:30:10:10] Monohydrate: NMT 2.0%
Spray reagent: Dissolve 2.5 g of ammonium molybdate
in 50 mL of 2 N sulfuric acid in a 100-mL volumetric OTHER REQUIREMENTS
flask. Add 1.0 g of ceric sulfate, swirl to dissolve, dilute • LABELING: Indicate whether the material is anhydrous or
to volume with 2 N sulfuric acid, and mix. the monohydrate.
Application volume: 5 µL
Analysis: Develop the chromatogram in the Developing
solvent system until the solvent front has moved about
Calcium Glycerophosphate
.
three-fourths of the length of the plate. Remove the

plate from the chamber and dry at 110° for 20 min. First Published: Prior to FCC 6
Allow to cool and spray with Spray reagent. After
spraying, heat the plate at 110° for about 10 min.
C3H7CaO6P Formula wt 210.14
Acceptance criteria: The principal spot obtained from
INS: 383 CAS: [27214-00-2]
the Sample solution corresponds in color, size, and RF
UNII: XWV9Z12C1C [calcium glycerophosphate]
value to that obtained from the Standard solution.
ASSAY DESCRIPTION
• PROCEDURE Calcium Glycerophosphate occurs as a fine, white powder.
Sample: 800 mg It is somewhat hygroscopic. One g dissolves in about 50
Analysis: Dissolve the Sample in 100 mL of water mL of water at 25°. It is more soluble in water at a lower
containing 2 mL of 2.7 N hydrochloric acid. While temperature, and citric acid increases its solubility in water.
stirring, preferably with a magnetic stirrer, add about It is insoluble in alcohol.
30 mL of 0.05 M disodium EDTA from a 50-mL buret. Function: Nutrient
Then, add 15 mL of 1 N sodium hydroxide and 300 mg Packaging and Storage: Store in tight containers.
of hydroxy naphthol blue indicator and continue the IDENTIFICATION
titration to a blue endpoint. Each mL of 0.05 M • CALCIUM, Appendix IIIA
disodium EDTA is equivalent to 21.52 mg of Sample solution: Saturated solution
C12H22CaO14 or 22.42 mg of C12H22CaO14 · H2O. Acceptance criteria: Passes tests
Acceptance criteria
Anhydrous: NLT 98.0% and NMT 102.0% of ASSAY
C12H22CaO14, calculated on the dried basis • PROCEDURE
Monohydrate: NLT 98.0% and NMT 102.0% of Sample: 2 g, previously dried
C12H22CaO14 · H2O, calculated on the as-is basis Analysis: Dissolve the Sample in 100 mL of water and 5
mL of 2.7 N hydrochloric acid. Transfer the solution
IMPURITIES into a 250-mL volumetric flask, dilute to volume with
Inorganic Impurities water, and mix well. Pipet 50.0 mL of this solution into
• LEAD, Lead Limit Test, Flame Atomic Absorption a suitable container and add 50 mL of water. While
Spectrophotometric Method, Appendix IIIB stirring, preferably with a magnetic stirrer, add about
Sample: 10 g 30 mL of 0.05 M disodium EDTA from a 50-mL buret.
194 / Calcium Glycerophosphate / Monographs FCC 9
Then, add 15 mL of 1 N sodium hydroxide and 300 mg solution into a 500-mL volumetric flask. Rinse the
of hydroxy naphthol blue indicator. Continue the beaker thoroughly and add the rinsings to the flask.
titration to a blue endpoint. Each mL of 0.05 M Dilute to volume with water and mix.
disodium EDTA is equivalent to 10.51 mg of Analysis: Transfer 50.0 mL of the Sample solution into a
C3H7CaO6P. suitable container and add 50 mL of water. While
Acceptance criteria: NLT 98.0% and NMT 100.5% stirring, preferably with a magnetic stirrer, add about
C3H7CaO6P, on the dried basis 30 mL of 0.05 M disodium EDTA from a 50-mL buret.
IMPURITIES of hydroxy naphthol blue indicator. Continue the
Inorganic Impurities titration to a blue endpoint. Each mL of 0.05 M
Monographs
• LEAD, Lead Limit Test, APDC Extraction Method, Appendix disodium EDTA is equivalent to 3.705 mg of Ca(OH)2.
IIIB Acceptance criteria: NLT 95.0% and NMT 100.5% of
Acceptance criteria: NMT 4 mg/kg Ca(OH)2
SPECIFIC TESTS IMPURITIES
• ALKALINITY Inorganic Impurities
Sample: 1 g • ARSENIC, Arsenic Limit Test, Appendix IIIB
Analysis: Dissolve the Sample in 60 mL of water. Titrate Sample solution: 1 g in 15 mL of 2.7 N hydrochloric
the solution with 0.1 N sulfuric acid to neutralization, acid
using 3 drops of phenolphthalein TS as indicator. Acceptance criteria: NMT 3 mg/kg
Acceptance criteria: Not more than 1.5 mL of acid is • CARBONATE
required. Sample solution: To 2 g of sample in 50 mL of water,
• LOSS ON DRYING, Appendix IIC: 150° for 4 h add an excess of 2.7 N hydrochloric acid.
Acceptance criteria: NMT 12.0% Acceptance criteria: No more than a slight
effervescence is observed.
Sample: 1.0 g
Calcium Hydroxide
.

First Published: Prior to FCC 6 • LEAD, Lead Limit Test, Appendix IIIB
Sample solution: 1 g in 15 mL of 2.7 N hydrochloric
Slaked Lime acid
Ca(OH)2 Formula wt 74.10 Solution)
INS: 526 CAS: [1305-62-0] Acceptance criteria: NMT 2 mg/kg
UNII: PF5DZW74VN [calcium hydroxide] • MAGNESIUM AND ALKALI SALTS
Sample solution: Dissolve 500 mg of sample in a
DESCRIPTION mixture of 30 mL of water and 10 mL of 2.7 N
Calcium Hydroxide occurs as a white powder. One g
hydrochloric acid.
dissolves in 630 mL of water at 25°, and in 1300 mL of
Analysis: Boil the Sample solution for 1 min, rapidly add
boiling water. It is soluble in glycerin and in a saturated
40 mL of oxalic acid TS, and stir vigorously until
solution of sucrose but insoluble in alcohol.
precipitation is well established. Immediately add 2
Function: Buffer; neutralizing agent; firming agent
drops of methyl red TS; then add 6 N ammonium
Packaging and Storage: Store in tight containers.
hydroxide, dropwise, until the mixture is just alkaline.
IDENTIFICATION Cool the mixture and transfer it into a 100-mL
• ALKALINITY graduated cylinder, dilute to 100 mL with water, and
Sample solution: Mix a sample with from 3 to 4 times let it stand for 4 h or overnight. Then decant the clear,
its weight of water. The sample forms a smooth supernatant liquid through a dry filter paper. Add 0.5
magma. Test the clear supernatant liquid from the mL of sulfuric acid to 50 mL of the clear filtrate
magma with litmus. contained in a tared platinum dish, and evaporate the
Acceptance criteria: Alkaline to litmus mixture on a steam bath to a small volume. Carefully
• CALCIUM, Appendix IIIA evaporate the remaining liquid to dryness over a free
Sample solution: Mix 1 g of sample with 20 mL of flame and continue heating until the ammonium salts
water and add sufficient glacial acetic acid to aid in have been completely decomposed and volatilized.
dissolution. Finally, ignite the residue at 800° ± 25° to constant
Acceptance criteria: Passes tests weight.
ASSAY
• PROCEDURE SPECIFIC TESTS
Sample solution: Transfer 1.5 g of sample into a beaker • ACID-INSOLUBLE SUBSTANCES
and gradually add 30 mL of 2.7 N hydrochloric acid. Sample: 2 g
When the sample has completely dissolved, transfer the Analysis: Dissolve the Sample in 30 mL of 1:3
hydrochloric acid, and heat to boiling. Filter the
FCC 9 Monographs / Calcium Lactate / 195
mixture through a suitable tared, porous-bottom

Calcium Lactate
.
porcelain crucible, and wash the residue with hot water

until the last washing is free from chloride. Ignite the First Published: Prior to FCC 6
residue at 800° ± 25° for 45 min, cool and weigh the
residue. [NOTE—Avoid exposing the crucible to sudden
2-Hydroxypropanoic Acid, Calcium Salt
temperature changes.]
Monographs
Calcium Iodate
.
C6H10CaO6 · xH2O Formula wt, anhydrous 218.22

First Published: Prior to FCC 6 INS: 327 CAS: [814-80-2]
UNII: 2URQ2N32W3 [calcium lactate]
Ca(IO3)2 · H2O Formula wt 407.90
INS: 916 CAS: [7789-80-2] DESCRIPTION
UNII: L8MN4Y57BR [calcium iodate] Calcium Lactate occurs as a white to cream-colored,
crystalline powder or granules. It contains up to five
DESCRIPTION molecules of water of crystallization. The pentahydrate is
Calcium Iodate occurs as a white powder. It is slightly somewhat efflorescent and at 120° becomes anhydrous. It
soluble in water and insoluble in alcohol. is soluble in water and practically insoluble in alcohol.
Function: Maturing agent; dough conditioner Function: Buffer; dough conditioner; yeast nutrient
Packaging and Storage: Store in well-closed containers. Packaging and Storage: Store in tight containers.
IDENTIFICATION IDENTIFICATION
• PROCEDURE • CALCIUM, Appendix IIIA
Sample: Saturated solution of the sample Sample solution: 50 mg/mL
Analysis: Add 1 drop of starch TS and a few drops of Acceptance criteria: Passes tests
20% hypophosphorous acid to 5 mL of the Sample. • LACTATE, Appendix IIIA
Acceptance criteria: A transient blue color appears. Sample solution: 50 mg/mL
Acceptance criteria: Passes test
ASSAY
• PROCEDURE ASSAY
Sample solution: Dissolve 600 mg of sample, in 10 mL • PROCEDURE
of 70% perchloric acid and 10 mL of water, heating Sample: Amount equivalent to 350 mg of C6H10CaO6
gently if necessary, and dilute with water to 250.0 mL. Analysis: Dissolve the Sample in 150 mL of water
[CAUTION—Handle perchloric acid in an appropriate containing 2 mL of 2.7 N hydrochloric acid. While
fume hood.] stirring, preferably with a magnetic stirrer, add about
Analysis: Transfer 50.0 mL of the Sample solution to a 30 mL of 0.05 M disodium EDTA from a 50-mL buret.
250-mL glass-stoppered Erlenmeyer flask, add 1 mL of Then, add 15 mL of 1 N sodium hydroxide and 300 mg
70% perchloric acid and 5 g of potassium iodide, of hydroxy naphthol blue indicator. Continue the
stopper the flask, and swirl briefly. Let the solution titration with the disodium EDTA to a blue endpoint.
stand for 5 min. Titrate with 0.1 N sodium thiosulfate, Each mL of 0.05 M disodium EDTA is equivalent to
adding starch TS just before the endpoint is reached. 10.91 mg of C6H10CaO6.
Each mL of 0.1 N sodium thiosulfate is equivalent to Acceptance criteria: NLT 98.0% and NMT 101.0% of
3.398 mg of Ca(IO3)2 · H2O. C6H10CaO6, calculated on the dried basis
Ca(IO3)2 · H2O IMPURITIES
IMPURITIES • FLUORIDE, Fluoride Limit Test, Method I or Method III,
Inorganic Impurities Appendix IIIB
• LEAD, Lead Limit Test, Flame Atomic Absorption Sample: 3.3 g for Method I or 1.0 g for Method III
Spectrophotometric Method, Appendix IIIB Acceptance criteria: NMT 0.0015%
Sample: 10 g • LEAD, Lead Limit Test, Flame Atomic Absorption
Acceptance criteria: NMT 4 mg/kg Spectrophotometric Method, Appendix IIIB
Sample: 3 g
Sample: 1 g
Analysis: Mix the Sample with 40 mL of water and
carefully add 1 mL of hydrochloric acid. Boil the
solution for 1 min and rapidly add 40 mL of oxalic acid
196 / Calcium Lactate / Monographs FCC 9
TS, followed immediately by 2 drops of methyl red TS. obtained by spray-drying, or the dihydrate when obtained
Then add 6 N ammonium hydroxide, dropwise from a by crystallization. It is freely soluble in water, but insoluble
buret, until the mixture is just alkaline. Cool the in alcohol and in ether. It decomposes at about 120°. The
mixture to room temperature and transfer it into a pH of a 1:10 aqueous solution is between 6.5 and 7.5.
100-mL graduate cylinder. Dilute with water to 100 Function: Firming agent in dry pudding mixes; nutrient
mL, mix, and allow the mixture to stand for 4 h or Packaging and Storage: Store in well-closed containers.
overnight. Decant the clear, supernatant liquid through
a dry filter paper, transfer 50 mL of the clear filtrate to IDENTIFICATION
a tared platinum dish, and add 0.5 mL of sulfuric acid. • CALCIUM, Appendix IIIA
Evaporate the contents of the dish to a small volume Acceptance criteria: Passes tests
Monographs
on a steam bath; then carefully heat over a free flame • INFRARED ABSORPTION, Spectrophotometric Identification
to dryness, and continue heating to complete Tests, Appendix IIIC
decomposition and volatilization of the ammonium Reference standard: USP Calcium Lactobionate RS
salts. Finally, ignite the residue to constant weight. Sample and Standard preparation: K (Sample
Acceptance criteria: The weight of the residue does previously dried at 105° for 8 h)
not exceed 5 mg. (NMT 1%) Acceptance criteria: The spectrum of the sample
SPECIFIC TESTS the spectrum of the Reference standard.
• ACIDITY (AS LACTIC ACID)
Sample solution: 1 g in 20 mL of water IMPURITIES
Analysis: Add 3 drops of phenolphthalein TS to the Inorganic Impurities
Sample solution and titrate with 0.1 N sodium • HALIDES, Chloride and Sulfate Limit Tests, Chloride Limit Test,
hydroxide. Appendix IIIB
Acceptance criteria: NMT 0.5 mL of titrant is required. Sample: 1.2 g
(About 0.45%, as lactic acid) Control: 0.7 mL of 0.020 N hydrochloric acid
• LOSS ON DRYING, Appendix IIC: 120° for 4 h Acceptance criteria: The Sample shows no more
Sample: 1.5 g turbidity than the Control (NMT 0.04%).
Acceptance criteria • LEAD, Lead Limit Test, Flame Atomic Absorption
Pentahydrate: Between 22.0% and 27.0% Spectrophotometric Method, Appendix IIIB
Trihydrate: Between 15.0% and 20.0% Sample: 3 g
Monohydrate: Between 5.0% and 8.0% Acceptance criteria: NMT 2 mg/kg
Dried Form: NMT 3.0% • SULFATE
Sample: 25 g
Analysis: Transfer the Sample to a 600-mL beaker,
dissolve it in 200 mL of water, adjust the solution to a
pH between 4.5 and 6.5 with 2.7 N hydrochloric acid,
Calcium Lactobionate
.
and filter, if necessary. Heat the filtrate or clear solution

First Published: Prior to FCC 6 to just below the boiling point. Then, while stirring
vigorously, add 10 mL of barium chloride TS, boil
gently for 5 min, and allow the solution to stand for at
Calcium 4-(β,D-Galactosido)-D-gluconate
least 2 h, or, preferably, overnight. Collect the
precipitate of barium sulfate on a suitable, tared
crucible, wash until free from chloride, dry, and ignite
at 600° to constant weight. The weight of barium
sulfate so obtained, multiplied by 0.412, represents the
weight of sulfate (SO4) in the sample taken.
Organic Impurities
• REDUCING SUBSTANCES (AS DEXTROSE)
Sample: 1.0 g
Analysis: Transfer the Sample to a 250-mL conical flask,
dissolve it in 20 mL of water, and add 25 mL of
alkaline cupric citrate TS. Cover the flask, boil the
contents gently for 5 min, accurately timed, and cool
C24H42CaO24 Formula wt, anhydrous 754.66 the flask rapidly to room temperature. Add 25 mL of
INS: 399 CAS: [5001-51-4] 0.6 N acetic acid, 10.0 mL of 0.1 N iodine, and 10 mL
UNII: 7D8YVA497F [calcium lactobionate] of 3 N hydrochloric acid. Titrate with 0.1 N sodium
thiosulfate, adding 3 mL of starch TS as the endpoint is
DESCRIPTION approached. Perform a blank determination (see
Calcium Lactobionate occurs as a white to cream-colored, General Provisions), make any necessary correction.
free-flowing powder. It readily forms double salts, such as Each mL of 0.1 N sodium thiosulfate consumed is
the chloride, bromide, and gluconate. It is anhydrous when
FCC 9 Monographs / Calcium Lignosulfonate / 197
equivalent to 2.7 mg of reducing substances (as Acceptance criteria: A peak is observed between 275
dextrose). and 280 nm.
ASSAY
SPECIFIC TESTS • SULFONATE SULFUR
• CALCIUM CONTENT Sample: 1.0 g
Sample: 1.5 g Analysis: Dissolve the Sample in 400 mL of water in a
Analysis: Dissolve the Sample in 100 mL of water beaker. Direct a gentle stream of nitrogen gas over the
containing 2 mL of 2.7 N hydrochloric acid. While liquid’s surface. Add 10 mL of nitric acid, and swirl the
stirring, preferably with a magnetic stirrer, add about solution thoroughly until the reaction subsides. Add 10
Monographs
30 mL of 0.05 M disodium EDTA from a 50-mL buret. mL of 70% perchloric acid, and swirl thoroughly again.
Then, add 15 mL of 1 N sodium hydroxide and 300 mg [CAUTION—Handle perchloric acid in an appropriate
of hydroxy naphthol blue indicator. Continue the fume hood.] Place the uncovered beaker on a hot plate,
titration with disodium EDTA to a blue endpoint. Each and heat the contents vigorously until the center of the
mL of 0.05 M disodium EDTA is equivalent to 2.004 mg bottom of the beaker becomes clear. Remove the
of calcium (Ca). beaker, and cool it to room temperature. Add 5 mL of
Acceptance criteria: NLT 5.05% and NMT 5.55%, hydrochloric acid, and heat it again until white fumes
calculated on the dried basis evolve. After cooling the beaker, dilute the solution to
• LOSS ON DRYING, Appendix IIC: 105° for 8 h approximately 100 mL with water, adjust to pH 6 ± 0.2
Acceptance criteria: NMT 8.0% with 10% sodium hydroxide, and heat the solution to
• OPTICAL (SPECIFIC) ROTATION, Appendix IIB boiling. Add 15 mL of 10% barium chloride solution,
Sample solution: 50 mg/mL (on the anhydrous basis) and leave the solution overnight in a fresh beaker in a
Acceptance criteria: [α]D25 between +23° and +25° steam bath at 90° to 95°. Pass through ashless filter
paper (Whatman No. 42, or equivalent), and wash the
OTHER REQUIREMENTS precipitate with 200 mL of warm water. Transfer the
• LABELING: Indicate whether the product has been paper and precipitate into a tared crucible. Heat the
obtained through spray-drying or from crystallization. crucible slowly on a Bunsen burner to expel moisture.
Place the crucible and contents in a muffle furnace at
850° for 1 h. Let the crucible cool in a desiccator, and
then weigh the residue to the nearest 0.0001 g.
Calcium Lignosulfonate
.
Calculate the percent sulfonate sulfur by the formula:

Result = (R/S) × 13.7
CAS: [8061-52-7] R = weight of the residue (g)

UNII: 33T2H9O73P [calcium lignosulfonate (20000 mw)] S = weight of the sample taken (g)
Acceptance criteria: NLT 5.0% sulfonate sulfur
DESCRIPTION
Calcium Lignosulfonate occurs as a brown, amorphous IMPURITIES
polymer. It is obtained from the spent sulfite and sulfate Inorganic Impurities
pulping liquor of wood or from the sulfate (Kraft) pulping • LEAD, Lead Limit Test, Atomic Absorption Spectrophotometric
process. It may contain up to 30% reducing sugars. It is Graphite Furnace, Method I, Appendix IIIB
soluble in water, but not in any of the common organic Acceptance criteria: NMT 1 mg/kg
solvents. The pH of a 1:100 aqueous solution is between
approximately 3 and 11. SPECIFIC TESTS
Function: Binder; dispersant • CALCIUM
Packaging and Storage: Store in well-closed containers. Strontium chloride solution: While stirring, add 164.7
g of 60% perchloric acid to 500 mL of water contained
IDENTIFICATION in a 1-L beaker. [CAUTION—Handle perchloric acid in an
• A. CALCIUM, Appendix IIIA appropriate fume hood.] Then, while stirring, add 15.2
Sample solution: 0.15 mg/mL g of strontium chloride hexahydrate, stirring until
Acceptance criteria: Passes tests solution is complete. Transfer the solution into a 1-L
• B. PROCEDURE volumetric flask, and dilute to volume at room
Sample: 100 mg temperature with water. Mix thoroughly.
Analysis: Dissolve the Sample in 50 mL of water. Add 1 Standard solution: 0.7 mg/mL of calcium, prepared
mL each of 10% acetic acid and 10% sodium nitrite from a certified Calcium Standard Solution (NIST, or
solution, and mix by swirling. Allow the solution to equivalent). [NOTE—Store the Standard solution in
stand for 15 min at room temperature. polyethylene bottles because of its instability in glass.]
Acceptance criteria: A brown color appears. Sample: 1 g, previously dried
• C. ULTRAVIOLET ABSORPTION Sample solution: Dilute the Sample to 10 mL, and mix.
Sample solution: 0.1 mg/mL (pH 5) If the solution is not particle-free, pass through a 0.45-
198 / Calcium Lignosulfonate / Monographs FCC 9
µm disposable Millipore filter, discarding the first few volume of 0.005 N sodium thiosulfate consumed as VD.
mL of filtrate. Pipet 5 mL of Strontium chloride solution Perform a corresponding blank titration using 5 mL of
into a 50-mL volumetric flask, and add 5.0 mL of the water and 5 mL of Copper reagent solution; record the
filtrate or clear solution. Dilute with water to volume, volume of 0.005 N sodium thiosulfate consumed as VB.
and mix well. Calculate the percent reducing sugars by the formula:
Analysis: Using a suitably calibrated atomic absorption
spectrophotometer, determine the absorbance of the Result = (A × F)/B
Standard solution and the Sample solution at 422.7 nm.
Acceptance criteria: The absorbance of the Sample
A = volume of 0.005 N sodium thiosulfate
solution is not greater than that of the Standard solution.
consumed by the 5-mL aliquot of Sample
Monographs
(NMT 7.0%)
solution, determined by VB − VS (mL)
F = factor, 35
B = volume of 0.005 N sodium thiosulfate
• REDUCING SUGARS
consumed by 5 mL of the Standard
Copper reagent solution: [NOTE—Solution must be
solution, determined by VB − VD (mL)
prepared several days in advance of use.] Dissolve 28 g
of anhydrous dibasic sodium phosphate and 40 g of
• RESIDUE ON IGNITION (SULFATED ASH), Appendix IIC
potassium sodium tartrate tetrahydrate in 700 mL of
Sample: 1 g
water. Add 100 mL of 1 N sodium hydroxide and 8 g of
copper sulfate pentahydrate, followed by 180 g of
• VISCOSITY OF A 50% SOLUTION
anhydrous sodium sulfate. Add 0.7134 g of potassium
Sample: 200 g, on the dried basis
iodate, and dilute to 1 L. Allow to stand for several
Analysis: Dissolve the Sample in 200 mL of water
days, then filter the clear top part of the solution
contained in a 500-mL beaker. Equilibrate the solution
through a medium-porosity, sintered-glass funnel.
at 25°, and measure its relative viscosity with a
Lead subacetate solution: Dissolve 80 g of lead
Brookfield viscometer (Model RVT, or equivalent), using
subacetate in 220 mL of water. Stir overnight, and pass
a number 2 spindle at 20 rpm.
through Whatman No. 42 filter paper, or equivalent.
Acceptance criteria: NMT 3000 centipoises
Dilute the supernatant solution to a specific gravity of
1.254 with freshly boiled water.
Dibasic sodium phosphate solution: 190 mg/mL
dibasic sodium phosphate heptahydrate, made to 100
Calcium Lignosulfonate (40–65)
.
mL
Standard solution: 280 µg/mL dried dextrose, made to First Published: First Supplement, FCC 7
500 mL
Sample solution: Dissolve 1 g of sample in 150 mL of Lignosulfonic Acid, Calcium Salt (40–65)
water, and adjust the pH to between 6.9 and 7.2 with INS: 1522
sodium hydroxide solution or acetic acid. UNII: 6HPP8U6S23 [calcium lignosulfonate (50000 mw)]
Analysis: To the Sample solution, add Lead subacetate
solution in increments until no further precipitation is DESCRIPTION
observed. Bring the volume to 250.0 mL with water, Calcium Lignosulfonate (40–65) occurs as a light yellow-
and mix well. Centrifuge the mixture, pipet 10 mL of brown to brown powder. It is an amorphous material
the supernatant into a 50-mL volumetric flask, and obtained from the sulfite pulping of softwood. The lignin
dilute with water to about 35 mL. Add 2 mL or more of framework is a sulfonated random polymer of three
Dibasic sodium phosphate solution until no further aromatic alcohols: coniferyl alcohol, p-coumaryl alcohol,
precipitation forms. Dilute with water to 50 mL, and and sinapyl alcohol, of which coniferyl alcohol is the
mix. Centrifuge at 2100× gravity for 10 min. Pipet 5 principal unit. After completion of the pulping, the water-
mL of supernatant solution into a test tube containing soluble calcium lignosulfonate is separated from the
exactly 5 mL of Copper reagent solution, and mix. cellulose, purified (ultrafiltration), and acidified. The
Loosely plug the tube, and place it in a boiling water recovered material is evaporated and spray dried. It is
bath for 40 min ± 10 s. At the end of the heating distinguished from Calcium Lignosulfonate by its
period, cool the tube immediately in cold water. Add 2 characteristic weight average molecular weight, its low
mL of 2.5% potassium iodide solution and 1.5 mL of degree of sulfonation, and its low level of reducing sugars.
2 N sulfuric acid. Mix well, and titrate with 0.005 N It is soluble in water and practically insoluble in organic
sodium thiosulfate, using starch as the indicator, and solvents.
note the volume of 0.005 N sodium thiosulfate Function: Carrier, encapsulating agent
consumed as VS. Perform a corresponding blank Packaging and Storage: Store in well-closed containers.
titration using 5 mL of water and 5 mL of Copper
reagent solution, and record the volume of 0.005 N IDENTIFICATION
sodium thiosulfate consumed as VB. • DEGREE OF SULFONATION: [NOTE—The degree of sulfonation
Repeat the entire procedure using 5 mL of Standard is determined as the content ratio of organic sulfur to
solution and 5 mL of Copper reagent solution, noting the methoxyl. The organic sulfur content is determined
FCC 9 Monographs / Calcium Lignosulfonate (40–65) / 199
indirectly as the difference between the total sulfur Standard solutions: 2.0, 5.0, 20.0, and 40.0 mg/L of
content (determined by elemental analysis) and sulfate, prepared by pipetting 0.1, 0.25, 1.0, and 2.0
inorganic sulfur content (determined by ion-exchange mL of Standard stock solution into separate 50-mL
chromatography).] volumetric flasks, adding 1 mL of 3% H2O2 to each,
Total sulfur determination and diluting with water to volume.
Calibration standards: Add approximately 0.2 mg of Sample solution: Transfer 30 mg of previously dried
vanadium pentoxide into each of four tin capsules. sample into a 50-mL volumetric flask, and dissolve in
Accurately weigh 0.5, 1.0, 1.5, and 2.0 mg of BBOT 10 mL of 10 mg/mL of NaOH. Add 5 mL of 3% H2O2,
(2,5-(bis(5-tert-butyl-2-benzo-oxazol-2-yl) thiophene)) and allow to stand overnight, then dilute with water
into the four capsules. to volume.
Monographs
System suitability standard: Add approximately 0.2 Chromatographic system, Appendix IIA
mg of vanadium pentoxide and 0.5–2.0 mg of BBOT Mode: High-performance liquid chromatography2
into a tin capsule. Detector: Ion detector with anion self-regenerating
Samples: Add approximately 0.2 mg of vanadium conductivity suppressor3
pentoxide into each of two tin capsules. Accurately Column: 25-cm × 4-mm, anion-exchange analytical
weigh 1–2 mg of sample, previously dried, into each column4, and 5-cm × 4-mm anion-exchange guard
capsule. column5
Equipment: Elemental analyzer capable of analyzing for Flow rate: 0.7 mL/min
sulfur1 Injection size: 10 µL
Equipment parameters System suitability
Carrier gas: Helium, 120 mL/min Sample: Sample solution
Combustion furnace temperature: 1000° Relative standard deviation: NMT 3.0%
Oven temperature: 70° Analysis: Separately inject equal volumes of the
Helium pressure: 150 kPa Standard solutions and Sample solution (previously
Oxygen pressure: 150 kPa filtered using a 0.2-µm syringe filter) into the
Oxygen loop: 5 mL chromatograph, and measure the responses for the
Run time: 300 s major peaks on the resulting chromatograms. [NOTE—
System suitability The approximate retention time for sulfate is 7 min.]
[NOTE—A system suitability check should be performed Prepare a standard curve for sulfate by plotting sulfate
after every fourth sample.] peak areas versus concentrations in mg/L. From the
Sample: System suitability standard standard curve, calculate the concentration (CU) of
Suitability requirement 1: The chromatogram sulfate in the Sample solution in mg/L. Calculate the
contains a sulfur peak. percentage of inorganic sulfur in the portion of the
Suitability requirement 2: The standard deviation for sample taken:
triplicate analyses is NMT 0.20.
Analysis: Run the four calibration standards and Result = CU/CSMP × F1/F2 × 100
construct a calibration curve. [NOTE—The correlation
CU = concentration of sulfate in the Sample
coefficient should be at least 0.999.] Run the Samples.
solution determined from the standard
Obtain the weight (mg) of total sulfur in the Sample
curve (mg/L)
using the calibration curve. Calculate the percentage
CSMP = concentration of the sample, on the dried
of total sulfur in the portion of the sample taken:
basis, in the Sample solution (mg/L)
Result = WU/WSMP × 100 F1 = formula weight of sulfur, 32
F2 = formula weight of sulfate, 96
WU = weight of total sulfur calculated from the Methoxyl (−OCH3) determination
standard curve (mg) Sample: 15–20 mg, previously dried and weighed onto
WSMP = weight of the sample taken, on the dried a small piece of aluminum foil
basis (mg) 0.025 N sodium thiosulfate: Dilute 0.1 N sodium
Inorganic sulfur determination thiosulfate VS with water (1:3).
Mobile phase: 0.1 N sodium hydroxide and water
(10:90)
Standard stock solution: 1 mg/mL of sulfate, prepared 2 Dionex Corporation (Sunnyvale, CA) ion exchange chromatograph, or
by dissolving 0.1479 g of sodium sulfate in 100 mL of equivalent.

3 ASRS-300 4mm (Dionex Corporation, Sunnyvale, CA), or equivalent.
water 4 IonPac AS11 (Dionex Corporation, Sunnyvale, CA), or equivalent.
1 Thermo Fisher Scientific, or equivalent. 5 IonPac AG11 (Dionex Corporation, Sunnyvale, CA), or equivalent.
200 / Calcium Lignosulfonate (40–65) / Monographs FCC 9
Monographs
Figure 1. (Reprinted from Analytica Chimica Acta, Vol 15, P.O. Bethge and O.T. Carlson, On the Semimicro Determination of
Methoxyl, Pages No. 279–283 (Fig. 1), Copyright (1956), with permission from Elsevier.)
Analysis See Figure 1 for apparatus setup. Wrap the foil add 10 mL of 1 M sulfuric acid and let the flask stand for 3
around the Sample and put it into the reaction flask (A) to min. Titrate the solution with 0.025 N sodium thiosulfate
which 5 mL of hydroiodic acid (min. 57%), approximately until the color changes from yellowish to colorless. Calculate
2 g of phenol, and a few glass beads have been added. Add the percentage of methoxyl:
5 mL of 50 mg/mL cadmium sulfate solution containing
about 0.3 mg of red phosphorus into the washer (G). Add Result = V × F1 × F2/(W × F3 × F4) × 100
10 mL of glacial acetic acid (saturated with sodium acetate)
V = volume of 0.025 N sodium thiosulfate used in
and 10 droplets of bromine to the receiver (D). Finally, fill
the titration (mL)
the U-trap (E) with sodium hydroxide or other suitable
F1 = concentration of the 0.025 N sodium
absorbant that will prevent bromine from leaving the
thiosulfate, 0.025
system. Pass nitrogen gas through a 30 mg/mL Na2CO3
F2 = formula weight of methoxyl, 31
solution and into the system through the side arm (I) of the
W = weight of the sample taken on the dried
air condensor (B). Heat the reaction flask (A) to 140°–145°
basis (mg)
for 1 h in a glycerin bath. Wash the contents of the receiver
F3 = stoichiometric conversion factor from titrant
(D) into a 250-mL Erlenmeyer flask containing 10 mL of
to methoxyl moiety, 6
acetic acid (saturated with sodium acetate). Rotate the flask
F4 = mL-to-L conversion factor for the 0.025 N
and add formic acid dropwise until the color disappears.
sodium thiosulfate, 1000
Add 5 mL of 10% potassium iodide solution, and mix. Then
FCC 9 Monographs / Calcium Lignosulfonate (40–65) / 201
Degree of sulfonation calculation: Calculate the degree Injection size: 200 µL

of sulfonation: System suitability
Sample: The Standard solution with the highest weight-
Result = (%OS)/(%M) average molecular weight.
Suitability requirement: The relative standard
%OS = % organic sulfur, as (% total sulfur) − (%
deviation of the lignosulfonate peak retention time for
inorganic sulfur), determined above in the
three injections is NMT 0.5%.
Total sulfur determination and Inorganic
Analysis: Run Mobile phase through the system for NLT
sulfur determination test procedures
2 h. [NOTE—Pressure should not exceed 1000 psi.]
%M = % methoxyl, determined above in the
Inject the Standards solutions, then the Sample solution
Methoxyl (−OCH3) determination test
Monographs
followed by another set of Standard solutions. Generate
procedure
a calibration curve using the Standard solutions.
Acceptance criteria: 0.3–0.7, on the dried basis
Calculate the weight-average molecular weight from
• INFRARED ABSORPTION, Spectrophotometric Identification
the chromatogram of the Sample solution using suitable
Tests, Appendix IIIC
software.9
Reference standard: USP Calcium Lignosulfonate (40-
Acceptance criteria: Between 40,000 and 65,000 with
65) RS
>90% of the sample ranging from 1,000–250,000
Sample and standard preparation: K
Acceptance criteria: The spectrum of the sample IMPURITIES
exhibits maxima at the same wavelengths as those in Inorganic Impurities
the spectrum of the Reference standard. • ARSENIC, Arsenic Limit Test, Appendix IIIB: Prepare as
• ULTRAVIOLET ABSORPTION directed for organic compounds.
Sample stock solution: 500 µg/mL [NOTE—Alternatively, the arsenic content may be
Sample solution: 50 µg/mL made from Sample stock determined by the following graphite furnace atomic
solution, and adjusted to a pH of 2.0–2.2 with 5 M absorption spectrophotometric method.]
hydrochloric acid. Standard solutions: 0–15 ng/mL of arsenic; prepared
Acceptance criteria: The Sample solution exhibits an from a commercially available 1000 mg/kg arsenic
absorption maximum at 280 nm. standard solution. [NOTE—Store this solution in
• WEIGHT-AVERAGE MOLECULAR WEIGHT polyethylene bottles due to instability in glass.]
Mobile phase: Combine 1600 g of water with 161.8 g Sample solution: [CAUTION—Wear proper eye
of dimethyl sulfoxide in a 2-L flask. Add 21.44 g of protection, protective clothing, and gloves during
dibasic sodium phosphate heptahydrate, and adjust the sample preparation. Closely follow the manufacturer’s
pH to 10.5 with NaOH. Add 1.6 g of sodium safety instructions for use of the microwave digestion
dodecylsulfate and pass through a 0.22-µm filter. apparatus.] Transfer 200 mg of sample into a Teflon
Standard solutions: Prepare two lignosulfonate digestion vessel liner. Add 3 mL of 65% nitric acid, 2
calibration standard6 solutions, each at 2 mg/mL in mL of 30% hydrogen peroxide, and cover. Heat for 20
Mobile phase. One should be prepared using a min in a microwave oven, and allow the vessel to cool
lignosulfonate standard with a weight-average to room temperature (a cool water bath may be used
molecular weight from 30,000–60,000 g/mol, and the to speed the cooling process), and carefully open in a
other using a lignosulfonate standard with a weight- ventilation hood. Dilute the cooled digest with water
average molecular weight from 5,000–10,000 g/mol. to 12 mL.
Filter each solution into a vial using a 0.2-µm syringe Reagent blank: Use the same quantities of reagents as
filter. used to prepare the Sample solution, but omitting the
Sample solution: 2 mg/mL using a previously dried sample.
sample. Filter into vial using a 0.2-µm syringe filter. Analysis: Use any suitable graphite furnace atomic
Chromatographic system, Appendix IIA absorption spectrophotometer. Optimize the
Mode: High-performance liquid chromatography instrument according to the manufacturer’s
Detector: UV 280 nm instructions. Determine the absorbance of each of the
Column: 50-cm × 10-mm glucose divinylbenzene Standard solutions, of the Sample solution, and of the
(DVB), 5-µm, 104 Å analytical column7, and 4-cm × 6- Reagent blank at 193.7 nm. Determine the corrected
mm, 7-µm, 300 Å guard column8 absorbance values by subtracting the Reagent blank
Oven temperature: 60° absorbance from each of the Standard solutions and
Flow rate: 1.0 mL/min from the Sample solution absorbances. Prepare a
standard curve by plotting the corrected absorbance of
the Standard solutions versus the concentration of
6 Lignosulfonate calibration standards available from Borregaard Industries
Limited, Borregaard LignoTech Research and Development, P.O. Box 162,

NO-1701 Sarpsborg, Norway. Phone no: +47 69118000; e-mail:
borregaard@borregaard.com.
7 Jordi Gel DVB Glucose (Jordi Labs, Bellingham, MA), or equivalent.
8 TSK-Gel PWXL (TOSOH Bioscience, Montgomeryville, PA), or equivalent. 9 Empower (Waters, Milford, MA), or equivalent.
202 / Calcium Lignosulfonate (40–65) / Monographs FCC 9
arsenic (ng/mL). Calculate the concentration (mg/kg) Organic Impurities

of arsenic in the sample taken: • REDUCING SUGARS
Standard solutions: 0.10, 1.0, and 2.0 mg/mL of
Result = C/W × F1 glucose
C = concentration of arsenic in the Sample
Equipment: Flow injection analyzer10 with flow set to
solution determined from the standard
“low” position on both pumps, and heater set at 90°.
curve (ng/mL)
[NOTE—The signal should be less than ± 1000 micro-
W = weight of the sample taken to prepare the
absorbance units before starting analysis.]
Sample solution (g)
Analysis: Introduce 100 µL each of the Sample solution
F1 = sample dilution factor, 12 mL
Monographs
and the Standard solutions into the analyzer. For each

analysis, air is introduced followed by addition of 2 mg
• LEAD, Lead Limit Test, Atomic Absorption Spectrophotometric
/mL of Brij-35 (polyoxyethyleneclycol dodecyl ether11),
Graphite Furnace Method, Method II, Appendix IIIB
at a continuous flow of 0.287 mL/min. The solutions
are then dialyzed through a cellulose membrane12.
• SULFITE
After dialysis add 1 M NaOH at 0.385 mL/min, and
Mobile phase: 0.1 M sodium hydroxide and water
CaCl2 and PHBH (p-hydroxylbenzoichydrazide), both at
(10:90)
0.074 mL/min, into the mixing chamber of the
Diluent: Dilute 0.5 mL of 37% formaldehyde with
analyzer. The mixture then enters the heater
water to 1000 mL.
(previously set at 90°) where bubbles are eliminated,
Standard stock solution: 1 mg/mL of sulfite in Diluent,
after which it reaches the detector (set at 410 nm).
prepared by dissolving 0.1574 g of sodium sulfite in
Run duplicate injections of every Sample solution.
and diluting with Diluent to 100 mL
Construct a calibration curve from the Standard
Standard solutions: 2.0, 5.0, 10.0, and 20.0 mg/L of
solutions. Calculate the percentage of reducing sugars
sulfite in Diluent, made from Standard stock solution
in the portion of the sample taken:
Sample solution: 3.0 mg/mL in Diluent
Chromatographic system, Appendix IIA Result = CU/CSMP × 100
Mode: High-performance liquid chromatography2
Detector: Ion detector with anion self-regenerating CU = concentration of reducing sugars, as glucose,
conductivity suppressor3 in the Sample solution determined from the
Column: 25-cm × 4-mm, anion-exchange analytical standard curve (mg/mL)
column4, and 5-cm × 4-mm anion-exchange guard CSMP = concentration of the sample, on the dried
column5 basis, in the Sample solution (mg/mL)
Flow rate: 0.7 mL/min Acceptance criteria: NMT 5.0% (as glucose), on the
Injection size: 10 µL dried basis
System suitability
Sample: Sample solution SPECIFIC TESTS
Relative standard deviation: NMT 3.0% for the • ASH (TOTAL), Appendix IIC
sulfite peak area Sample: 0.5–1 g, previously dried
Analysis: Separately inject equal volumes of the Analysis: Proceed as directed, but igniting at 550° for 1
Standard solutions and Sample solution (previously h and then 900° for 10 min until all dark particles have
filtered using a 0.2-µm syringe filter) into the disappeared and the ash is white.
chromatograph, and measure the responses for the Acceptance criteria: NMT 14%, on the dried basis
major peaks on the resulting chromatograms. [NOTE— • CALCIUM
The approximate retention time for sulfite is 6 min.] Standard stock solution: 3.00 µg/mL of calcium,
Prepare a standard curve for sulfite by plotting sulfite prepared from a certified Calcium Standard Solution13.
peak areas versus concentrations in mg/L. From the [NOTE—Store in polyethylene bottles because of its
standard curve, calculate the concentration (CU) of instability in glass.]
sulfite in the Sample solution in mg/L. Calculate the Standard solutions: 0.750, 1.50, 2.25, and 3.00 µg/mL
percentage of sulfite in the portion of the sample of calcium, made from Standard stock solution. [NOTE—
taken: Store in polyethylene bottles because of its instability in
glass.]
Result = CU/(CSMP × F1) × 100 Sample stock solution: 4000 µg/mL prepared as
follows. Transfer 0.2 g of a previously dried sample into
CU = concentration of sulfite in the Sample solution a graduated flask. Add 5 mL of 65% nitric acid and 2
determined from the standard curve (mg/L) mL of 30% hydrogen peroxide. Boil for 1 h in a
CSMP = concentration of the sample, on the dried microwave oven. Dilute with water to 50 mL.
basis, in the Sample solution (mg/mL) Sample solution: 40 µg/mL from Sample stock solution
F1 = mL-to-L conversion factor, 1000
Acceptance criteria: NMT 0.5%, on the dried basis 10 O.I. Analytical (College Station, TX), or equivalent.
11 Ultra grade, O.I. Analytical (College Station, TX), or equivalent.
12 Type C 25 MM (Astoria-Pacific, Inc, Clackamas, OR), or equivalent.
13 NIST or equivalent.
FCC 9 Monographs / Calcium L-5-Methyltetrahydrofolate / 203
Analysis: Using a suitably calibrated atomic absorption IDENTIFICATION

spectrophotometer, determine the absorbance of the • A. INFRARED ABSORPTION, Spectrophotometric Identification
Standard solutions and Sample solution at 422.7 nm. Tests, Appendix IIIC
Prepare a standard curve for calcium by plotting Reference standard: USP Calcium D,L-5-
calcium peak areas versus concentrations in mg/L. From Methyltetrahydrofolate RS
the standard curve, determine the concentration (CU) of Sample and standard preparation: K
calcium in the Sample solution in µg/mL. Calculate the Acceptance criteria: The spectrum of the sample
percentage of calcium in the portion of the sample exhibits maxima at the same wavelengths as those in
taken: the spectrum of the Reference standard.
[NOTE—If the spectrum obtained shows differences,
Monographs
Result = CU/CSMP × 100 dissolve the substance to be examined and the USP
CU = concentration of calcium in the Sample Calcium D,L-5-Methyltetrahydrofolate RS separately in
solution determined from the standard the minimum quantity of water, and add dropwise
curve (µg/mL) sufficient acetone to produce a precipitate. Allow to
CSMP = concentration of the sample in the Sample stand for 15 min, centrifuge to collect the precipitate,
solution (µg/mL) wash the precipitate twice with a minimum quantity
Acceptance criteria: NMT 5.0%, on the dried basis of acetone, and dry. Record new spectrum using the
• LOSS ON DRYING, Appendix IIC: 105° for 24 h residues.]
Acceptance criteria: NMT 8.0% • B. CALCIUM
Solution A: 30 g of acetic acid in 100 mL
Solution B: 5.3 g of potassium ferrocyanide in 100 mL
Sample: 20 mg
Analysis: Add the Sample and 0.5 mL of Solution B to 5
Calcium L-5-Methyltetrahydrofolate
.
mL of Solution A. Mix, and add 50 mg of ammonium

First Published: Third Supplement, FCC 8 chloride.
Acceptance criteria: A white crystalline precipitate is
N-[4-[[(2-Amino-1,4,5,6,7,8-hexahydro-5-methyl-4-oxo-(6S)- formed.
pteridinyl)methyl]amino]benzoyl]-L-glutamic Acid, Calcium • C. PROCEDURE
Salt (1:1) Acceptance criteria: The retention time of the major
N-{4-[[((6S)-2-Amino-1,4,5,6,7,8-hexahydro-5-methyl-4-oxo- peak in the chromatogram of the Sample solution
6-pteridinyl)methyl]amino]benzoyl}-L-glutamic Acid, corresponds to that of the Standard solution in the Assay
Calcium Salt (1:1) and to the L-isomer of the Standard solution in the test
L-5-Methyltetrahydrofolic Acid, Calcium Salt for Enantiomeric Purity.
L-Methyltetrahydrofolate, Calcium Salt
L-Methylfolate, Calcium ASSAY
L-5-MTHF-Ca • PROCEDURE
Solution A: Prepare a 0.05 M solution of sodium
dihydrogen phosphate dihydrate, and adjust to a pH of
6.5 using a 32% (w/v) sodium hydroxide solution.
Solution B: Add 350 mL of methanol to 650 mL of 0.05
M sodium dihydrogen phosphate solution, then adjust
to a pH of 8.0 using a 32% (w/v) sodium hydroxide
solution.
Mobile phase: See Table 1.
C20H23CaN7O6 · xH2O Formula wt, anhydrous 497.52 Table 1

CAS: [151533-22-1]
Time Solution A Solution B
UNII: A9R10K3F2F [calcium l-5-methyltetrahydrofolate] (min) (%) (%)
0 100 0
DESCRIPTION
Calcium L-5-Methyltetrahydrofolate occurs as a white to 14 45 55
light yellow or beige crystalline powder. The commercial 17 0 100
product contains variable amounts of water of 24 0 100
crystallization. It is soluble in alkaline solutions; sparingly 24.01 100 0
soluble in water; very slightly soluble or insoluble in most
33 100 0
organic solvents.
Function: Nutrient [NOTE—The final gradient of 100% Solution A to 100%
Packaging and Storage: Store in a tight container, in a Solution B (from 24.01–33 min) is post-analysis to
cool and dry place. recondition the column. After analysis, the column
should be flushed and stored in a mixture of methanol
and water (85:15).]
204 / Calcium L-5-Methyltetrahydrofolate / Monographs FCC 9
System suitability solution: Transfer 25.0 mg of USP [NOTE—For the System suitability solution the relative
Folic Acid RS and 25.0 mg of USP 4- retention times of the component peaks are listed in
Aminobenzoylglutamic Acid RS to a 100-mL volumetric Table 2. The L- and D-isomers of 5-
flask. Add about 15 mg of each of sodium hydrogen methyltetrahydrofolate co-elute as a single peak. The
carbonate and sodium carbonate to the flask, add 4α-hydroxy-5-methyltetrahydrofolic acid, 5-
sufficient water, sonicate to dissolve, and dilute to methyltetrahydropteroic acid, and
volume. Transfer 1.0 mL of this solution to a second dimethyltetrahydrofolic acid are included as minor
100-mL volumetric flask containing 50.0 mg of USP components in USP Calcium D,L-5-
Calcium D,L-5-Methyltetrahydrofolate RS, dissolve, and Methyltetrahydrofolate RS.]
dilute to volume. Suitability requirements
Monographs
[NOTE—The following Standard solution and Sample Suitability requirement 1: The resolution, R,
solution must be injected immediately after preparation between 4-aminobenzoylglutamic acid and 4α-
and injected only once.] hydroxy-5-smethyltetrahydrofolic acid is NLT 6 from
Standard solution: 0.50 mg/mL of USP Calcium D,L-5- the System suitability solution.
Methyltetrahydrofolate RS Suitability requirement 2: The resolution, R,
Sample solution: 0.50 mg/mL between folic acid and 5-methyltetrahydropteroic
Chromatographic system, Appendix IIA acid is NLT 8 from the System suitability solution.
Mode: High-performance liquid chromatography Suitability requirement 3: The resolution, R,
Detector: UV 280 nm between 5-methyltetrahydropteroic acid and
Column: 250-mm × 4.6-mm; contains 5-µm dimethyltetrahydrofolic acid is NLT 15 from the
octadecylsilane chemically bonded to porous silica or System suitability solution.
ceramic micro-particles1 Suitability requirement 4: The relative standard
Column temperature: 32° deviation, RSD, for three separately prepared
Flow rate: 1.1 mL/min Standard solutions is NMT 2.0%.
Injection volume: 10 µL Analysis: Separately inject equal volumes of the Standard
System suitability solution and Sample solution into the chromatograph,
Samples: System suitability solution and Standard and measure the responses for the major peaks of the
solution resulting chromatograms.
1 Purospher STAR RP 18e (Merck KGaA); or equivalent.
Table 2
Approximate Relative Relative Acceptance
Retention Retention Response Criteria,
Name Time Time Factor NMT (%)
4-Aminobenzoylglutamic acida 4.4 0.29 0.91 0.5
4α-Hydroxy-5-Methyltetrahydrofolic acidb 5.6 0.37 1.09 1.0
(6R)-Mefoxc,d — 0.49 1.05 —
1.0 (sum of
—
(6S)-Mefoxc,d 0.50 1.05 6R and 6S)
Tetrahydrofolic acide — 0.65 1.00f 0.5
7,8-Dihydrofolic acidg — 0.83 0.95 0.5
Folic acidh 12.8 0.85 0.83 0.5
5,10-Methylenetetrahydrofolic acidi — 0.88 1.00 f 0.5
D,L-5-Methyltetrahydrofolic acid 15.1 1.00 1.00 —
5-Methyltetrahydropteroic acidj — 1.10 0.67 0.5
Dimethyltetrahydrofolic acidk 18.8 1.25 1.00f 0.15
Total impurities — — — 2.5
a N-(4-Aminobenzoyl)-L-glutamic acid.
b N-[4-({[(6S)-2-Amino-4α-hydroxy-5-methyl-4-oxo-1,4,4α,5,6,7,8,8α-octahydropteridin-6-yl]methyl}amino)benzoyl]-L-glutamic acid.
c 2-Amino-8-methyl-4,9-dioxo-7-methyl-p-aminobenzoyl-glutamate-6,7,8,9-tetrahydro-4H-pyrazino-(1,2-α)-s-triazine.
d Report the impurity Mefox as sum of 6S- and 6R-Mefox.
e N-[4-({[(S)-2-Amino-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl]methyl}amino)benzoyl]-L-glutamic acid.
f Estimated factor.
g N-(4-{[(2-Amino-4-oxo-1,4,7,8-tetrahydropteridin-6-yl]methyl]amino}benzoyl)-L-glutamic acid.
h N-(4-{[(2-Amino-4-oxo-1,4-dihydropteridin-6-yl]methyl]amino}benzoyl)-L-glutamic acid.
i N-(4-(3-Amino-1-oxo-5,6,6α,7-tetrahydroimidazo[1,5-f]pteridin-8(1H,4H,9H)-yl)benzyl)-L-glutamic acid.
j (S)-4-{[(2-Amino-5-methyl-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl]methyl]amino}benzoic acid].
k N-[4-({[(S)-5-Methyl-2-(methylamino)-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl]methyl}amino)benzoyl]-L-glutamic acid.
FCC 9 Monographs / Calcium L-5-Methyltetrahydrofolate / 205
Calculate the percentage of calcium D,L-5-methyl- Internal standard stock solution: Transfer 1.0 mL of
tetrahydrofolate (C20H23CaN7O6) in the sample taken: USP 1-Propanol RS to a 100-mL volumetric flask, and
dilute with Dilution solution to volume.
Result = (rU/rS) × (CS/CU) × 100 Internal standard solution 1: Transfer 1.0 mL of
Internal standard stock solution to a 100-mL volumetric
rU = peak area of D,L-5-methyltetrahydrofolic acid
flask, and dilute to volume with Dilution solution.
from the chromatogram of the Sample
Internal standard solution 2: Transfer 2.0 mL of
solution
Internal standard stock solution to a 100-mL volumetric
rS = peak area of D,L-5-methyltetrahydrofolic acid
flask, and dilute to volume with Dilution solution.
from the chromatogram of the Standard
Ethanol standard solution: Transfer 2.5 mL of USP
solution
Monographs
Alcohol Determination-Alcohol RS to a 100-mL
CS = concentration of USP Calcium D,L-5-
volumetric flask, and dilute to volume with Dilution
Methyltetrahydrofolate RS in the Standard
solution.
solution (mg/mL)
2-Propanol standard solution: Transfer 15 mL of USP
CU = concentration of the Sample solution (mg/
2-Propanol RS to a 100-mL volumetric flask, and dilute
mL)
to volume with Dilution solution.
Acceptance criteria: 95.0%–102.0% of calcium D,L-5-
Mixed standard solution: Dilute a portion of the 2-
methyltetrahydrofolate, calculated on the anhydrous
Propanol standard solution 1:25 with Dilution solution.
and solvent-free basis
Combine 3.0 mL of the resulting solution with 1.0 mL
[NOTE—The criteria for Enantiomeric Purity must also be
of the Ethanol standard solution in a 100-mL volumetric
met in the portion of D,L-5-methyltetrahydrofolate and
flask, then dilute with Dilution solution to volume. Add
NMT 1.0% of D-5-methyltetrahydrofolate.]
5 mL of the resulting solution and 5 mL of Internal
IMPURITIES standard solution 2 to a headspace vial, and seal.
Inorganic Impurities [NOTE—The headspace vial is 20 mL, glass with PTFE/
• CHLORIDE silicone septa or equivalent.]
Sample: 300 mg Sample: 250 mg
0.005 M Silver nitrate: Dissolve 849 mg silver nitrate in Sample solution: Weigh the Sample into a headspace
sufficient water to make 1000 mL. vial, then add 10 mL of Internal standard solution 1 to
Analysis: Dissolve the Sample in 75 mL of water (heat the vial, and seal.
to a maximum of 40° if necessary for dissolution), add Chromatographic system, Appendix IIA
1 mL of nitric acid, and titrate with 0.005 M Silver Mode: Gas chromatography
nitrate, determining the endpoint potentiometrically. Detector: Flame-ionization
Perform a blank determination (see General Provisions). Column: 30-m × 0.32-mm (id) capillary coated with
Calculate the percentage of chloride in the Sample 6% cyanopropylphenyl and 94%
taken: dimethylpolysiloxane; 3 µm film thickness2
Temperatures
Result = [(VS − VB) × M × F/W] × 100 Injector: 180°
Column: See the temperature program table below.
VS = volume of 0.005 M Silver nitrate consumed
by the Sample (mL) Initial End
VB = volume of 0.005 M Silver nitrate consumed Temperature Rate Temperature Hold Time
by the blank (mL) (°) (°/min) (°) (min)
M = actual molarity of the 0.005 M Silver nitrate 40 0 40 8
titrant used (mmol/mL) 40 30 100 1
F = equivalency factor, 35.45 mg/mmol
postrun — 200 5
W = Sample weight (mg)
Acceptance criteria: NMT 0.5% Detector: 320°
• ELEMENTAL IMPURITIES, Elemental Impurities by ICP, Method Carrier gas: Helium
I, Appendix IIIC Flow rate: 1.4 mL/min constant flow
Acceptance criteria Injection volume: About 3 mL
Boron: NMT 50 µg/g Headspace parameters
Platinum: NMT 10 µg/g Oven temperature: 80°
Arsenic: NMT 1.5 µg/g Temperature loop: 110°
Cadmium: NMT 0.5 µg/g Temperature transfer line: 130°
Lead: NMT 1.0 µg/g Vial equilibration time: 10 min
Mercury: NMT 1.5 µg/g Vial pressurization time: 0.10 min
Organic Impurities Loop fill time: 0.20 min
• ETHANOL AND 2-PROPANOL (RESIDUAL SOLVENTS) Loop volume: 3 mL
Dilution solution: 5 g of trisodium citrate dihydrate in Loop equilibration time: 0.05 min
100 mL water Sample injection time: 1.50 min
2 Optima 624 (Macherey-Nagel); or equivalent.
206 / Calcium L-5-Methyltetrahydrofolate / Monographs FCC 9
Shaking: Low rS = peak response for the D,L-5-

Vial pressure: 55 kPa methyltetrahydrofolic acid from the
Analysis: Separately inject the Mixed standard solution chromatogram of the Standard solution
and the Sample solution into the chromatograph, and CS = concentration of USP Calcium D,L-5-
record the resulting chromatograms. From the Methyltetrahydrofolate RS in the Standard
chromatogram of the Mixed standard solution, solution (mg/mL)
separately calculate the response factors for ethanol CU = concentration of the Sample solution (mg/
and 2-propanol. mL)
[NOTE—The approximate retention times for ethanol, F = relative response factor for the
2-propanol, and 1-propanol are 6.3 min, 7.9 min, corresponding impurity peak (see Table 2)
Monographs
and 10.1 min.] M1 = molecular weight of L-5-

methyltetrahydrofolic acid, 459.5
RFS = (AIS × VS × ρS)/(AS × DFS) M2 = molecular weight of calcium L-5-
methyltetrahydrofolate, 497.5
Acceptance criteria
RFS = response factor for ethanol or 2-propanol
Individual and total impurities: See Table 2.
AIS = area of the 1-propanol peak from the
[NOTE—Disregard any impurity peak less than 0.05%.]
corresponding internal standard solution
• ENANTIOMERIC PURITY
VS = volume of the corresponding USP Reference
Buffer: 4.54 g/L of sodium dihydrogen phosphate,
Standard taken (USP 2-Propanol RS or USP
dihydrate in water
Alcohol Determination-Alcohol RS, mL)
Mobile phase: Acetonitrile and Buffer (3:97), adjust with
ρS = density of the corresponding USP Reference
32% (w/v) sodium hydroxide to a pH of 6.8
Standard (USP 2-Propanol RS or USP
Standard solution: 0.5 mg/mL of USP Calcium D,L-5-
Alcohol Determination-Alcohol RS, mg/mL)
Methyltetrahydrofolate RS in water
AS = area of the peak for the corresponding USP
Sample solution: 0.5 mg/mL in water
Reference Standard (USP 2-Propanol RS or
System suitability solution: Transfer 1.0 mL of the
USP Alcohol Determination-Alcohol RS)
Standard solution to a 50-mL volumetric flask, and dilute
DFS = dilution factor of the corresponding standard
to volume with the Sample solution.
solution (2500/6 for 2-Propanol standard
solution, 200 for Ethanol standard solution)
From the chromatogram of the Sample solution,
Detector: UV 280 nm
calculate the mass percentage of ethanol and 2-
Column: 4.0-mm × 15-cm; 5-µm packing of human
propanol in the Sample taken:
serum albumin, which has been immobilized onto
R% = (AS × RFS × 100)/(AIS × W) spherical particles3
Flow rate: 1.0 mL/min
AS = area of the peak for the corresponding USP Injection volume: 10 µL
Reference Standard (USP 2-Propanol RS or System suitability
USP Alcohol Determination-Alcohol RS) Sample: System suitability solution
RFS = response factor for ethanol or 2-propanol [NOTE—The relative retention times of L-5-
AIS = area of the 1-propanol peak from the methyltetrahydrofolate and D-5-methyltetrahydrofolate
corresponding internal standard solution are 1 and about 1.5, respectively.]
W = weight of sample taken (mg) Suitability requirements: The resolution between L-5-
Acceptance criteria: NMT 0.5% ethanol and NMT methyltetrahydrofolate and D-5-
0.5% 2-propanol methyltetrahydrofolate is NLT 1.5.
• RELATED COMPOUNDS Analysis: Separately inject equal volumes of the Sample
Solution A, Solution B, Mobile phase, System solution and the System suitability solution into the
suitability solution, Standard solution, Sample chromatograph, record the chromatograms, and
solution, and Chromatographic system: Proceed as identify the peaks of the D- and L-enantiomers in the
directed in the Assay. chromatogram of the Sample solution by comparing
Analysis: Proceed as directed under Analysis in the with those in the chromatogram of the System
Assay. suitability solution. Measure the peak area response.
Calculate the percentage of each impurity as listed in Calculate the percentage of D-5-methyltetrahydrofolate
Table 2 (with the exception of D,L-5- in the portion of D,L-5-Methyltetrahydrofolate taken:
methyltetrahydrofolic acid), as free acid, in the sample
taken: Result = [(rD/(rD + rL) × 100]
Result = (rU/rS) × (CS/CU) × F × (M1/M2) × 100 rD = peak response of D-5-methyltetrahydrofolate

from the chromatogram of the Sample
rU = peak response for the corresponding solution
impurity from the chromatogram of the
Sample solution 3 Chiral-HSA 34714 (Chromtech), or equivalent.
FCC 9 Monographs / Calcium Oxide / 207
rL = peak response of L-5-methyltetrahydrofolate IDENTIFICATION

from the chromatogram of the Sample • CALCIUM, Appendix IIIA
solution Sample solution: Slake 1 g of sample with 20 ml of
Acceptance criteria: NMT 1.0% of D-5- water and add glacial acetic acid until the sample is
methyltetrahydrofolate dissolved.
SPECIFIC TESTS
• CALCIUM ASSAY
0.05 M Edetate disodium: Dissolve 18.612 g of edetate • PROCEDURE
disodium in sufficient water to make 1000 mL. Sample: 1 g of sample ignited to a constant weight (See
Monographs
Sample: 250 mg Loss on Ignition below.)
Analysis: Transfer Sample to a suitable flask. Dissolve the Analysis: Dissolve the Sample in 20 mL of 2.7 N
Sample in 150 mL of water. Add 15 mL of 1 N sodium hydrochloric acid. Cool the solution, dilute to 500.0 mL
hydroxide and 300 mg of hydroxy naphthol blue to the with water, and mix. Pipet 50.0 mL of this solution into
flask, and mix. Titrate with 0.05 M Edetate disodium a suitable container, and add 50 mL of water. While
until the solution is deep blue in color. Perform a blank stirring, preferably with a magnetic stirrer, add about
determination (see General Provisions). 30 mL of 0.05 M disodium EDTA from a 50-mL buret.
Calculate the percentage of calcium in the Sample taken: Then, add 15 mL of 1 N sodium hydroxide and 300 mg
of hydroxy naphthol blue indicator. Continue the
Result = [(VS − VB) × M × F/W] × 100 titration with disodium EDTA to a blue endpoint. Each
VS = volume of 0.05 M Edetate disodium mL of 0.05 M disodium EDTA is equivalent to 2.804 mg
consumed by the Sample (mL) of CaO.
VB = volume of 0.05 M Edetate disodium Acceptance criteria: NLT 95.0% and NMT 100.5% of
consumed by the blank (mL) CaO, on the ignited basis
M = actual molarity of 0.05 M Edetate disodium IMPURITIES
titrant used (mmol/mL) Inorganic Impurities
F = equivalency factor, 40.08 mg/mmol • ALKALIES OR MAGNESIUM
W = Sample weight (mg) Sample: 500 mg
Acceptance criteria: 7.0%–8.5%, calculated on the Analysis: Dissolve the Sample in 30 mL of water and 15
anhydrous and solvent-free basis mL of 2.7 N hydrochloric acid. Heat the solution, boil
• WATER, Water Determination, Method Ic, Appendix IIB for 1 min, and rapidly add 40 mL of oxalic acid TS,
Sample: 40 mg and stir vigorously. Add 2 drops of methyl red TS, and
Analysis: Transfer Sample to a 20-mL headspace vial, neutralize the solution with 6 N ammonium hydroxide
and cap tightly. Heat the vial in a suitable Karl Fischer to precipitate the calcium completely. Heat the mixture
oven at 250°. Transfer the released and evaporated on a steam bath for 1 h and allow it to cool. Dilute the
water into the titration-cell in a stream of dry nitrogen mixture to 100 mL with water, mix well, and filter.
at a flow rate of about 40 mL/min as directed in the Add 0.5 mL of sulfuric acid to 50 mL of the filtrate.
referenced method. Then evaporate to dryness and ignite to constant
Acceptance criteria: 6.0%–17.0% weight in a tared platinum crucible at 800° ± 25°.
Calcium Oxide
.
acid
First Published: Prior to FCC 6 Acceptance criteria: NMT 3 mg/kg
Lime Sample: 1.0 g
CaO Formula wt 56.08 • LEAD, Lead Limit Test, Appendix IIIB
INS: 529 CAS: [1305-78-8]
UNII: C7X2M0VVNH [lime (cao)] acid
DESCRIPTION
Solution)
Calcium Oxide occurs as hard, white or gray-white masses
or granules or as a white to gray-white powder. One g
dissolves in about 840 mL of water at 25° and in about SPECIFIC TESTS
1740 mL of boiling water. It is soluble in glycerin but • ACID-INSOLUBLE SUBSTANCES
insoluble in alcohol. Sample solution: Slake 5 g of sample, and then mix it
Function: pH control agent; nutrient; dough conditioner; with 100 mL of water and sufficient hydrochloric acid,
yeast food added dropwise, to dissolve it.
Packaging and Storage: Store in tight containers. Analysis: Boil the Sample solution, cool, add hydrochloric
acid, if necessary, to make the solution distinctly acid,
208 / Calcium Oxide / Monographs FCC 9
and filter through a tared glass filter crucible. Wash the Mobile phase: Transfer 2.0 mL phosphoric acid into a
residue with water until free of chlorides, dry at 105° 2–L volumetric flask and dilute to volume with water.
for 1 h, cool, and weigh. Filter the solution through a 0.45–µm pore-size disk.
Acceptance criteria: NMT 1% Internal standard solution: Transfer 80 mg of p-
• LOSS ON IGNITION hydroxybenzoic acid into a 1–L volumetric flask,
Sample: 1 g dissolve in 5 mL of alcohol, dilute to volume with
Analysis: Ignite the Sample to constant weight in a tared Mobile phase, and mix.
platinum crucible at 1100° ± 50°. Standard solution: Transfer 15 mg of USP Calcium
Acceptance criteria: NMT 10.0% Pantothenate RS, previously dried, into a 25-mL
volumetric flask, dilute to volume with Internal standard
Monographs
solution, and mix.

Sample solution: Transfer 15 mg of sample, previously
Calcium Pantothenate
.
dried, into a 25-mL volumetric flask, dilute to volume

with Internal standard solution, and mix.
D-Calcium Pantothenate Detector: UV 210 nm
Dextro Calcium Pantothenate Column: 15-cm × 3.9-mm (id) column packed with
octadecylsilanized silica (10-µm µBondapak C 18, or
equivalent)
Flow rate: About 1.5 mL/min
Injection volume: About 10 µL
System Suitability
C18H32CaN2O10 Formula wt 476.54 Analysis: Chromatograph 3 replicate injections of the
CAS: [137-08-6] Standard solution and record the peak responses.
UNII: 568ET80C3D [calcium pantothenate] [NOTE—The relative retention times are 0.5 for
calcium pantothenate and 1.0 for p-hydroxybenzoic
DESCRIPTION acid.]
Calcium Pantothenate occurs as a slightly hygroscopic,
Suitability requirement: The relative standard
white powder. It is the calcium salt of the dextrorotatory
deviation is NMT 2.0%.
isomer of pantothenic acid. It is stable in air. One g
Analysis: Separately inject equal volumes of the Standard
dissolves in about 3 mL of water. It is soluble in glycerin,
solution and the Sample solution into the
but is practically insoluble in alcohol, in chloroform, and in
ether.
measure the peak responses obtained for each solution.
Function: Nutrient
[NOTE—The relative retention times are 0.5 for calcium
Packaging and Storage: Store in tight containers.
pantothenate and 1.0 for p-hydroxybenzoic acid.]
IDENTIFICATION Calculate the quantity (mg), of C18H32CaN2O10 in the
• A. CALCIUM, Appendix IIIA sample taken by the formula:
Sample: 50 mg/mL
Result = 25C(RU/RS)
• B. INFRARED ABSORPTION, Spectrophotometric Identification
Tests, Appendix IIIC C = concentration (mg/mL) of USP Calcium
Reference standard: USP Calcium Pantothenate RS Pantothenate RS in the Standard solution
Sample and standard preparation: K (Sample RU = ratio of the peak responses obtained for
previously dried at 105° for 3 h) calcium pantothenate and p-
Acceptance criteria: The spectrum of the sample hydroxybenzoic acid from the Sample
exhibits maxima at the same wavelengths as those in solution
the spectrum of the Reference standard. RS = ratio of the peak responses obtained for
• C. PROCEDURE calcium pantothenate and p-
Sample: 50 mg hydroxybenzoic acid from the Standard
Analysis: Boil the Sample in 5 mL of 1 N sodium solution
hydroxide for 1 min, and allow the solution to cool. Acceptance criteria: NLT 97.0% and NMT 103.0% of
Then add 5 mL of 1 N hydrochloric acid and 2 drops of calcium pantothenate (C18H32CaN2O10), on the dried
ferric chloride TS. basis
Acceptance criteria: A strong yellow color appears
IMPURITIES
ASSAY Inorganic Impurities
• PROCEDURE • LEAD, Lead Limit Test, Flame Atomic Absorption
[NOTE—Use low-actinic glassware throughout this Spectrophotometric Method, Appendix IIIB
procedure.] Sample: 10 g
FCC 9 Monographs / Calcium Pantothenate, Calcium Chloride Double Salt / 209
• ORGANIC IMPURITIES Packaging and Storage: Store in tight containers.

• ALKALOIDS
Sample: 200 mg IDENTIFICATION
Analysis: Dissolve the Sample in 5 mL of water, add 1 • A. CALCIUM, Appendix IIIA
mL of 2.7 N hydrochloric acid and 2 drops of Sample solution: 50 mg/mL
mercuric–potassium iodide TS. Acceptance criteria: Passes tests
Acceptance criteria: No turbidity develops within 1 • B. PROCEDURE
min. Sample: 50 mg
Analysis: Dissolve the Sample in 5 mL of 1 N sodium
SPECIFIC TESTS hydroxide, and filter. Add 1 drop of cupric sulfate TS to
Monographs
• ALKALINITY the filtrate.
Sample: 1 g Acceptance criteria: A deep blue color appears.
Analysis: Dissolve the Sample in 15 mL of recently • C. UNCOMPLEXED MATERIAL
boiled and cooled water in a small flask. As soon as the Sample: 1.0 g, previously dried
Sample is completely dissolved, add 1.6 mL of 0.1 N Analysis: Stir the Sample with 15 mL of
hydrochloric acid to the Sample solution, then add 0.05 dimethylformamide for 5 min. Centrifuge the mixture,
mL of phenolphthalein TS, and mix. transfer 2.0 mL of the clear supernatant liquid to a
Acceptance criteria: No pink color appears within 5 s. weighing dish, evaporate the liquid under vacuum on a
• CALCIUM CONTENT steam bath, and dry the residue in an oven at 105° for
Sample: 950 mg, previously dried 1 h. The weight (g) of the residue (composed of
Analysis: Dissolve the Sample in 100 mL of water uncombined calcium pantothenate and calcium
containing 2 mL of 2.7 N hydrochloric acid. While chloride) multiplied by 750 equals the percentage of
stirring, preferably with a magnetic stirrer, add about uncomplexed material in the sample.
30 mL of 0.05 M disodium EDTA from a 50-mL buret. Acceptance criteria: NMT 10.0%
of hydroxy naphthol blue indicator. Continue the ASSAY
titration with disodium EDTA to a blue endpoint. Each • PROCEDURE
mL of 0.05 M disodium EDTA is equivalent to 2.004 mg [NOTE—Use low-actinic glassware throughout this
of calcium (Ca). procedure.]
Acceptance criteria: NLT 8.2% and NMT 8.6% of Mobile phase: Transfer 2.0 mL phosphoric acid into a 2-
calcium (Ca), on the dried basis L volumetric flask and dilute to volume with water.
• LOSS ON DRYING, Appendix IIC: 105° for 3 h Filter the solution through a 0.45 µm pore-size disk.
Acceptance criteria: NMT 5.0% Internal standard solution: Transfer 80 mg of p-
• OPTICAL (SPECIFIC) ROTATION, Appendix IIB hydroxybenzoic acid into a 1-L volumetric flask, dissolve
Sample: 50 mg/mL, prepared using a previously dried in 5 mL of alcohol, dilute to volume with Mobile phase,
sample and mix.
Acceptance criteria: [α]D25 between +25.0° and +27.5°, Standard solution: Transfer 15 mg of USP Calcium
on the dried basis Pantothenate RS, previously dried, into a 25-mL
volumetric flask, dilute to volume with Internal standard
solution, and mix.
Sample solution: Transfer 15 mg of sample, previously
dried, into a 25-mL volumetric flask, dilute to volume
Calcium Pantothenate, Calcium
.
with Internal standard solution, and mix.

Chloride Double Salt Chromatographic system, Appendix IIA
First Published: Prior to FCC 6 Mode: High-performance liquid chromatography
Detector: UV 210 nm
Calcium Chloride Double Salt of DL- or D-Calcium Column: 15-cm × 3.9-mm (id) column packed with
Pantothenate octadecylsilanized silica (10-µm µBondapak C 18, or
C18H32CaN2O10 · CaCl2 Formula wt 587.52 equivalent)
CAS: [6363-38-8] Flow rate: About 1.5 mL/min
UNII: 43A575C21D [calcium pantothenate chloride] Injection volume: About 10 µL
System Suitability
DESCRIPTION Sample: Standard solution
Calcium Pantothenate, Calcium Chloride Double Salt occurs Suitability requirement: The relative standard
as a white, free-flowing, fine powder. It is a chemical deviation is NMT 2.0% for replicate injections.
complex composed of approximately equimolar quantities Analysis: Separately inject equal volumes of the Standard
of dextrorotatory (D) or racemic (DL) calcium pantothenate solution and the Sample solution into the
and calcium chloride. It is freely soluble in water, but chromatograph, record the chromatograms, and
insoluble in alcohol. Its solutions in water are alkaline to measure the peak responses obtained for each solution.
litmus. [NOTE—The relative retention times are 0.5 for Calcium
Function: Nutrient Pantothenate and 1.0 for p-hydroxybenzoic acid.]
210 / Calcium Pantothenate, Calcium Chloride Double Salt / Monographs FCC 9
Calculate the quantity (mg), of C18H32CaN2O10 in the

Calcium Pantothenate, Racemic
.
sample taken by the formula:

Result = 25C(RU/RS)
C18H32CaN2O10 Formula wt 476.54

C = concentration (mg/mL) of USP Calcium CAS: [6381-63-1]
Pantothenate RS in the Standard solution UNII: 2KC899R47Q [calcium pantothenate, racemic]
RU = ratio of the peak responses obtained for
calcium pantothenate and p- DESCRIPTION
hydroxybenzoic acid from the Sample Calcium Pantothenate, Racemic occurs as a white, slightly
Monographs
solution hygroscopic powder. It is a mixture of the calcium salts of

RS = ratio of the peak responses obtained for the dextrorotatory (D) and levorotatory (L) isomers of
calcium pantothenate and p- pantothenic acid. It is optically inactive. It is stable in air
hydroxybenzoic acid from the Standard and freely soluble in water. It is soluble in glycerin, and is
solution practically insoluble in alcohol, in chloroform, and in ether.
Acceptance criteria: NLT 45.0% and NMT 55.0% of Its solutions are neutral or alkaline to litmus. [NOTE—The
calcium pantothenate (C18H32CaN2O10), on the dried physiological activity of racemic Calcium Pantothenate is
basis approximately one-half that of the dextrorotatory isomer.]
Function: Nutrient
IMPURITIES Packaging and Storage: Store in tight containers.
• ARSENIC, Arsenic Limit Test, Appendix IIIB IDENTIFICATION
Sample solution: 1 g in 25 mL • A. CALCIUM, Appendix IIIA
Acceptance criteria: NMT 3 mg/kg Sample: 50 mg/mL
• LEAD, Lead Limit Test, APDC Extraction Method, Appendix Acceptance criteria: Passes tests
IIIB • B. INFRARED ABSORPTION, Spectrophotometric Identification
Acceptance criteria: NMT 2 mg/kg Tests, Appendix IIIC
Reference standard: USP Calcium Pantothenate RS
SPECIFIC TESTS Sample and Standard preparation: K (Sample
• CALCIUM CONTENT previously dried at 105° for 3 h)
Sample: 950 mg, previously dried Acceptance criteria: The spectrum of the sample
Analysis: Dissolve the Sample in 100 mL of water exhibits maxima at the same wavelengths as those in
containing 2 mL of 2.7 N hydrochloric acid. While the spectrum of the Reference Standard.
stirring, preferably with a magnetic stirrer, add about • C. PROCEDURE
30 mL of 0.05 M disodium EDTA from a 50-mL buret. Sample: 50 mg
Then, add 15 mL of 1 N sodium hydroxide and 300 mg Analysis: Boil the Sample in 5 mL 1 N sodium
of hydroxy naphthol blue indicator. Continue the hydroxide for 1 min, cool, and add 5 mL of 1 N
titration with disodium EDTA to a blue endpoint. Each hydrochloric acid and 2 drops of ferric chloride TS.
mL of 0.05 M disodium EDTA is equivalent to 2.004 mg Acceptance criteria: A strong yellow color appears.
of calcium (Ca).
Acceptance criteria: NLT 12.4% and NMT 13.6% of ASSAY
calcium (Ca), on the dried basis • PROCEDURE
• CHLORIDE CONTENT (as Cl) [NOTE—Use low-actinic glassware throughout this
Sample: 1 g, previously dried procedure.]
Analysis: Transfer the Sample to a 250-mL beaker, and Mobile phase: Transfer 2.0 mL phosphoric acid into a 2-
add sufficient water to make 100 mL. Equip a pH meter L volumetric flask and dilute to volume with water.
with glass and silver electrodes, and set it on the + Filter the solution through a 0.45-µm pore-size disk.
millivolt scale. Insert the electrodes and a motor-driven Internal standard solution: Transfer 80 mg of p-
glass stirring rod into the sample beaker. Add 1 to 2 hydroxybenzoic acid into a 1-L volumetric flask, dissolve
drops of methyl orange TS, stir, and add, dropwise, in 5 mL of alcohol, dilute to volume with Mobile phase,
10% nitric acid until a pink color appears; then add 10 and mix.
mL in excess. Titrate the solution with 0.1 N silver Standard solution: Transfer 15 mg of USP Calcium
nitrate to a reading of +1.0 millivolt. Each mL of 0.1 N Pantothenate RS, previously dried, into a 25-mL
silver nitrate is equivalent to 3.545 mg of chloride. volumetric flask, dilute to volume with Internal standard
Acceptance criteria: Between 10.5% and 12.1% of solution, and mix.
chloride, on the dried basis Sample solution: Transfer 15 mg of sample, previously
• LOSS ON DRYING, Appendix IIC: in vacuum, 100° for 1 h dried, into a 25-mL volumetric flask, dilute to volume
Acceptance criteria: NMT 5.0% with Internal standard solution, and mix.
Detector: UV 210 nm
FCC 9 Monographs / Calcium Peroxide / 211
Column: 15-cm × 3.9-mm (id) column packed with stirring, preferably with a magnetic stirrer, add about
octadecylsilanized silica (10-µm µBondapak C 18, or 30 mL of 0.05 M disodium EDTA from a 50-mL buret.
equivalent) Then, add 15 mL of 1 N sodium hydroxide and 300 mg
Flow rate: About 1.5 mL/min of hydroxy naphthol blue indicator. Continue the
Injection volume: About 10 µL titration with disodium EDTA to a blue endpoint. Each
System suitability mL of 0.05 M disodium EDTA is equivalent to 2.004 mg
Sample: Standard solution of calcium (Ca).
Suitability requirement: The relative standard Acceptance criteria: NLT 8.2% and NMT 8.6% of
deviation for three replicate injections is NMT 2.0%. calcium (Ca), on the dried basis
Analysis: Separately inject equal volumes of the Standard • LOSS ON DRYING, Appendix IIC: 105° for 3 h
Monographs
solution and the Sample solution into the Acceptance criteria: NMT 5.0%
chromatograph, record the chromatograms, and • OPTICAL (SPECIFIC) ROTATION, Appendix IIB
measure the peak responses obtained for each solution. Sample solution: 50 mg/mL, prepared using a
[NOTE—The relative retention times are 0.5 for calcium previously dried sample
pantothenate and 1.0 for p-hydroxybenzoic acid.] Acceptance criteria: [α]D25 between −0.05° and +0.05°,
Calculate the quantity (mg), of C18H32CaN2O10 in the on the dried basis
Result = 25C(RU/RS)
Calcium Peroxide
.
C = concentration (mg/mL) of USP Calcium First Published: Prior to FCC 6

Pantothenate RS in the Standard solution
RU = ratio of the peak responses obtained for CaO2 Formula wt 72.08
calcium pantothenate and p- INS: 930 CAS: [1305-79-9]
hydroxybenzoic acid from the Sample UNII: 7FRO2ENO91 [calcium peroxide]
solution
RS = ratio of the peak responses obtained for DESCRIPTION
calcium pantothenate and p- Calcium Peroxide occurs as a white or yellow powder or
hydroxybenzoic acid from the Standard granular material. It decomposes in moist air, is practically
solution insoluble in water, and dissolves in acids, forming
Acceptance criteria: NLT 97.0% and NMT 103.0% of hydrogen peroxide. A 1:100 aqueous slurry has a pH of
calcium pantothenate, on the dried basis about 12.
Function: Dough conditioner; oxidizing agent
IMPURITIES Packaging and Storage: Store in tight containers, and
Inorganic Impurities avoid contact with readily oxidizable materials. Observe the
• LEAD, Lead Limit Test, Flame Atomic Absorption safety precautions printed on the label of the original
Spectrophotometric Method, Appendix IIIB container.
Sample: 10 g
Acceptance criteria: NMT 2 mg/kg IDENTIFICATION
Organic Impurities • CALCIUM, Appendix IIIA
• ALKALOIDS Sample solution: Cautiously dissolve 250 mg of sample
Sample: 200 mg in 5 mL of glacial acetic acid and add a few drops of a
Analysis: Dissolve the Sample in 5 mL of water, add 1 saturated solution of potassium iodide. Iodine is
mL of 2.7 N hydrochloric acid and 2 drops of liberated. Add 20 mL of water and sufficient sodium
mercuric-potassium iodide TS. thiosulfate TS to remove the iodine color.
Acceptance criteria: No turbidity develops within 1 Acceptance criteria: Passes tests
min.
ASSAY
SPECIFIC TESTS • PROCEDURE
• ALKALINITY Sample: 1 g
Sample: 1 g Analysis: Transfer the Sample into an Erlenmeyer flask,
Analysis: Dissolve the Sample in 15 mL of recently add 30 mL of water and 30 mL of 1:1 (v/v) 85%
boiled and cooled water in a small flask. As soon as the phosphoric acid:water, and titrate immediately with 0.5
Sample is completely dissolved, add 1.6 mL of 0.1 N N potassium permanganate to the first faint pink color
hydrochloric acid to the Sample solution, then add 0.05 that persists for 1 min. Each mL of 0.5 N potassium
mL of phenolphthalein TS, and mix. permanganate is equivalent to 18.02 mg of CaO2.
Acceptance criteria: No pink color appears within 5 s. Acceptance criteria: NLT 60.0% of CaO2
• CALCIUM CONTENT
Sample: 950 mg, previously dried IMPURITIES
Analysis: Dissolve the Sample in 100 mL of water Inorganic Impurities
containing 2 mL of 2.7 N hydrochloric acid. While • FLUORIDE, Fluoride Limit Test, Appendix IIIB
212 / Calcium Peroxide / Monographs FCC 9
Sample: 1.0 g equipped with a magnetic stirrer, and cautiously add

Acceptance criteria: NMT 0.005% 125 mL of water. With constant stirring, add, in the
• LEAD, Lead Limit Test, Appendix IIIB order named, 0.5 mL of triethanolamine, 300 mg of
Sample solution: Transfer 4.0 g of sample, into a 250- hydroxy naphthol blue indicator, and, from a 50-mL
mL beaker, cautiously add 50 mL of nitric acid, and buret, about 23 mL of 0.05 M disodium EDTA. Then,
evaporate just to dryness on a steam bath. Add 20 mL add a 45:100 sodium hydroxide solution until the initial
of nitric acid, repeat the evaporation, cool, and dissolve red color changes to clear blue and continue to add it
the residue in sufficient water containing 4 drops of dropwise until the color changes to violet; then, add an
nitric acid to make 40.0 mL. additional 0.5 mL. The pH is between 12.3 and 12.5.
Control: 4 µg of Pb (4 mL of Diluted Standard Lead Continue the titration, dropwise, with the disodium
Monographs
Solution) EDTA to the appearance of a clear blue endpoint that

Analysis: Use 10 mL of the Sample solution. persists for NLT 60 s. Each mL of 0.05 M disodium
Acceptance criteria: NMT 4 mg/kg EDTA is equivalent to 6.803 mg of CaHPO4 or to 8.604
mg of CaHPO4 · 2H2O.
Acceptance criteria: NLT 97.0% and NMT 105.0%
(Anhydrous or dihydrate)
Calcium Phosphate, Dibasic
.
IMPURITIES
First Published: Prior to FCC 6 Inorganic Impurities
• ARSENIC, Appendix IIIB
Dicalcium Phosphate Sample solution: 1 g in 5 mL 2.7 N hydrochloric acid
CaHPO4 Formula wt, anhydrous 136.06 Acceptance criteria: NMT 3 mg/kg
CaHPO4 · 2H2O Formula wt, dihydrate 172.09 • FLUORIDE
INS: 341(ii) CAS: anhydrous [7757-93-9] [NOTE—Prepare and store all solutions in plastic
dihydrate [7789-77-7] containers.]
UNII: L11K75P92J [calcium phosphate, dibasic, anhydrous] Buffer solution: Dissolve 73.5 g sodium citrate in
UNII: O7TSZ97GEP [calcium phosphate, dibasic, dihydrate] water, made to 250 mL.
Standard stock solution: 1.1052 mg/mL USP Sodium
DESCRIPTION Fluoride RS
Calcium Phosphate, Dibasic occurs as a white powder. It is Standard solution: Transfer 20.0 mL of the Standard
anhydrous or contains two molecules of water of stock solution to a 100-mL volumetric flask containing
hydration. It is stable in air. It is insoluble in alcohol, is 50 mL of Buffer solution, dilute to volume with water,
practically insoluble in water, but is readily soluble in dilute and mix. (100 µg/mL fluoride ion)
hydrochloric and nitric acids. Sample solution: Transfer 2.0 g of sample to a beaker
Function: Leavening agent; dough conditioner; nutrient; containing a plastic-coated stirring bar, add 20 mL of
yeast food water and 2.0 mL of hydrochloric acid, and stir until
Packaging and Storage: Store in tightly closed the sample is dissolved. Add 50.0 mL of Buffer solution
containers. and sufficient water to make 100 mL.
Electrode system: Use a fluoride-specific, ion-indicating
IDENTIFICATION electrode and a silver-silver chloride reference electrode
• A. PROCEDURE connected to a pH meter capable of measuring
Sample: 100 mg potentials with a minimum reproducibility of ±0.2 mV.
Analysis: Dissolve the Sample by warming it with 5 mL Standard response line: Transfer 50.0 mL of Buffer
of 2.7 N hydrochloric acid and 5 mL of water. While solution and 2.0 mL of hydrochloric acid into a beaker
shaking the solution, add 2.5 mL of 6 N ammonium and add water to make 100 mL. Add a plastic-coated
hydroxide, dropwise, and then add 5 mL of ammonium stirring bar, insert the electrodes into the solution, stir
oxalate TS. for 15 min, and read the potential (mV). Continue
Acceptance criteria: A white precipitate forms. stirring, and at 5-min intervals, add 100 µL, 100 µL,
• B. PROCEDURE 300 µL, and 500 µL of Standard solution, reading the
Sample solution: 10 mg/mL potential 5 min after each addition. Plot the logarithms
Analysis: Add 10 mL ammonium molybdate TS to 10 of the cumulative fluoride ion concentrations (0.1, 0.2,
mL of warm Sample solution in a slight excess of nitric 0.5, and 1.0 µg/mL) versus potential, in mV.
acid. Analysis: Rinse and dry the electrodes, insert them into
Acceptance criteria: A yellow precipitate of ammonium the Sample solution, stir for 5 min, and read the
phosphomolybdate forms. potential (mV). From the measured potential and the
Standard response line, determine the concentration, C
ASSAY (µg/mL), of fluoride ion in the Sample solution.
• PROCEDURE
Calculate the percentage of fluoride in the sample
Sample: 250 mg
taken by the formula:
Analysis: Dissolve the Sample, with the aid of gentle
heat if necessary, in a mixture of 5 mL of hydrochloric Result = C × 0.005
acid and 3 mL of water contained in a 250-mL beaker
FCC 9 Monographs / Calcium Phosphate, Monobasic / 213
Acceptance criteria: NMT 0.005% ASSAY

• LEAD, Lead Limit Test, APDC Extraction Method, Appendix • CALCIUM
IIIB Sample: 450 mg, dried
Acceptance criteria: NMT 2 mg/kg Analysis: In a 250-mL beaker equipped with a magnetic
stirrer, dissolve the Sample in a mixture of 5 mL of
SPECIFIC TESTS water and 1 mL of hydrochloric acid, with the aid of
• LOSS ON IGNITION gentle heat if necessary, and add 50 mL of water. With
Sample: 3 g constant stirring, add exactly 25 mL of 0.1 M disodium
Analysis: Ignite the Sample, preferably in a muffle EDTA to the solution. The solution should turn cloudy
furnace, at 800° to 825° to constant weight. indicating that enough EDTA has been added. Add to
Monographs
Acceptance criteria the solution, ammonium hydroxide dropwise until it
Anhydrous: Between 7.0% and 8.5% becomes clear, then add 20 more drops of ammonium
Dihydrate: Between 24.5% and 26.5% hydroxide and 10 mL ammonia–ammonium chloride
OTHER REQUIREMENTS buffer TS. Add to the mixture 0.1 mL eriochrome black
• LABELING: Indicate whether the product is anhydrous or TS to obtain a clear blue color. Titrate this solution with
the dihydrate. 0.1 M zinc sulfate until the clear blue color changes to
dark red. To obtain the amount of 0.1 M disodium
EDTA consumed (mL), subtract the volume (mL) of zinc
sulfate used from the initial volume of 0.1 M disodium
EDTA that was added (25 mL). Each mL of 0.1 M
Calcium Phosphate, Monobasic
.
disodium EDTA consumed is equivalent to 4.008 mg

First Published: Prior to FCC 6 Ca.
Last Revision: Second Supplement, FCC 8 Acceptance criteria
Anhydrous: NLT 16.8% and NMT 18.3% of Ca
Monocalcium Phosphate Monohydrate: NLT 15.9% and NMT 17.7% of Ca
Calcium Biphosphate
Acid Calcium Phosphate IMPURITIES
Ca(H2PO4)2 Formula wt anhydrous 234.05 • ARSENIC, Arsenic Limit Test, Appendix IIIB
Ca(H2PO4)2 · H2O Formula wt monohydrate 252.07 Sample solution: 1 g in 5 mL of 2.7 N hydrochloric
INS: 341(i) CAS: anhydrous [7758-23-8]
acid
monohydrate [10031-30-8]
UNII: 0N4E6L5449 [calcium phosphate, monobasic, • FLUORIDE
monohydrate] [NOTE—Prepare and store all solutions in plastic
UNII: 701EKV9RMN [calcium phosphate, monobasic, containers.]
anhydrous] Anhydrous material: Fluoride Limit Test, Method II,
Appendix IIIB
DESCRIPTION
Monohydrate
Calcium Phosphate, Monobasic, occurs as white crystals or
Buffer solution: Dissolve 73.5 g of sodium citrate in
granules, or as a granular powder. It is anhydrous or
water, made to 250 mL.
contains one molecule of water of hydration, but because
Standard stock solution: 1.1052 mg/mL of USP
of its deliquescent nature, more than the calculated
Sodium Fluoride RS
amount of water may be present. It is sparingly soluble in
Standard solution: Transfer 20.0 mL of the Standard
water, and insoluble in alcohol.
stock solution to a 100-mL volumetric flask containing
Function: Buffer; dough conditioner; firming agent;
50 mL of Buffer solution, dilute with water to volume,
leavening agent; nutrient; yeast food; sequestrant
and mix. (100 µg/mL fluoride ion)
Sample solution: Transfer 2.0 g of sample to a beaker
IDENTIFICATION containing a plastic-coated stirring bar, add 20 mL of
• A. PROCEDURE water and 2.0 mL of hydrochloric acid, and stir until
Sample: 100 mg the sample is dissolved. Add 50.0 mL of Buffer
Analysis: Dissolve the Sample by warming it in a mixture solution and sufficient water to make 100 mL.
of 2 mL of 2.7 N hydrochloric acid and 8 mL of water. Electrode system: Use a fluoride-specific, ion-
Add 5 mL of ammonium oxalate TS. indicating electrode and a silver–silver chloride
Acceptance criteria: A white precipitate forms. reference electrode connected to a pH meter capable
• B. PROCEDURE of measuring potentials with a minimum
Analysis: Add ammonium molybdate TS to a warm reproducibility of ±0.2 mV.
solution of sample in a slight excess of nitric acid. Standard response line: Transfer 50.0 mL of Buffer
Acceptance criteria: A yellow precipitate of ammonium solution and 2.0 mL of hydrochloric acid into a
phosphomolybdate forms. beaker, and add water to make 100 mL. Add a
plastic-coated stirring bar, insert the electrodes into
the solution, stir for 15 min, and read the potential
214 / Calcium Phosphate, Monobasic / Monographs FCC 9
(mV). Continue stirring, and at 5-min intervals, add IDENTIFICATION

100 µL, 100 µL, 300 µL, and 500 µL of the Standard • A. PROCEDURE
solution, reading the potential 5 min after each Analysis: Add ammonium molybdate TS to a warm
addition. Plot the logarithms of the cumulative solution of sample in a slight excess of nitric acid.
fluoride ion concentrations (0.1 µg/mL, 0.2 µg/mL, Acceptance criteria: A yellow precipitate of ammonium
0.5 µg/mL, and 1.0 µg/mL) versus potential, in mV. phosphomolybdate forms.
Analysis: Rinse and dry the electrodes, insert them • B. PROCEDURE
into the Sample solution, stir for 5 min, and read the Sample: 100 mg
potential (mV). From the measured potential and the Analysis: Dissolve the Sample by warming it with 5 mL
Standard response line, determine the concentration, of 2.7 N hydrochloric acid and 5 mL of water. While
Monographs
C (µg/mL), of fluoride ion in the Sample solution. shaking the solution, add 1 mL of 6 N ammonium
Calculate the percentage of fluoride in the sample hydroxide, dropwise; then add 5 mL of ammonium
taken by the formula: oxalate TS.
Acceptance criteria: A white precipitate forms.
Result = C × 0.005
ASSAY
Acceptance criteria: NMT 0.005% • CALCIUM
• LEAD, Lead Limit Test, APDC Extraction Method, Appendix Sample: 150 mg
IIIB Analysis: Dissolve the Sample, with the aid of gentle
Acceptance criteria: NMT 2 mg/kg heat if necessary, in a mixture of 5 mL of hydrochloric
SPECIFIC TESTS acid and 3 mL of water contained in a 250-mL beaker
• LOSS ON DRYING, Appendix IIC: 60° for 3 h equipped with a magnetic stirrer, and cautiously add
[NOTE—Monohydrate only.] 125 mL of water. With constant stirring, add, in the
Acceptance criteria: NMT 1% order named, 0.5 mL of triethanolamine, 300 mg of
• LOSS ON IGNITION hydroxy naphthol blue indicator, and, from a 50-mL
[NOTE—Anhydrous material only.] buret, about 23 mL of 0.05 M disodium EDTA. Then,
Sample: 3 g add a 45:100 sodium hydroxide solution until the initial
Analysis: Ignite the Sample, preferably in a muffle red color changes to clear blue and continue to add it
furnace, at 800° for 30 min. dropwise until the color changes to violet; then, add an
Acceptance criteria: 14.0%–15.5% additional 0.5 mL. The pH is between 12.3 and 12.5.
Continue the titration, dropwise, with the disodium
OTHER REQUIREMENTS EDTA to the appearance of a clear blue endpoint that
• LABELING: Indicate the state of hydration. persists for not less than 60 s. Each mL of 0.05 M
disodium EDTA is equivalent to 2.004 mg of Ca.
Acceptance criteria: NLT 34.0% and NMT 40.0% of Ca
IMPURITIES
Calcium Phosphate, Tribasic
.
First Published: Prior to FCC 6 • ARSENIC, Arsenic Limit Test, Appendix IIIB
Tricalcium Phosphate acid
Precipitated Calcium Phosphate Acceptance criteria: NMT 3 mg/kg
Calcium Hydroxyapatite • FLUORIDE
Ca3(PO4)2 Formula wt 310.18 [NOTE—Prepare and store all solutions in plastic
Ca5OH(PO4)3 Formula wt 502.31 containers.]
Ca10(OH)2(PO4)6 Formula wt 1004.61 Buffer solution: Dissolve 73.5 g sodium citrate in
INS: 341(iii) CAS: [7758-87-4] water, made to 250 mL.
[1306-06-5] Standard stock solution: 1.1052 mg/mL USP Sodium
[62974-97-4] Fluoride RS
UNII: 91D9GV0Z28 [tribasic calcium phosphate] Standard solution: Transfer 20.0 mL of the Standard
UNII: K4C08XP666 [tricalcium phosphate] stock solution to a 100-mL volumetric flask containing
50 mL of Buffer solution, dilute to volume with water,
DESCRIPTION and mix. (100 µg/mL fluoride ion)
Calcium Phosphate, Tribasic occurs as a white powder that Sample solution: Transfer 2.0 g of sample to a beaker
is stable in air. It consists of a variable mixture of calcium containing a plastic-coated stirring bar, add 20 mL of
phosphates. It is insoluble in alcohol and almost insoluble water and 3.0 mL of hydrochloric acid, and stir until
in water, but it dissolves readily in dilute hydrochloric and the sample is dissolved. Add 50.0 mL of Buffer solution
nitric acids. and sufficient water to make 100 mL.
Function: Anticaking agent; buffer; nutrient; clouding Electrode system: Use a fluoride-specific, ion-indicating
agent electrode and a silver-silver chloride reference electrode
FCC 9 Monographs / Calcium Propionate / 215
connected to a pH meter capable of measuring IDENTIFICATION

potentials with a minimum reproducibility of ±0.2 mV. • A. CALCIUM, Appendix IIIA
Standard response line: Transfer 50.0 mL of Buffer Sample solution: 50 mg/mL
solution and 3.0 mL of hydrochloric acid into a beaker Acceptance criteria: Passes tests
and add water to make 100 mL. Add a plastic-coated • B. PROCEDURE
stirring bar, insert the electrodes into the solution, stir Analysis: Ignite a sample at a relatively low temperature.
for 15 min, and read the potential (mV). Continue Acceptance criteria: An alkaline residue that effervesces
stirring, and at 5-min intervals, add 100 µL, 100 µL, with acids is formed.
300 µL, and 500 µL of Standard solution, reading the
potential 5 min after each addition. Plot the logarithms ASSAY
Monographs
of the cumulative fluoride ion concentrations (0.1, 0.2, • PROCEDURE
0.5, and 1.0 µg/mL) versus potential, in mV. Sample solution: Dissolve 400 mg of sample in 75 mL
Analysis: Rinse and dry the electrodes, insert them into of hot water (>82°) and filter through a 30 medium
the Sample solution, stir for 5 min, and read the filtering crucible; wash the filtering crucible with 25 mL
potential (mV). From the measured potential and the of hot water (>82°). Combine all collected filtrate and
Standard response line, determine the concentration, C use as the Sample solution.
(µg/mL), of fluoride ion in the Sample solution. Analysis: While stirring the Sample solution, preferably
Calculate the percentage of fluoride in the sample with a magnetic stirrer, add about 30 mL of 0.05 M
taken by the formula: disodium EDTA from a 50-mL buret. Then, add 15 mL
of 1 N sodium hydroxide and 300 mg of hydroxy
Result = C × 0.005 naphthol blue indicator. Continue the titration with the
disodium EDTA to a blue endpoint. Each mL of 0.05 M
Acceptance criteria: NMT 0.0075% disodium EDTA is equivalent to 9.311 mg of C6H10CaO4.
• LEAD, Lead Limit Test, APDC Extraction Method, Appendix Acceptance criteria: NLT 98.0% and NMT 100.5% of
IIIB C6H10CaO4, calculated on the anhydrous basis
IMPURITIES
SPECIFIC TESTS Inorganic Impurities
• LOSS ON IGNITION • FLUORIDE, Fluoride Limit Test, Method III, Appendix IIIB
Sample: 3 g Sample: 1.0 g
Analysis: Ignite the Sample, preferably in a muffle Acceptance criteria: NMT 0.003%
furnace, at 800° to 825° to constant weight. • LEAD, Lead Limit Test, Flame Atomic Absorption
Acceptance criteria: NMT 10.0% Spectrophotometric Method, Appendix IIIB
Sample: 10 g
• MAGNESIUM (as MgO)
Calcium Propionate
.
Sample: 400.0 mg
First Published: Prior to FCC 6 Magnesium standard solution: 50 µg/mL (see Standard
Last Revision: Second Supplement, FCC 6 Solutions for the Preparation of Controls and Standards,
Solutions and Indicators)
Calcium Propanoate Analysis: Place the Sample, 5 mL of 2.7 N hydrochloric
acid, and about 10 mL of water in a small beaker, and
dissolve the sample by heating it on a hot plate.
Evaporate the solution to a volume of about 2 mL, and
cool. Transfer the residual liquid into a 100-mL
volumetric flask, dilute with water to volume, and mix.
C6H10CaO4 Formula wt 186.22
Dilute 7.5 mL of this solution with water to 20 mL, add
INS: 282 CAS: [4075-81-4]
2 mL of 1 N sodium hydroxide and 0.05 mL of a
UNII: 8AI80040KW [calcium propionate]
1:1000 solution of Titan yellow (Clayton yellow), mix,
DESCRIPTION allow the mixture to stand for 10 min, and shake. Note
Calcium Propionate occurs as white crystals or as a any color produced, and repeat the preceding using
crystalline solid. It is produced by reacting calcium 1.0 mL of the Magnesium standard solution in the same
hydroxide (lime) with propionic acid. The pH of a 1:10 volume as that of a control solution containing 2.5 mL
aqueous solution is between 7.5 and 10.5. One g dissolves of the sample solution (corresponding to 10 mg of
in about 3 mL of water. sample) and the quantities of the reagents used in the
Function: Preservative; mold inhibitor test.
Packaging and Storage: Store in tightly closed Acceptance criteria: Any color produced by the Sample
containers. does not exceed that produced by the control solution
(about 0.4%).
216 / Calcium Propionate / Monographs FCC 9
SPECIFIC TESTS few drops of methyl orange TS and boil for 30 min.
• ACID-INSOLUBLE SUBSTANCES Keep the volume and pH of the solution constant
Sample: 2 g during the boiling period by adding hydrochloric acid
Analysis: Dissolve the Sample in 30 mL of 1:3 or water if necessary. Add 2 drops of methyl red TS and
hydrochloric acid, and heat to boiling. Filter the 30 mL of ammonium oxalate TS. Then add, dropwise,
mixture through a suitable tared, porous-bottom with constant stirring, a mixture of equal volumes of
porcelain crucible, and wash the residue with hot water 6 N ammonium hydroxide and water until the pink
until the last washing is free from chloride. Ignite the color of the indicator just disappears. Digest the
residue at 800° ± 25° for 45 min, cool, and weigh the solution on a steam bath for 30 min, cool it to room
residue. [NOTE—Avoid exposing the crucible to sudden temperature, allow the precipitate to settle, and filter
Monographs
temperature changes.] the supernatant liquid through a sintered-glass filter

Acceptance criteria: NMT 0.2% crucible using gentle suction. Wash the precipitate in
• WATER, Water Determination, Appendix IIB the beaker with about 30 mL of cold (below 20°) Wash
Acceptance criteria: NMT 5.0% solution. Allow the precipitate to settle and pour the
supernatant liquid through the filter. Repeat this
washing by decantation three more times. Using the
Wash solution, transfer the precipitate as completely as
Calcium Pyrophosphate possible to the filter. Finally, wash the beaker and the
.
filter with two 10-mL portions of cold (below 20°)

water. Place the sintered-glass filter crucible in the
beaker and add 100 mL of water and 50 mL of cold,
Ca2P2O7 Formula wt 254.10 1:6 sulfuric acid. Add 35 mL of 0.1 N potassium
INS: 450(vi) CAS: [7790-76-3] permanganate from a buret and stir until the color
UNII: X69NU20D19 [calcium pyrophosphate] disappears. Heat to about 70°, and complete the
titration with 0.1 N potassium permanganate. Each mL
DESCRIPTION of 0.1 N potassium permanganate is equivalent to 6.35
Calcium Pyrophosphate occurs as a fine, white powder. It is
mg of Ca2P2O7.
insoluble in water, but is soluble in dilute hydrochloric and
Acceptance criteria: NLT 96.0% of Ca2P2O7
nitric acids.
Function: Buffer; neutralizing agent; nutrient IMPURITIES
Packaging and Storage: Store in well-closed containers. Inorganic Impurities
IDENTIFICATION Sample: 1 g in 5 mL of 2.7 N hydrochloric acid
• A. PROCEDURE
Sample: 100 mg
Analysis: Dissolve the Sample by warming it with a
Sample: 1 g
mixture of 5 mL of 2.7 N hydrochloric acid and 5 mL
of water. While shaking the solution, add 2.5 mL of 6 N
ammonium hydroxide, dropwise. Then add 5 mL of
IIIB
ammonium oxalate TS.
• B. PROCEDURE SPECIFIC TESTS
Sample: 100 mg • LOSS ON IGNITION
Analysis: Dissolve the Sample in 100 mL of 1.7 N nitric Sample: 1 g
acid and add 0.5 mL of this solution (reserving the Analysis: Ignite the Sample, preferably in a muffle
remainder for Identification test C) to 30 mL of furnace, at 800° to 825° for 30 min.
quimociac TS. Acceptance criteria: NMT 1.0%
Acceptance criteria: A yellow precipitate does not form.
• C. PROCEDURE
Sample: Reserved solution from Procedure B
Analysis: Heat the Sample for 10 min at 95°, and then Calcium Saccharin
.
add 0.5 mL of the solution to 30 mL of quimociac TS.

Acceptance criteria: A yellow precipitate forms First Published: Prior to FCC 6
immediately. Last Revision: FCC 9
ASSAY 1,2-Benzisothiazolin-3-one-1,1-Dioxide, Calcium Salt

• PROCEDURE
Sample: 300 mg
Wash solution: Dilute 10 mL of ammonium oxalate TS
to 1000 mL with water.
Analysis: Dissolve the Sample in 10 mL of 2.7 N
hydrochloric acid, add about 120 mL of water and a
FCC 9 Monographs / Calcium Saccharin / 217
C14H8CaN2O6S2 · 31/2H2O Formula wt 467.48 • SELENIUM, Selenium Limit Test, Method I, Appendix IIIB
INS: 954 CAS: [6485-34-3] Sample: 200 mg
UNII: 5101OP7P2I [saccharin calcium] Acceptance criteria: NMT 0.003%
DESCRIPTION Change to read:

Calcium Saccharin occurs as white crystals or as a white, Organic Impurities
crystalline powder. One g is soluble in 1.5 mL of water. • BENZOATE AND SALICYLATE
Function: Non-nutritive sweetener Sample solution: 50 mg/mL
Packaging and Storage: Store in well-closed containers. Analysis: Add 3 drops of ferric chloride TS to 10 mL of
Sample solution previously acidified with 5 drops of
IDENTIFICATION
Monographs
glacial acetic acid.
Change to read: Acceptance criteria: No precipitate or violet color
appears.
• A. ▲ INFRARED ABSORPTION, Spectrophotometric Identification
• TOLUENESULFONAMIDES
Tests, Appendix IIIC ▲
▲ FCC9
Sample: Dry at 105° to constant weight.
Internal standard ▲ ▲ FCC9 solution: ▲ 0.25 mg/mL
Reference standard: USP Saccharin Calcium RS
caffeine in methylene chloride▲ FCC9
Sample and standard preparation: K▲ FCC9
Standard stock solution: ▲ 20 µg/mL USP o-
Acceptance criteria: ▲ The spectrum of the sample
Toluenesulfonamide RS and 20 µg/mL USP p-
Toluenesulfonamide RS in methylene chloride
the spectrum of the Reference standard.▲ FCC9
Standard solution: Evaporate 5.0 mL of Standard stock
• B. PROCEDURE
solution to dryness in a stream of nitrogen. Dissolve the
Sample: 20 mg
residue in 1.0 mL of the Internal standard solution.
Analysis: Mix the Sample with 40 mg of resorcinol,
Sample stock solution: 200 mg/mL in water. If
cautiously add 10 drops of sulfuric acid, and heat the
necessary, adjust the pH to 7–8 with 1 N sodium
mixture in a liquid bath at 200° for 3 min. After
hydroxide or 1 N hydrochloric acid before final
cooling, add 10 mL of water and an excess of 1 N
dilution.
sodium hydroxide.
Sample solution: Shake 50 mL of the Sample stock
Acceptance criteria: A fluorescent green liquid is
solution with four quantities each of 50 mL of
produced.
methylene chloride. Combine the lower layers, dry over
• C. CALCIUM, Appendix IIIA
anhydrous sodium sulfate, and filter. Wash the filter
and the sodium sulfate with 10 mL of methylene
▲
chloride. Combine the solution and the washings, and
▲ FCC9
evaporate almost to dryness in a water bath at a
ASSAY temperature not exceeding 40°. Using a small quantity
• PROCEDURE of methylene chloride, quantitatively transfer the
Sample: 500 mg residue into a suitable 10-mL tube, evaporate to
Analysis: With the aid of 10 mL of water, quantitatively dryness in a stream of nitrogen, and dissolve the
transfer the Sample into a separatory funnel. Add 2 mL residue in 1.0 mL of the Internal standard solution.
of 2.7 N hydrochloric acid, and extract the precipitated Blank solution: Evaporate 200 mL of methylene
saccharin, first with 30 mL, then with five 20-mL chloride to dryness in a water bath at a temperature
portions of solvent comprising 9:1 (v/v) not exceeding 40°. Dissolve the residue in 1 mL of
chloroform:alcohol. Filter each extract through a small methylene chloride.
filter paper moistened with the solvent mixture, and Chromatographic system, Appendix IIA
evaporate the combined filtrates to dryness on a steam Mode: Gas chromatography
bath with the aid of a current of air. Dissolve the Detector: Flame ionization
residue in 75 mL of hot water, cool, add Column: 0.53-mm × 10-m fused silica column coated
phenolphthalein TS, and titrate with 0.1 N sodium with a 2-µm film of 50% phenyl–50%
hydroxide. Perform a blank determination (see General methylpolysiloxane1
Provisions), and make any necessary correction. Each mL Temperatures
of 0.1 N sodium hydroxide is equivalent to 20.22 mg Column: 180°
of C14H8CaN2O6S2. Injection port: 250°
Acceptance criteria: 98.0%–101.0% of C14H8CaN2O6S2, Detector: 250°
calculated on the anhydrous basis Carrier gas: Nitrogen
Flow rate: 10 mL/min
IMPURITIES Injection volume: 1 µL
Inorganic Impurities Split ratio: 2:1
• LEAD, Lead Limit Test, Flame Atomic Absorption System suitability
Spectrophotometric Method, Appendix IIIB Samples: Standard solution and Blank solution
Sample: 10 g
Acceptance criteria: NMT 2 mg/kg 1 DB-17 (J & W Scientific), or equivalent; available from www.agilent.com.
218 / Calcium Saccharin / Monographs FCC 9
Suitability requirements: [NOTE—The substances are IDENTIFICATION

eluted in the following order: o-toluenesulfonamide, • CALCIUM, Appendix IIIA
or p-toluenesulfonamide, and caffeine.] Sample solution: Mix 500 mg of sample with 10 mL of
Suitability requirement 1: The resolution, R, 2.7 N hydrochloric acid, filter, and neutralize the filtrate
between o-toluenesulfonamide and p- to litmus paper with 6 N ammonium hydroxide.
toluenesulfonamide is NLT 1.5 in the chromatogram Acceptance criteria: Passes tests
of the Standard solution. • SILICA
Suitability requirement 2: No peaks are exhibited at Analysis: Prepare a bead by fusing a few crystals of
the retention times for caffeine, o- sodium ammonium phosphate on a platinum loop in
toluenesulfonamide, or p-toluenesulfonamide in the the flame of a Bunsen burner. Place the hot,
Monographs
chromatogram of the Blank solution. transparent bead in contact with a sample, and again
Analysis: Separately inject the Standard solution and fuse.
the Sample solution into the chromatograph, record the Acceptance criteria: Silica floats about in the bead,
chromatograms, and measure responses for the major producing, upon cooling, an opaque bead with a
peaks on the resulting chromatograms. weblike structure.
Acceptance criteria: If any peaks due to o-
toluenesulfonamide and p-toluenesulfonamide appear ASSAY
in the chromatogram obtained with the Sample • SILICON DIOXIDE
solution, the ratio of their areas to that of the Internal Sample: 400 mg
standard solution is NMT the corresponding ratio in the Analysis: Transfer the Sample into a beaker, add 5 mL of
chromatogram obtained with the Standard solution water and 10 mL of perchloric acid, and heat until
(NMT 10 ppm o-toluenesulfonamide and NMT 10 ppm dense, white fumes of perchloric acid evolve.
p-toluenesulfonamide).▲ FCC9 [CAUTION—Handle perchloric acid in an appropriate
fume hood.] Cover the beaker with a watch glass, and
SPECIFIC TESTS continue to heat for 15 min longer. Allow to cool, add
• READILY CARBONIZABLE SUBSTANCES, Appendix IIB 30 mL of water, filter, and wash the precipitate with
Sample: 200 mg 200 mL of hot water. Retain the combined filtrate and
Analysis: Dissolve the Sample in 5 mL of 95% sulfuric washings for use in the Assay for Calcium Oxide. Transfer
acid, and hold the solution at 48°–50° for 10 min. the filter paper and its contents into a platinum
Acceptance criteria: The color of the resulting solution crucible, heat slowly to dryness, and then heat
is no darker than that of Matching Fluid A. sufficiently to char the filter paper. After cooling, add a
• WATER, Water Determination, Appendix IIB few drops of sulfuric acid, and then ignite at about
Acceptance criteria: NMT 15.0% 1300° to constant weight. Moisten the residue with 5
drops of sulfuric acid, add 15 mL of hydrofluoric acid,
heat cautiously on a hot plate until all of the acid is
driven off, and ignite to constant weight at a
Calcium Silicate
.
temperature not lower than 1000°. [CAUTION—Handle

First Published: Prior to FCC 6 hydrofluoric acid in an appropriate fume hood.] Cool in
Last Revision: First Supplement, FCC 6 a desiccator, and weigh. The loss in weight is
equivalent to the amount of SiO2 in the Sample taken.
Acceptance criteria: The result should conform to the
INS: 552 CAS: [1344-95-2]
representations of the vendor.
UNII: S4255P4G5M [calcium silicate]
• CALCIUM OXIDE
DESCRIPTION Sample solution: The retained combined filtrate and
Calcium Silicate occurs as a white to off-white, free-flowing washings from the Assay for Silicon Dioxide above
powder that remains so after absorbing relatively large Analysis: Using 1 N sodium hydroxide, neutralize the
amounts of water or other liquids. It is a hydrous or Sample solution to litmus and add, while stirring, about
anhydrous silicate with varying proportions of CaO and 30 mL of 0.05 M disodium EDTA from a 50-mL buret.
SiO2. It is manufactured by two distinct processes identified Add 15 mL of 1 N sodium hydroxide and 300 mg of
by the form of silica used, either diatomaceous earth or hydroxy naphthol blue indicator. Continue the titration
precipitated silica. Diatomaceous earth-based products are with the disodium EDTA to a blue endpoint. Each mL of
produced through hydrothermal reaction processes, which 0.05 M disodium EDTA is equivalent to 2.804 mg of
combine natural, or flux-calcined diatomaceous earth with CaO.
hydrated lime to produce synthetic mineral forms of Acceptance criteria: The result should conform to the
gyrolite and tobermorite. Precipitated or other silica-based representations of the vendor.
products are produced by reacting sodium silicate and IMPURITIES
calcium oxide. It is insoluble in water, but it forms a gel Inorganic Impurities
with mineral acids. The pH of 1:20 aqueous slurry is • FLUORIDE
between 8.4 and 12.5. [NOTE—Store all fluoride solutions in plastic containers.]
Function: Anticaking agent; filter aid
FCC 9 Monographs / Calcium Sorbate / 219
0.2 N EDTA/0.2 N TRIS solution: Transfer 18.6 g of Acceptance criteria: NMT 10 mg/kg
disodium ethylenediaminetetraacetate (EDTA) and 6.05 • LEAD
g of tris-(hydroxymethyl)aminomethane (TRIS), into a Standard stock solution: (100 µg/mL lead ion) [NOTE—
single 250-mL beaker. Add 200 mL of hot, deionized Prepare and store this solution in glass containers that
water, and stir until dissolved. Adjust the pH to 7.5 to are free from lead salts.] Dissolve 159.8 mg of ACS
7.6 by adding 5 N sodium hydroxide. Cool the reagent-grade lead nitrate in 100 mL of water
solution, and adjust the pH to 8.0 with 5 N sodium containing 1 mL of nitric acid. Dilute with water to
hydroxide. Transfer the solution into a 250-mL 1000.0 mL, and mix.
volumetric flask, and dilute with deionized water to Standard solution: 0.25 µg/mL of lead, prepared on
volume. Mix well, and store in a plastic container. the day of use from the Standard stock solution
Monographs
Standard stock solution: (1000 mg/kg fluoride) Sample solution: Transfer 5.0 g of sample into a 250-
Dissolve 2.210 g of sodium fluoride in 50 mL of mL beaker, add 50 mL of 0.5 N hydrochloric acid,
deionized water. Transfer the solution into a 1-L cover with a watch glass, and heat slowly to boiling.
volumetric flask, and dilute to volume. Boil gently for 15 min, cool, and let the undissolved
Standard solutions: (1 mg/kg and 10 mg/kg fluoride) material settle. Decant the supernatant through
[NOTE—Prepare on the day of use.] Pipet 10 mL of the Whatman No. 4, or equivalent, filter paper into a 100-
Standard stock solution into a 100-mL volumetric flask, mL volumetric flask, retaining as much as possible of
dilute with deionized water to volume, and mix. Pipet the insoluble material in the beaker. Wash the slurry
10 mL and 1 mL of this solution into separate 100-mL and beaker with three 10-mL portions of hot water,
volumetric flasks, and dilute each with deionized water decanting each washing through the filter paper into
to volume. the flask. Finally, wash the filter paper with 15 mL of
Sample solution hot water, cool the filtrate to room temperature, dilute
Precipitated or other silica-based product: Transfer with water to volume, and mix.
5 g of sample into a 150-mL Teflon beaker. Add 40 Analysis: Using a suitable atomic absorption
mL of deionized water and 20 mL of 1 N spectrophotometer set at 217 nm, separately aspirate
hydrochloric acid. Heat to near boiling for 1 min and read the absorbances of the Standard solution and
while stirring continuously. Cool the beaker in an ice Sample solution which has been zeroed with water.
bath, transfer its contents into a 100-mL volumetric Acceptance criteria: The absorbance of the Sample
flask, and dilute with deionized water to volume. solution is not more than that of the Standard solution.
[NOTE—The sample does not dissolve completely.] (NMT 5 mg/kg)
Diatomaceous earth-based product: Transfer 5 g of
sample into a 150-mL Teflon beaker. Add 60 mL of SPECIFIC TESTS
deionized water and stir for 1 min. Transfer the • LOSS ON DRYING, Appendix IIC: 105° for 2 h
beaker contents into a 100-mL volumetric flask, and Acceptance criteria: The result should conform to the
dilute with deionized water to volume. [NOTE—The representations of the vendor.
sample does not dissolve completely.] Decant the • LOSS ON IGNITION
supernatant into two 50-mL centrifuge tubes and Sample: 1 g, previously dried at 105° for 2 h
centrifuge until the solution is clear, usually less than Analysis: Transfer the Sample into a suitable tared
30 min. crucible, and ignite at 900° to constant weight.
Calibration curve: Pipet 20 mL of each of the two Acceptance criteria: The result should conform to the
Standard solutions into separate 100-mL plastic beakers. representations of the vendor.
Add 10 mL of 0.2 N EDTA/0.2 N TRIS solution to each
OTHER REQUIREMENTS
beaker. Measure the potential (mV) of each solution
• LABELING: If it is the Diatomaceous earth-based product, it
with a suitable fluoride-selective, ion-indicating
is so labeled.
electrode and a calomel reference electrode connected
to a pH meter capable of measuring potentials with a
reproducibility of ±0.2 mV (Orion model 96-09
combination fluoride electrode, or equivalent).
Calcium Sorbate
.
Generate a standard curve by plotting the logarithms

of the fluoride ion concentrations (mg/kg) of the First Published: Prior to FCC 6
Standard solutions versus the potential (mV) or calibrate
an Orion Expandable Ion Analyzer EA-940 (or an 2,4-Hexadienoic Acid, Calcium Salt
equivalent instrument) for a direct concentration
reading.
Analysis: Pipet a 20-mL aliquot of Sample solution into a
100-mL plastic beaker, add 10 mL of 0.2 N EDTA/0.2 N
TRIS solution, and measure the solution potential as
C12H14CaO4 Formula wt 262.32
described for the Calibration curve (above). From the
INS: 203 CAS: [7492-55-9]
measured potential of the Sample solution, calculate the
UNII: 2P47R6817F [calcium sorbate]
concentration (mg/kg) of fluoride ion using the
Calibration curve.
220 / Calcium Sorbate / Monographs FCC 9
DESCRIPTION • LOSS ON DRYING, Appendix IIC: 105° for 3 h

Calcium Sorbate occurs as a fine, white crystalline powder. Acceptance criteria: NMT 1.0%
It decomposes at about 400°. It is sparingly soluble in
water and practically insoluble in organic solvents, in fats,
and in oils.
Calcium Stearate
.
Function: Antimicrobial agent; preservative

Packaging and Storage: Store in tight containers. First Published: Prior to FCC 6
IDENTIFICATION
• A. CALCIUM, Appendix IIIA CAS: [1592-23-0]
UNII: 776XM7047L [calcium stearate]
Monographs
Sample: 1 g
Analysis: Ignite the Sample at 800°. Cool, and slake with
10 mL of water. Add glacial acetic acid until the sample
DESCRIPTION
Calcium Stearate occurs as a fine, white to yellow-white,
is dissolved, and filter if necessary.
bulky powder. It is a compound of calcium with a mixture
of solid organic acids obtained from edible sources and
• B. PROCEDURE
consists chiefly of variable proportions of calcium stearate
Sample: 200 mg
and calcium palmitate. It is unctuous and free from
Analysis: Place the Sample in 5 mL of methanol. Add
grittiness. It is insoluble in water, in alcohol, and in ether.
0.1 mL of 1 N sodium hydroxide, and dissolve in 95 mL
Function: Anticaking agent; binder; emulsifier
of water. Add a few drops of bromine TS.
Acceptance criteria: The color disappears.
ASSAY IDENTIFICATION
• PROCEDURE
Sample: 1 g
Sample: 150 mg
Sample solution: The water layer on which floats an oily
Analysis: Dissolve the Sample in 50 mL of glacial acetic
layer of fatty acids that are liberated by heating the
acid in a 250-mL glass-stoppered Erlenmeyer flask,
Sample with a mixture of 25 mL of water and 5 mL of
warming if necessary to dissolve the Sample. Cool the
hydrochloric acid
flask to room temperature, add 2 drops of crystal violet
Acceptance criteria: The water layer passes tests.
TS, and titrate with 0.1 N perchloric acid in glacial
• SOLIDIFICATION POINT, Appendix IIB
acetic acid to a blue-green endpoint that persists for at
Sample: 25 g
least 30 s. [CAUTION—Handle perchloric acid in an
Analysis: Mix the Sample with 200 mL of hot water, add
appropriate fume hood.] Perform a blank determination
60 mL of 2 N sulfuric acid, and heat the mixture, while
(see General Provisions) and make any necessary
stirring frequently, until the fatty acids separate cleanly
correction. Two mL of 0.1 N perchloric acid is
as a transparent layer. Wash the fatty acids with boiling
equivalent to 26.23 mg of C12H14CaO4.
water until free from sulfate, collect them in a small
Acceptance criteria: NLT 98.0% and NMT 101.0%
beaker, and warm them on a steam bath until the
C12H14CaO4, calculated on the dried basis
water has separated and the fatty acids are clear. Allow
IMPURITIES the acids to cool, pour off the water layer, then melt
Inorganic Impurities the acids, filter into a dry beaker, and dry at 105° for
• LEAD, Lead Limit Test, Flame Atomic Absorption 20 min.
Spectrophotometric Method, Appendix IIIB Acceptance criteria: The solidification point of the fatty
Sample: 10 g acids so obtained is not below 54°.
ASSAY
SPECIFIC TESTS • PROCEDURE
• ACIDITY OR ALKALINITY Sample: 1.2 g
Sample: 1 g Analysis: Boil the Sample with 50 mL of 0.1 N
Analysis: Add some drops of methanol, 30 mL of water, hydrochloric acid for 10 min or until the fatty acid layer
and several drops of phenolphthalein TS to the Sample. is clear, adding water if necessary to maintain the
If the mixture is colorless, titrate with 0.1 N sodium original volume. Cool the mixture, filter, and wash the
hydroxide to a pink color that persists for 15 s. If the filter and flask thoroughly with water until the last
mixture is pink, titrate with 0.1 N hydrochloric acid washing is not acid to litmus. Neutralize the filtrate to
until pink color is discharged. litmus with 1 N sodium hydroxide. While stirring,
Acceptance criteria preferably with a magnetic stirrer, add about 30 mL of
Acidity (as sorbic acid): NMT 1.0 mL of 0.1 N sodium 0.05 M disodium EDTA from a 50-mL buret. Then, add
hydroxide is required to reach a persistent pink color 15 mL of 1 N sodium hydroxide and 300 mg of
(∼ 1%). hydroxy naphthol blue indicator. Continue the titration
Alkalinity (as Ca(OH)2): NMT 1.35 mL of hydrochloric with the disodium EDTA to a blue endpoint. Each mL of
acid is required to discharge the pink color (∼0.5%). 0.05 M disodium EDTA is equivalent to 2.804 mg of
CaO.
FCC 9 Monographs / Calcium Stearoyl Lactylate / 221
Acceptance criteria: NLT 9.0% and NMT 10.5% of Analysis: Mix the Sample with 200 mL of hot water, add
CaO, calculated on the dried basis 60 mL of 2 N sulfuric acid, and heat the mixture, while
stirring frequently, until the fatty acids separate cleanly
IMPURITIES as a transparent layer. Wash the fatty acids with boiling
Inorganic Impurities water until free from sulfate, collect them in a small
• LEAD, Lead Limit Test, Flame Atomic Absorption beaker, and warm them on a steam bath until the
Spectrophotometric Method, Appendix IIIB water has separated and the fatty acids are clear. Allow
Sample: 10 g the acids to cool, pour off the water layer, then melt
Acceptance criteria: NMT 2 mg/kg the acids, filter into a dry beaker, and dry at 105° for
20 min.
SPECIFIC TESTS
Monographs
Acceptance criteria: The solidification point of the fatty
• FREE FATTY ACIDS (AS STEARIC ACID)
acids so obtained is not below 54°.
Sample: 2g
Analysis: Transfer the Sample into a dry 125-mL IMPURITIES
Erlenmeyer flask containing 50 mL of acetone, fit an air- Inorganic Impurities
cooled reflux condenser onto the neck of the flask, boil • LEAD, Lead Limit Test, Flame Atomic Absorption
the mixture on a steam bath for 10 min, and cool. Spectrophotometric Method, Appendix IIIB
Filter through two layers of Whatman No. 42, or Sample: 10 g
equivalent, filter paper and wash the flask, residue, and Acceptance criteria: NMT 2 mg/kg
filter with 50 mL of acetone. Add phenolphthalein TS
and 5 mL of water to the filtrate, and titrate with 0.1 N SPECIFIC TESTS
sodium hydroxide. Perform a blank determination (see • ACID VALUE
General Provisions) using 100 mL of acetone and 5 mL Sample: 1 g
of water, and make any necessary correction. Each mL Analysis: Transfer the Sample into a 125-mL conical
of 0.1 N sodium hydroxide is equivalent to 28.45 mg flask, add 25 mL of alcohol, previously neutralized in
of stearic acid (C18H36O2). phenolphthalein TS, and heat on a hot plate until the
Acceptance criteria: NMT 3.0% sample is dissolved. Cool the flask, add 5 drops of
• LOSS ON DRYING, Appendix IIC: 105° using 2 h increments phenolphthalein TS, and titrate rapidly with 0.1 N
of heating, to constant weight sodium hydroxide to the appearance of the first pink
Acceptance criteria: NMT 4.0% color that persists for at least 30 s. [NOTE—Retain the
neutralized solution for the determination of Ester Value
below.] Calculate the Acid value by the formula:
Result = 56.1V × N/W

Calcium Stearoyl Lactylate
.

V = volume (mL) of 0.1 N sodium hydroxide
Calcium Stearoyl-2-Lactylate used in the titration
Calcium Stearoyl Lactate N = normality of the sodium hydroxide used in
INS: 482(i) CAS: [5793-94-2] the titration
UNII: 30MXH4012A [calcium stearoyl lactylate] W = weight (g) of the sample taken
Acceptance criteria: Between 50 and 86
DESCRIPTION • CALCIUM CONTENT
Calcium Stearoyl Lactylate occurs as a cream-colored Lanthanum stock solution: Transfer 5.86 g of
powder. It is a mixture of calcium salts of stearoyl lactic lanthanum oxide (La2O3) into a 100-mL volumetric
acid, with minor proportions of other salts of related acids. flask, wet with a few mL of water, slowly add 25 mL of
It is slightly soluble in hot water. hydrochloric acid, and swirl until the lanthanum oxide
Function: Dough conditioner; stabilizer; whipping agent is completely dissolved. Dilute to volume with water
Packaging and Storage: Store in tight containers in a and mix.
cool, dry place. Calcium stock solution: Transfer 124.8 mg of calcium
carbonate, previously dried at 200° for 2 h, into a 100-
IDENTIFICATION mL volumetric flask, carefully dissolve in 2 mL of 2.7 N
• CALCIUM, Appendix IIIA hydrochloric acid, dilute to volume with water, and
Sample: 1 g mix. [NOTE—This 500-mg/kg calcium solution is
Sample solution: The water layer on which floats an oily commercially available.]
layer of fatty acids that are liberated by heating the Standard solutions: Transfer 10.0 mL of the Lanthanum
Sample with a mixture of 25 mL of water and 5 mL of stock solution into each of three 50-mL volumetric flasks.
hydrochloric acid. Using a microliter syringe, transfer 0.20 mL of Calcium
Acceptance criteria: The water layer passes tests. stock solution into the first flask, 0.40 mL into the
• SOLIDIFICATION POINT, Appendix IIB second flask, and 0.50 into the third flask. Dilute the
Sample: 25 g contents of each flask with water to volume and mix.
222 / Calcium Stearoyl Lactylate / Monographs FCC 9
The flasks contain, respectively, 2.0, 4.0, and 5.0 µg/mL acid, heat until the fatty acids are melted, then cool to
of Ca. [NOTE—Prepare these solutions fresh daily.] about 60°, and add 25 mL of petroleum ether. Swirl
Sample solution: Transfer 250 mg of sample, into a 30- the mixture gently, and transfer quantitatively to a
mL beaker. While heating, dissolve the sample in 10 mL separatory funnel. Collect the water layer in a 100-mL
of alcohol, and quantitatively transfer the solution into a volumetric flask, and wash the petroleum ether layer
25-mL volumetric flask. Wash the beaker with two 5-mL with two 20-mL portions of water, adding the washings
portions of alcohol, adding the washings to the flask, to the volumetric flask. Dilute to volume with water,
dilute to volume with alcohol, and mix. To a second and mix.
25-mL volumetric flask, transfer 5.0 mL of the Sample solution: Transfer 1.0 mL of the Sample stock
Lanthanum stock solution and, using a microliter syringe, solution into a 100-mL volumetric flask, dilute to volume
Monographs
0.25 mL of the alcoholic solution of the sample, dilute with water, and mix.
to volume, and mix. Standard stock solution: 1.067 mg/mL of lithium
Analysis: Determine the absorbance of each Standard lactate (LiC3H5O3), made to 1000.0 mL
solution and of the Sample solution at 422.7 nm using a Standard solutions: 1, 2, 4, 6, and 8 µg/mL of lactic
suitable atomic absorption spectrophotometer. Plot the acid prepared by first transferring 10.0 mL of the
absorbance of the Standard solutions versus Standard stock solution into a 100-mL volumetric flask,
concentration of calcium (µg/mL). From the curve so diluting to volume and mixing, and then transferring
obtained, determine the concentration, C (µg/mL), of 1.0, 2.0, 4.0, 6.0, and 8.0 mL of this solution into
calcium in the Sample solution. Calculate the quantity separate 100-mL volumetric flasks, and diluting each
(mg) of calcium in the sample taken by the formula: flask to volume and mixing.
Analysis: Transfer 1.0 mL of each Standard solution and
Result = 2.5C the Sample solution into separate test tubes. Similarly
transfer 1.0 mL of water to a test tube to serve as the
blank. Treat each tube as follows: Add 1 drop of cupric
C = concentration (µg/mL) of Ca in the Sample
sulfate TS, swirl gently, and then rapidly add 9.0 mL of
solution
sulfuric acid from a buret. Loosely stopper the tube,
Acceptance criteria: Between 4.2% and 5.2%
and heat in a water bath at 90° for exactly 5 min. Cool
• ESTER VALUE
immediately to below 20° in an ice bath for 5 min, add
Alcoholic potassium hydroxide solution: Dissolve 11.2
3 drops of p-phenylphenol TS, shake the tube
g of potassium hydroxide in 250 mL of alcohol, diluting
immediately, and heat in a water bath at 30° for 30
with 25 mL of water.
min, shaking the tube twice during this time to disperse
Sample solution: Neutralized solution retained from
the reagent. Heat the tube in a water bath at 90° for
determination of Acid Value above
exactly 90 s, and then cool immediately to room
Analysis: Add 10.0 mL of the Alcoholic potassium
temperature in an ice water bath. Using a suitable
hydroxide solution to the Sample solution. Add 5 drops of
spectrophotometer set at 570 nm and the blank to zero
phenolphthalein TS, connect a suitable condenser, and
the instrument, determine the absorbance of each
reflux for 2 h. Cool the flask, add 5 additional drops of
solution in a 1-cm cell. Construct a standard curve by
phenolphthalein TS, and titrate the excess alkali with
plotting the absorbance values for the Standard
0.1 N sulfuric acid. Perform a blank determination (see
solutions versus the amount of lactic acid (µg) in each
General Provisions) using 10.0 mL of the Alcoholic
solution. Use the curve so obtained to obtain the
potassium hydroxide solution, and make any necessary
weight (µg) of lactic acid in the portion of the Sample
correction. Calculate the ester value by the formula:
solution used in this Analysis.
Result = 56.1(B − S) × (N/W) Acceptance criteria: Between 32.0% and 38.0%
B = volume (mL) of 0.1 N sulfuric acid used in

the titration of the blank Calcium Sulfate
.
S = volume (mL) of 0.1 N sulfuric acid used in

the titration of the Sample solution
N = normality of the sulfuric acid used in this
titration CaSO4 Formula wt, anhydrous 136.14
W = weight (g) of the sample taken CaSO4 · 2H20 Formula wt, dihydrate 172.18
Acceptance criteria: Between 125 and 164 INS: 516 CAS: anhydrous [7778-18-9]
• TOTAL LACTIC ACID dihydrate [10101-41-4]
Sample stock solution: Transfer 200 mg of sample into UNII: 4846Q921YM [calcium sulfate dihydrate]
a 125-mL Erlenmeyer flask, add 10 mL of 0.5 N UNII: E934B3V59H [calcium sulfate anhydrous]
alcoholic potassium hydroxide and 10 mL of water,
attach an air condenser, and reflux gently for 45 min.
DESCRIPTION
Calcium Sulfate occurs as a fine, white to slightly yellow-
Wash the sides of the flask and the condenser with
white powder. It is anhydrous or contains two molecules of
about 40 mL of water, and heat on a steam bath until
water of hydration.
no odor of alcohol remains. Add 6 mL of 1:2 sulfuric
FCC 9 Monographs / (+)-Camphor / 223
Function: Nutrient; yeast food; dough conditioner; firming

Camphene
.
agent; sequestrant
Packaging and Storage: Store in well-closed containers. First Published: Prior to FCC 6
IDENTIFICATION
• A. PROCEDURE
Sample solution: Dissolve 200 mg of sample by
warming it with a mixture of 4 mL of 2.7 N
hydrochloric acid and 16 mL of water.
Analysis: Add 5 mL of ammonium oxalate TS to 10 mL
Monographs
of the Sample solution. Retain the remainder of the
solution for Identification Procedure B below. C10H16 Formula wt 136.24
Acceptance criteria: A white precipitate forms. FEMA: 2229
• B. PROCEDURE UNII: G3VG94Z26E [camphene]
Analysis: Add barium chloride TS to the retained 10 mL
of Sample solution prepared for Procedure A above. DESCRIPTION
Acceptance criteria: A white precipitate forms that is Camphene occurs as a colorless crystalline mass. It may
insoluble in hydrochloric and nitric acids. contain a suitable antioxidant.
Odor: Camphoraceous-oily
ASSAY Solubility: Soluble in alcohol; miscible in most fixed oils;
• PROCEDURE insoluble or practically insoluble in water
Sample solution: Disperse 250 mg of sample in 100 mL Boiling Point: ∼159°
of water and 4 mL of 2.7 N hydrochloric acid. Boil to Function: Flavoring agent
dissolve the sample and cool the solution.
Analysis: While stirring the Sample solution, preferably ASSAY
with a magnetic stirrer, add about 30 mL of 0.05 M • PROCEDURE: Proceed as directed under M-1a, Appendix
disodium EDTA from a 50-mL buret. Then, add 25 mL XI.
of 1 N sodium hydroxide and 300 mg of hydroxy Acceptance criteria: NLT 80.0% of C10H16
naphthol blue indicator. Continue the titration with
disodium EDTA to a blue endpoint. Each mL of 0.05 M OTHER REQUIREMENTS
disodium EDTA is equivalent to 6.807 mg of CaSO4. • SOLIDIFICATION POINT, Appendix IIB
Acceptance criteria: NLT 98.0% of CaSO4, calculated Acceptance criteria: NLT 40°
on the dried basis
IMPURITIES
Inorganic Impurities (+)-Camphor
.

Sample: 1.67 g
IIIB d-Camphor
• SELENIUM, Selenium Limit Test, Method II, Appendix IIIB
Sample: 200 mg
SPECIFIC TESTS
• LOSS ON DRYING, Appendix IIC: 250° to constant weight C10H16O Formula wt 152.24
Acceptance criteria FEMA: 2230
Anhydrous: NMT 1.5% UNII: N20HL7Q941 [camphor (natural)]
Dihydrate: Between 19.0% and 23.0%
DESCRIPTION
(+)-Camphor occurs as a white to gray translucent
crystalline or fused mass.
Odor: Minty, ethereal
Solubility: Soluble in alcohol; insoluble or practically
insoluble in most fixed oils, propylene glycol, water
Boiling Point: ~204°
Solubility in Alcohol, Appendix VI: One mL dissolves in 1
mL of 95% alcohol.
224 / (+)-Camphor / Monographs FCC 9
IDENTIFICATION soluble in most fixed oils and in mineral oil, but it is

• INFRARED ABSORPTION, Spectrophotometric Identification practically insoluble in glycerin and in propylene glycol.
Tests, Appendix IIIC Function: Flavoring agent
Reference standard: USP Camphor RS Packaging and Storage: Store in a cool place protected
Sample and standard preparation: M from light in full, tight containers that are made from steel
Acceptance criteria: The spectrum of the sample or aluminum and that are suitably lined.
the spectrum of the Reference standard. IDENTIFICATION
• INFRARED SPECTRA, Spectrophotometric Identification Tests,
OTHER REQUIREMENTS Appendix IIIC
Monographs
• MELTING RANGE OR TEMPERATURE DETERMINATION, Appendix Acceptance criteria: The spectrum of the sample
IIB exhibits relative maxima at the same wavelengths as
Acceptance criteria: Between 174° and 179° those of the spectrum below.
• ANGULAR ROTATION, Optical (Specific) Rotation, Appendix
IIB SPECIFIC TESTS
Acceptance criteria: Between +41° and +43° • ANGULAR ROTATION, Optical (Specific) Rotation, Appendix
IIB: Use 100 mm tube.
Acceptance criteria: Between −15° and −30°
• REFRACTIVE INDEX, Appendix IIB
[NOTE—Use an Abbé or other refractometer of equal or
Cananga Oil
.
greater accuracy.]
First Published: Prior to FCC 6 Acceptance criteria: Between 1.495 and 1.505 at 20°
• SOLUBILITY IN ALCOHOL, Appendix VI
CAS: [68606-83-7] Acceptance criteria: One mL of sample dissolves in 0.5
UNII: 8YOY78GNNX [cananga oil] mL of 95% alcohol, usually becoming cloudy on further
dilution.
DESCRIPTION • SPECIFIC GRAVITY: Determine by any reliable method (see
Cananga Oil occurs as a light to deep yellow liquid with a General Provisions).
harsh, floral odor suggestive of ylang ylang. It is the oil Acceptance criteria: Between 0.904 and 0.920
obtained by distillation from the flowers of the tree
Cananga odorata Hook f. et Thoms (Fam. Anonaceae). It is
Cananga Oil
FCC 9 Monographs / Canola Oil / 225

Candelilla Wax
.
exhibits relative maxima at the same wavelengths as

First Published: Prior to FCC 6 those of the spectrum below.
IMPURITIES
INS: 902 CAS: [8006-44-8] Inorganic Impurities
UNII: WL0328HX19 [candelilla wax] • LEAD, Sample Solution for Lead Limit Test, Appendix IV
DESCRIPTION Solution)
Candelilla Wax occurs as a hard, yellow-brown, opaque to
translucent wax. It is a purified wax obtained from the
Monographs
leaves of the candelilla plant, Euphorbia antisyphilitica (Fam. SPECIFIC TESTS
Euphorbiaceae). Its specific gravity is about 0.983. It is • ACID VALUE [FATS AND RELATED SUBSTANCES], Method I,
soluble in chloroform and in toluene, but insoluble in Appendix VII
water. Acceptance criteria: Between 12 and 22
Function: Masticatory substance in chewing gum base; • MELTING RANGE OR TEMPERATURE DETERMINATION, Procedure
surface-finishing agent for Class II, Appendix IIB
Packaging and Storage: Store in well-closed containers. Acceptance criteria: Between 68.5° and 72.5°
• SAPONIFICATION VALUE, Appendix VII
IDENTIFICATION Acceptance criteria: Between 43 and 65
Appendix IIIC
Sample preparation: Melted sample on a potassium
bromide plate
Candelilla Wax
plant Brassica juncea, Brassica napus, or Brassica rapa (Fam.

Canola Oil
.
Cruciferae). The plant varieties are those producing oil-

First Published: Prior to FCC 6 bearing seeds with a low erucic acid (C22:1) content. It is a
mixture of triglycerides composed of both saturated and
unsaturated fatty acids. It is refined, bleached, and
Low Erucic Acid Rapeseed Oil
deodorized to substantially remove free fatty acids,
LEAR Oil
CAS: [120962-03-0] phospholipids, color, odor and flavor components, and
UNII: 331KBJ17RK [canola oil] miscellaneous, other non-oil materials. It can be
hydrogenated to reduce the level of unsaturated fatty acids
DESCRIPTION for functional purposes in foods. It is a liquid at 0° and
Canola Oil occurs as a light yellow oil. It is typically above.
obtained by a combination of mechanical expression Function: Cooking or salad oil; component of margarine
followed by n-hexane extraction, from the seed of the or shortening; coating agent; texturizer
226 / Canola Oil / Monographs FCC 9
Packaging and Storage: Store in tightly closed samples; the samples contain 0, 3, 6, 9, and 12 mg/kg of
containers, ensuring no contact with metals, filled to the sulfur, respectively.
top or flushed with nitrogen gas. Raney nickel preparation: [CAUTION—Raney nickel is
pyrophoric when dry.] Raney nickel is produced by
IDENTIFICATION reacting nickel–aluminum alloy with sodium hydroxide.
• FATTY ACID COMPOSITION, Appendix VII Each Raney nickel pellet is prepared individually, and each
Acceptance criteria: A sample exhibits the following is enough catalyst for one determination. To produce one
composition profile of fatty acids: Raney nickel pellet, accurately weigh 1 g of
nickel–aluminum alloy powder (50% Ni, 50% Al), place it
Weight %
in a 50-mL centrifuge tube, and chill it in an ice bath.
Fatty Acid (Range)
Monographs
Slowly add 5 mL of water to the tube, and let it stand for

<14 <0.1
10 min. Then, slowly add 10 mL of 2.5 N sodium
14:0 <0.2 hydroxide, and allow the mixture to react for 30 min.
16:0 <6.0 Cap the tube, and place it in a 50° water bath for 2 h.
16:1 <1.0 Centrifuge the mixture at 1000 rpm for 10 min, and
18:0 <2.5
discard the supernatant liquid. Wash the pellet twice with
15 mL of water and twice with 15 mL of isopropanol,
18:1 >50
centrifuging between each wash. Store the catalyst under
18:2 <40.0 isopropanol for no longer than 2 weeks. [NOTE—Properly
18:3 <14 dispose of unused Raney nickel preparation by transferring
20:0 <1.0 it to a 250-mL Erlenmeyer flask, and placing it in a fume
20:1 <2.0 hood. Add 20 mL of 60% (w/v) hydrochloric acid, and
allow complete digestion of the catalyst.] [CAUTION—
22:0 <0.5
Hydrogen gas evolves during the digestion process.]
22:1 <2.0
Dithizone indicator solution: 1 mg/mL of dithizone
24:0 <0.2 (diphenylthiocarbazone) in acetone
24:1 <0.2 Mercuric acetate titrant: [CAUTION—Mercuric acetate is a
strong irritant when ingested or inhaled or upon dermal
exposure.] Transfer 3.82 g of mercuric acetate into a
IMPURITIES 1000-mL volumetric flask containing 950 mL of water.
Inorganic Impurities Add 12.2 mL of glacial acetic acid, dilute to volume with
• LEAD, Lead Limit Test, Atomic Absorption Spectrophotometric water, and mix. Transfer 10.0 mL of this solution into a
Graphite Furnace Method, Method II, Appendix IIIB 100-mL volumetric flask, dilute to volume with water, and
Acceptance criteria: NMT 0.1 mg/kg mix. The titrant solution contains 0.0012 M mercuric
Organic Impurities acetate.
• SULFUR COMPOUNDS Titration reagent blank: Add 50.0 mL of 1 N sodium
[NOTE—Organosulfur compounds present in the sample hydroxide and 50.0 mL of acetone to a 250-mL beaker,
react with Raney nickel to produce nickel sulfides. and mix. Add 0.5 mL of the Dithizone indicator solution,
Nickel sulfides are treated with a strong acid to produce and titrate with Mercuric acetate titrant until the color
hydrogen sulfide, which is trapped and titrated with changes from bright amber to red. Record the volume of
mercuric acetate using a dithizone indicator.] titrant used.
[CAUTION—This test requires the use of the following Sample: 15 to 20 g
hazardous substances: mercuric acetate, spongy nickel, Analysis: Place the Sample on the bottom of the boiling
and dibenzyl disulfide. Conduct the test in a fume flask. Discard the isopropanol from the Raney nickel
hood.] preparation, add 10 mL of 95% isopropanol, mix, and add
Apparatus: Fit a 125-mL, round-bottom boiling flask with the mixture to the sample. Attach the water condenser
a cylindrical filling funnel (20 mL with open top), an ST and the nitrogen line to the boiling flask, and adjust the
PTFE metering valve stopcock, and a gas inlet tube [see gas flow through the sample to 4 psi. Place a heating
the figure for Raney Nickel Reduction Apparatus, Sulfur mantle under the flask. Immerse the bubbler in a 250-mL
(by Oxidative Microcoulometry), Appendix IIIC]. Fit a water- beaker containing 50.0 mL of 1 N sodium hydroxide, and
jacketed distillation column with hooks on top of the stir slowly. Boil the sample for 90 min. Add 50 mL of
boiling flask. Fit a piece of glass tubing with ground ST acetone and 0.5 mL of Dithizone indicator solution to the
inner joints with hooks to the distillation column, and 250-mL beaker. Add 20 mL of 60% hydrochloric acid into
connect the distillation column and a gas dispersion tube the filling funnel fitted onto the boiling flask, and adjust
with ST outer joints with hooks. the nitrogen flow to 2 to 3 psi. Position the stir bar
Dibenzyl disulfide solution: 3 mg/mL dibenzyl disulfide in directly under the bubbler for maximum dispersion of the
methyl isobutyl ketone hydrogen sulfide bubbles. Slowly add the solution of 60%
Sulfur standard: Accurately weigh five 250.0-g samples of hydrochloric acid to the boiling flask. Begin the titration
food-grade peanut oil. Transfer 0.0, 1.0, 2.0, 3.0, and 4.0 with Mercuric acetate titrant until the bright amber color
mL of the Dibenzyl disulfide solution into the peanut oil changes to red. Add enough hydrochloric acid to turn the
solution in the boiling flask green, and then let it boil for
FCC 9 Monographs / Canthaxanthin / 227
15 min. Continue the titration throughout the boiling determination (see General Provisions), and make any
stage, making sure to rinse the inside of the bubbler with necessary correction. Calculate the peroxide value, as
the solution in the beaker by turning off the nitrogen flow mEq of peroxide per kg of sample, by the formula:
until the solution rises to the top of the vertical tube (the
solution usually returns to amber during the first rinse). Result = S × N × 1000/W
Rinse the tube a second time. Continue the titration, and
record the volume of titrant used to the nearest 0.01 mL.
S = net volume of sodium thiosulfate solution
Calculate the concentration of sulfur in the sample, in mg
required for the sample (mL)
/kg, by the following formula:
N = exact normality of the sodium thiosulfate
solution
Monographs
Result = (VU − VB) × K / W
W = weight of the sample taken (g)
Acceptance criteria: NMT 10 mEq/kg
VU = volume (mL) of titrant to the endpoint for • REFRACTIVE INDEX, Appendix IIB
the Sample [NOTE—Use an Abbé or other refractometer of equal or
VB = volume (mL) of titrant to the endpoint for greater accuracy.]
the blank (usually about 0.10 mL) Acceptance criteria: Between 1.465 and 1.467 at 40°
K = constant determined (below) from the • SAPONIFIABLE VALUE, Saponification Value, Appendix VII
calibration of the Sulfur standard (µg of Acceptance criteria: Between 178 and 193
sulfur per mL of titrant) • STABILITY, Appendix VII
W = weight of the sample (g) taken Acceptance criteria: NLT 7 h
Analyze the Sulfur standards, in duplicate, to determine the • UNSAPONIFIABLE MATTER, Appendix VII
constant, K, using the following formula: Acceptance criteria: NMT 1.5%
• WATER, Water Determination, Appendix IIB
K = W × C/(VS − VRB) Analysis: In place of 35 to 40 mL of methanol, use 50
mL of 1:1 chloroform:methanol mixture to dissolve the
sample.
W = weight of the Sulfur standard (g)
C = concentration of the Sulfur standard (mg/kg)
VS = volume of titrant for the Sulfur standard OTHER REQUIREMENTS
(mL) • LABELING: Hydrogenated Canola Oil less than fully
VRB = volume of titrant for the Titration reagent hydrogenated must be labeled as Partially Hydrogenated
blank Canola Oil.
SPECIFIC TESTS
• ACID VALUE [FATS AND RELATED SUBSTANCES], Method II,
Canthaxanthin
.
Appendix VII
Acceptance criteria: NMT 6 First Published: Prior to FCC 6
• COLD TEST, Appendix VII
Acceptance criteria: Passes test 4,4′-Diketo-β-carotene
• COLOR (FATS AND RELATED SUBSTANCES), Appendix VII: Use Cantha
a 133.4-mm cell. β-Carotene-4,4′-dione
Acceptance criteria: NMT 1.5 red/15 yellow
• ERUCIC ACID, Fatty Acid Composition, Appendix VII
• FREE FATTY ACIDS (AS OLEIC ACID), Appendix VII
Analysis: Use 28.2 for the equivalence factor (e).
• IODINE VALUE, Appendix VII
C40H52O2 Formula wt 564.85
• LINOLENIC ACID, Fatty Acid Composition, Appendix VII
INS: 161g CAS: [514-78-3]
UNII: 4C3C6403MU [canthaxanthin]
• PEROXIDE VALUE
Sample: 10 g DESCRIPTION
Analysis: To the Sample, add 30 mL of a 3:2 mixture of Canthaxanthin occurs as a dark, crystalline powder. It is
glacial acetic acid: chloroform, and mix. Add 1 mL of a soluble in chloroform, very slightly soluble in acetone, but
saturated solution of potassium iodide, and mix for 1 insoluble in water. It melts at about 207° to 212° with
min. Add 100 mL of water, begin titrating with 0.05 N decomposition.
sodium thiosulfate, adding starch TS as the endpoint is Function: Color
approached, and continue the titration until the blue Packaging and Storage: Store in tight, light-resistant
starch color has just disappeared. Perform a blank containers under inert gas.
228 / Canthaxanthin / Monographs FCC 9
IDENTIFICATION DESCRIPTION
• PROCEDURE Caramel usually occurs as a dark brown to black liquid or
Acceptance criteria: The Sample solution (prepared solid. It is a complex mixture of compounds, some of
below under Assay) exhibits a maximum absorbance which are in the form of colloidal aggregates. Caramel is
near 470 nm. manufactured by heating carbohydrates, either alone or in
the presence of food-grade acids, alkalies, and/or salts.
ASSAY Caramel is produced from commercially available food-
• PROCEDURE: [NOTE—Carry out all work in low-actinic grade nutritive sweeteners consisting of fructose, dextrose
glassware and in subdued light.] (glucose), invert sugar, sucrose, malt syrup, molasses, and
Sample stock solution: Transfer 50 mg of sample into a starch hydrolysates and fractions thereof. The acids that
Monographs
100-mL volumetric flask, add 10 mL of acid-free may be used are food-grade sulfuric, sulfurous, phosphoric,
chloroform to dissolve the sample, dilute to volume acetic, and citric acids; the alkalies are ammonium, sodium,
with cyclohexane, and mix. Pipet 5 mL of this solution potassium, and calcium hydroxides; and the salts are
into a second 100-mL volumetric flask, dilute to volume ammonium, sodium, and potassium carbonate,
with cyclohexane, and mix. bicarbonate, phosphate (including mono- and dibasic),
Sample solution: Pipet 5 mL of Sample stock solution sulfate, and bisulfite. Food-grade antifoaming agents, such
into a 50-mL volumetric flask, dilute to volume with as polyglycerol esters of fatty acids, may be used as
cyclohexane, and mix. processing aids during its manufacture. Caramel is soluble
Analysis: Using a suitable spectrophotometer and a 1- in water.
cm cell, measure the absorbance of the Sample solution Four distinct classes of Caramel can be distinguished by the
at the wavelength maximum of about 470 nm; use reactants used in their manufacture and by specific
cyclohexane for the blank. Calculate the weight (mg) of identification tests:
C40H52O2 in the sample taken by the formula: Class I (Plain Caramel, Caustic Caramel): Prepared by
heating carbohydrates with or without acids or alkalis; no
Result = 20,000 A/a
ammonium or sulfite compounds are used.
A = absorbance of Sample solution Class II (Caustic Sulfite Caramel): Prepared by heating
a = absorptivity (220) of pure canthaxanthin carbohydrates with or without acids or alkalis in the
Acceptance criteria: NLT 96.0% and NMT 101.0% of presence of sulfite compounds; no ammonium compounds
C40H52O2 are used.
Class III (Ammonia Caramel): Prepared by heating
IMPURITIES carbohydrates with or without acids or alkalis in the
Inorganic Impurities presence of ammonium compounds; no sulfite compounds
• ARSENIC, Arsenic Limit Test, Appendix IIIB are used.
Sample solution: Prepare as directed for organic Class IV (Sulfite Ammonia Caramel): Prepared by heating
compounds. carbohydrates with or without acids or alkalis in the
Acceptance criteria: NMT 3 mg/kg presence of both sulfite and ammonium compounds.
• LEAD, Lead Limit Test, Appendix IIIB All of these Caramels shall meet the criteria established for
Sample solution: Prepare as directed for organic Caramel in this monograph.
compounds. Function: Color
Control: 10 µg Pb (10 mL of Diluted Standard Lead Packaging and Storage: Store in well-closed containers
Solution) and avoid exposure to excessive heating and, for solid
Acceptance criteria: NMT 10 mg/kg products, excessive humidity.
• MERCURY, Mercury Limit Test, Appendix IIIB
Acceptance criteria: NMT 1 mg/kg IMPURITIES
SPECIFIC TESTS • ARSENIC, Arsenic Limit Test, Appendix IIIB
• RESIDUE ON IGNITION (SULFATED ASH), Appendix IIC Sample solution: Prepare as directed for organic
Sample: 1 g compounds.
Analysis: Proceed as directed, using a silica crucible and Control: 1 µg As (1.0 mL of Standard Arsenic Solution)
moistening the residue with 2 mL of nitric acid and 1 Acceptance criteria: NMT 1 mg/kg
mL of sulfuric acid. • LEAD
[NOTE—Use reagent-grade chemicals with as low a lead
content as is practicable as well as high-purity water
and gases. Before use, rinse all glassware and
plasticware twice with 10% nitric acid and twice with
Caramel
.
10% hydrochloric acid. Then rinse them thoroughly

First Published: Prior to FCC 6 with high-purity water, preferably obtained from a
mixed-bed, strong-acid, strong-base ion-exchange
Caramel Color cartridge capable of producing water with an electrical
INS: 150 CAS: [8028-89-5] resistivity of 12 to 15 megohms.]
UNII: T9D99G2B1R [caramel]
FCC 9 Monographs / Caramel / 229
Lead nitrate solution: 100 µg Pb/mL, prepared by ashing. [NOTE—Do not ash nitric acid in a furnace
transferring 159.8 mg of ACS reagent-grade lead because the lead contaminant will be lost.] Evaporate
nitrate, Pb(NO3)2, into a 1000-mL volumetric flask, nitric acid to dryness in an ashing vessel on a steam
adding 100 mL of water containing 1 mL of nitric acid bath or hot plate. Dissolve the residue in 5 mL of 1 N
to dissolve the lead nitrate, diluting to volume with nitric acid, warming on a steam bath or hot plate for 2
water, and mixing. [NOTE—Prepare and store this to 3 min to aid solution. Filter, if necessary, and decant
solution in glass containers that are free from lead through S&S 589 Black Ribbon paper, or equivalent,
salts.] into a 50-mL volumetric flask. Repeat with two 5-mL
Standard stock solution: 10 µg Pb/mL prepared by portions of 1 N nitric acid, filter, and add the washings
transferring 50.0 mL of Lead nitrate solution into a 500- to the original filtrate. Dilute to volume with 1 N nitric
Monographs
mL volumetric flask containing 50 mL of water, adding acid, and mix to prepare each reagent blank.
5 mL of nitric acid, diluting to volume with water, and
[NOTE—Complete the sample preparation (above) and
mixing. [NOTE—Prepare on the day of use.]
the analysis (below) on the same day.]
Standard solutions: 0.20, 0.50, 1.00 and 2.00 µg Pb/
Aqueous butyl acetate: Use spectral-grade butyl
mL, prepared by pipeting 2, 5, 10, and 20 mL of
acetate and saturate it with water.
Standard stock solution into separate 100-mL volumetric
APDC solution: 20 mg/mL of APDC (ammonium
flasks, adding 1 mL of nitric acid to each, diluting to
1-pyrrolidinedithiocarbamate, Aldrich Chemical, or
volume with water, and mixing.
equivalent) made to 100 mL. Remove insoluble free
Sample solution: Transfer 25 g of sample into an
acid and other impurities normally present by two to
ashing vessel. [NOTE—Suitable ashing vessels have
three extractions with 10-mL portions of Aqueous butyl
approximately a 100-mL capacity and are flat-
acetate.
bottomed platinum crucibles or dishes, Vycor or quartz
Citric acid solution, lead-free: Dissolve 10 g of citric
tall-form beakers, or Vycor evaporating dishes (Corning
acid in 30 mL of water. While stirring, slowly add
Glass Works No. 13180, or equivalent). Discard Vycor
ammonium hydroxide until the pH is between 8.0 and
vessels when the inner surfaces become etched.] Dry
8.5, using short-range pH paper as an external
the sample overnight at 120° in a forced draft oven.
indicator. Transfer the solution into a separatory funnel,
[NOTE—The sample must be absolutely dry to prevent
and extract with 10-mL portions of Dithizone Extraction
flowing or spattering in the furnace.] Place the sample
Solution (see Lead Limit Test, Appendix IIIB), until the
in a furnace equipped with a pyrometer to control the
dithizone solution retains its green color or remains
temperature over a range of 260° to 600°, with a
unchanged. Drain the final dithizone layer, plus about
variation of less than 10° and set the temperature at
1 mL of the aqueous layer, into a beaker and, while
250°. Slowly, in 50° increments, raise the temperature
stirring, slowly add 1:1 nitric acid until the pH is
to 350°, and hold at this temperature until smoking
between 3.5 and 4, again using short-range pH paper
ceases. Increase the temperature to 500° in
as an external indicator. Transfer this solution into a
approximately 75° increments; the sample must not
100-mL volumetric flask (through a filter, if necessary),
ignite. Ash for 16 h or overnight at 500°. Remove the
dilute to volume with water, and mix thoroughly.
sample from the furnace and allow it to cool. The ash
Test solutions: Pipet 20 mL each of the Standard
should be white and essentially carbon free. If the ash
solutions, the Sample solution, and the appropriate
still contains excess carbon particles (i.e., the ash is
Reagent blanks into separate 60-mL separatory funnels.
gray rather than white), proceed as follows: Wet the
Treat each solution as follows: Add 4 mL of Citric acid
ash with a minimal amount of water followed by the
solution, lead-free and 2 to 3 drops of bromocresol
dropwise addition of 0.5 to 3 mL of nitric acid. Dry on
green TS. The solution should be yellow. Adjust the pH
a hot plate. Transfer the ash to a furnace set at 250°,
to about 5.4, using ammonium hydroxide initially and
slowly increase the temperature to 500°, and continue
then ammonium hydroxide diluted with 4 volumes of
heating for 1 to 2 h. Repeat the nitric acid treatment
water in the vicinity of the color change (the first
and ashing, if necessary, to obtain a carbon-free
permanent appearance of light blue). Add 4 mL of
residue. [NOTE—Local overheating or deflagration may
APDC solution, stopper, and shake the funnel for 30 to
result if the sample still contains much intermingled
60 s. Pipet 5.0 mL of Aqueous butyl acetate, stopper the
carbon and especially if much potassium is present in
funnel, and shake the funnel vigorously for 30 to 60 s.
the ash.]
Let stand until the layers separate clearly, drain, and
Dissolve the residue in 5 mL of 1 N nitric acid, warming
discard the lower aqueous phase. If an emulsion forms
on a steam bath or hot plate for 2 to 3 min to aid
or the solvent layer is cloudy, drain the solvent layer
solution. Filter, if necessary, and decant through S&S
into a 15-mL centrifuge tube, cover the tube with
589 Black Ribbon paper, or equivalent, into a 50-mL
aluminum foil or Parafilm (or equivalent), and
volumetric flask. Repeat with two 5-mL portions of 1 N
centrifuge it for about 1 min at 2000 rpm.
nitric acid, filter, and add the washings to the original
Analysis: Use an atomic absorption spectrophotometer
filtrate. Dilute to volume with 1 N nitric acid and mix
equipped with a 4-in., single-slot burner head. Set the
to prepare the Sample solution.
instrument to previously determined optimum
Reagent blanks: Prepare duplicate blanks for each
conditions for organic solvent aspiration (3 to 5 mL/
Standard solution and for the Sample solution, including
min) and at a wavelength of 283.3 nm. Use an
any additional water and nitric acid if used for sample
230 / Caramel / Monographs FCC 9
air–acetylene flame adjusted for maximum lead • SULFUR DIOXIDE, Sulfur Dioxide Determination, Appendix X
absorption with a fuel-lean flame. Aspirate each of the Sample: 0.5 g
Test solutions, flushing with water and then with Analysis: Determine as directed, and calculate on an
Aqueous butyl acetate between measurements and equivalent color basis expressed in terms of a caramel
record their absorbance. Correct the absorbances of having a Color Intensity of 0.1 a.u. (absorbance unit).
the Test solutions prepared from the Standard solutions, Acceptance criteria: NMT 0.2%, calculated on the
and the Sample solution, with the readings of the Test equivalent color basis
solutions prepared from the Reagent blanks. Prepare a Organic Impurities
standard curve by plotting the absorbance of each of • 4-METHYLIMIDAZOLE
the Test solutions prepared from the Standard solutions 4-Methylimidazole stock solution: Purify reagent-grade
Monographs
against their concentrations (µg Pb/mL). (These 4-methylimidazole (Aldrich, or equivalent) by

concentrations, in butyl acetate, are four times those in redistillation (b.p. 92° to 93°, 0.05 mm Hg). Transfer
the aqueous standard.) From the standard curve, 50 mg of the distillate, into a 50-mL volumetric flask,
determine the concentration, C, (µg/mL), of the dilute to volume with tetrahydrofuran (acetone is also
Sample solution. Calculate the quantity, in mg/kg, of acceptable), and mix thoroughly. Store in a refrigerator
lead in the sample by the formula: until ready for use.
Standard solutions: 100, 150, 200, 250, 300, 350,
Result = 12.5 × C/W 400, and 500 µg/mL prepared by pipetting,
respectively 1.0-, 1.5-, 2.0-, 2.5-, 3.0-, 3.5-, 4.0-, and
5.0-mL portions of 4-Methylimidazole stock solution into
separate 10-mL volumetric flasks, diluting each to
volume with the same solvent used to prepare the
• MERCURY, Mercury Limit Test, Method I, Appendix IIIB
stock solution, and mixing. Store the solutions in a
Standard preparation: Prepare as directed transferring
refrigerator; use them within 1 month.
1.0 mL of the stock solution (1 µg Hg) into a 50-mL
Sample solution: Transfer 10.0 g of sample into a 250-
beaker rather than the specified 2.0 mL.
mL polypropylene beaker, and mix thoroughly with 5.0
Sample preparation: Transfer 5 g of sample into a 250-
g of 3 N sodium hydroxide; the pH of the mixture
mL Erlenmeyer flask and add 5 mL of sulfuric acid and
should exceed 12. Add 20.0 g of chromatographic
a few glass beads. Digest the mixture at a temperature
siliceous earth (Johns-Manville Celite 545, or
not exceeding 120° until charring begins, preferably
equivalent) to the beaker and thoroughly mix with a
using a hot plate in a fume hood. [NOTE—Additional
wide-blade, stainless steel spatula until a
sulfuric acid may be necessary to completely wet some
homogeneous, semidry mixture is obtained.
samples, but the total volume added should not
Homogeneity is obtained when the color is uniform
exceed about 10 mL.] After the acid has initially
and no dark clumps are seen. Place a plug of fine glass
decomposed the sample, cautiously add, dropwise,
wool in the base of a 300-× 22-mm (id)
hydrogen peroxide (30%), allowing the reaction to
chromatography tube having a Teflon stopcock.
subside and reheating the sample between drops. Add
Quantitatively transfer the mixture into the column.
the first few drops very slowly with sufficient mixing to
The column bed, approximately 150 mm tall, should
prevent a rapid reaction and discontinue heating if
be of uniform consistency, yet open enough to allow
foaming becomes excessive. Swirl the solution in the
elution to occur readily. Place a plug of glass wool on
flask to prevent unreacted substance from caking on
top of the column, and then allow the column to fall a
the walls or bottom of the flask during digestion.
short distance vertically to help settle the contents.
[NOTE—Maintain oxidizing conditions at all times
Rinse the sample beaker with methylene chloride and
during the digestion by adding small quantities of the
pour the contents into the column with the stopcock
peroxide whenever the mixture turns brown or
open. Allow the methylene chloride to pass down the
darkens.] Continue the digestion until the organic
column until it reaches the stopcock. Close the
matter is destroyed, gradually raising the temperature
stopcock and allow the methylene chloride to remain
of the hot plate to 250° to 300° until fumes of sulfur
in contact with the column bed for 5 min. Open the
trioxide are copiously evolved and the solution
stopcock and pass methylene chloride through the
becomes colorless or retains only a light straw color.
column at a rate of 5 mL/min. Collect 200 mL of
Cool the flask, cautiously add 10 mL of water, heat
eluate in a 300-mL round-bottom flask. While
again to strong fuming, and cool. Cautiously add 10
maintaining the flask at 35° in a water bath, remove
mL of water, mix, wash the sides of the flask with a
the bulk of the solvent from the eluate by rotary
few milliliters of water, and dilute to 35 mL. Add 1 mL
vacuum evaporation (350 to 390 mm Hg). Reduce the
of a 1:25 solution of potassium permanganate, and
volume to about 1 mL. During the concentration step,
mix.
watch the flask carefully to ensure that no loss of
Analysis: Continue as directed in the Procedure, using a
sample occurs by bumping. Use a disposable Pasteur
suitable atomic absorption spectrophotometer.
pipet to quantitatively transfer the extract residue to a
Acceptance criteria: Any absorbance produced by the
5-mL volumetric flask, rinsing the flask several times
Sample preparation is not more than half that produced
with small (approximately 0.7 mL) portions of the
by the Standard preparation, indicating NMT 0.1 mg/
same solvent used to prepare the original solutions
kg.
(tetrahydrofuran or acetone); transfer the rinsings to of water, collecting the washings in the receiving flask.
the volumetric flask until the 5-mL dilution mark is Add 4 or 5 drops of methyl red TS, titrate with 0.1 N
reached. Mix thoroughly. sodium hydroxide, and record the volume (mL) as S.
Chromatographic system, Appendix IIA Conduct a blank determination (see General Provisions)
Mode: Gas chromatography and record the mL of 0.1 N sodium hydroxide required
Detector: Hydrogen flame-ionization as B. Calculate the percent Ammoniacal Nitrogen (on an
Column: 1-m × 4-mm (id) silanized glass column, or equivalent color basis) by the formula:
equivalent, packed with 90- to 100-mesh Anakrom
ABS, or equivalent, containing 7.5% Carbowax 20M Result = [(B – S) × WN × 100/W] × F/A610
and 2% potassium hydroxide, or equivalent
Monographs
WN = the mEq weight of nitrogen for 0.1 N
Injection port temperature: 200°
sodium hydroxide, 0.0014
Detector temperature: 250°
W = weight of sample taken (g)
Carrier gas: Nitrogen
F = the basis of color equivalency, 0.1
Flow rate: 50 mL/min
A610 = absorbance of the 0.1% solution prepared
Injection volume: 5.0 µL
for the determination of Color intensity
Analysis: [NOTE—Preferably, use an autosampler to
(below)
inject the Standard solutions. If using manual injections,
[NOTE—The above formula gives the result on an
avoid fractionation in the syringe needle, and ensure
equivalent color basis that permits expression in terms
that 5.0 µL is injected by using the solvent-flush
of a caramel having a color intensity standardized to 1
technique with the solvent used to prepare the
a.u.]
Standard solutions.]
Acceptance criteria: NMT 0.6%, calculated on the
Inject the Standard solutions into the chromatograph
equivalent color basis
and obtain the chromatograms. From each
• COLOR INTENSITY
chromatogram, obtain the corrected peak area. If not
Sample solution: 1 mg/mL (0.1%) [NOTE—Centrifuge if
using an integrator, calculate the corrected peak area
the solution is cloudy.]
by multiplying the peak height (mm) by the peak
Analysis: Using a suitable spectrophotometer and a 1-
width at one-half height (mm), and by the proper
cm cell, determine the absorbance (A610) of the clear
attenuation and range factors, depending on the
Sample solution at 610 nm; use water as the blank.
particular apparatus and operating parameters used.
Calculate the Color intensity by the formula:
Plot each corrected peak area versus its respective
concentration of 4-methylimidazole to obtain the Result = (A610 × 100)/S
standard curve. In the same manner, chromatograph a
5.0-µL portion of the Sample solution, calculate the
peak area corresponding to any 4-methylimidazole S = the percent Total solids (see test below)
contained in the sample, and, by reference to the Acceptance criteria: Between 0.01 and 0.6 absorbance
standard curve, obtain the content of the 4- units (a.u.)
methylimidazole in the sample. Calculate the percent • TOTAL NITROGEN, Nitrogen Determination, Method II,
4-methylimidazole on an equivalent color basis Appendix IIIC
expressed in terms of a Caramel having a Color Analysis: Determine as directed and calculate on an
Intensity of 0.1 a.u. equivalent color basis expressed in terms of a caramel
Acceptance criteria: NMT 0.025%, calculated on the having a Color Intensity of 0.1 a.u.
equivalent color basis Acceptance criteria: NMT 3.3%, calculated on the
SPECIFIC TESTS • TOTAL SOLIDS
• AMMONIACAL NITROGEN Liquid samples
Sample: 2 g Sample: 1.5-2.0 g
Analysis: Transfer 25.0 mL of 0.1 N sulfuric acid into a Analysis: Mix the Sample with 30.0 g of fine quartz
500-mL receiving flask. Connect the flask to a sand that passes a No. 40, but not a No. 60 sieve, and
distillation apparatus consisting of a Kjeldahl connecting that has been digested with hydrochloric acid, washed
bulb and a condenser, making certain that the acid-free, dried, and ignited. Dry the mixture to
condenser delivery tube is immersed beneath the constant weight at 60° under reduced pressure (50
surface of the acid solution in the receiving flask. mm Hg). Record the final weight of the sand plus the
Transfer the Sample into an 800-mL long-neck Kjeldahl sample solids and calculate the percent solids as
digestion flask and add 2 g of carbonate-free follows:
magnesium oxide, 200 mL of water, and several boiling
chips to the flask. Swirl the digestion flask to mix the Result = [(WF – WS)/WC] × 100
contents, and quickly connect it to the distillation
apparatus. Heat the contents of the flask to boiling and
collect about 100 mL of distillate in the receiving flask. WF = weight of the sand and sample solids (g)
Wash the tip of the delivery tube with a few milliliters WS = weight of the prepared sand taken (g)
WC = weight of the sample taken (g)
Use the calculated percent Total solids in the calculation S = weight of the sample taken (g)
for Color intensity (see above). F = the basis of color equivalency, 0.1
Solid samples (powdered or granular) A610 = absorbance of the 0.1% solution prepared
Analysis: Determine as directed under Loss on Drying, for the determination of Color intensity
Appendix IIC, drying a sample at 60° under reduced (above)
pressure (50 mm Hg) to constant weight. Calculate [NOTE—The above formula gives the result on an
the percent solids as follows: equivalent color basis that permits expression in terms
of a caramel having a color intensity standardized to 1
Result = [(WD – WB)/(WS – WB)] × 100 a.u.]
Acceptance criteria: NMT 3.5%, calculated on the
Monographs

WD = weight of the bottle and sample after
drying (g) ADDITIONAL INFORMATION
WB = weight of the empty bottle (g) • IDENTIFICATION OF CLASSES
WS = weight of the bottle and sample before [NOTE—The four classes of Caramel may be distinguished
drying (g) from each other by the methods below.]
Use the calculated percent Total solids in the calculation Class I: Not more than 50% of the color is bound by
for Color intensity (see above). DEAE (diethylaminoethyl) cellulose, and not more than
• TOTAL SULFUR 50% of the color is bound by phosphoryl cellulose.
Sample: 5 g when the expected amount of sulfur is Class II: More than 50% of the color is bound by DEAE
2.5% or less; or 1 g when the expected amount of cellulose and it exhibits an Absorbance Ratio (see below)
sulfur is greater than 2.5% of more than 50.
Analysis: Into the largest casserole available that fits in Class III: Not more than 50% of the color is bound by
an electric muffle furnace, place: 1 to 3 g of magnesium DEAE cellulose, and more than 50% of the color is
oxide or an equivalent quantity of magnesium nitrate, bound by phosphoryl cellulose.
hexahydrate (6.4 to 19.2 g); 1 g of powdered sucrose; Class IV: More than 50% of the color is bound by DEAE
and 50 mL of nitric acid. Transfer the Sample into the cellulose and it exhibits an Absorbance Ratio (see below)
casserole. Place the same quantities of reagents in of not more than 50.
another casserole for the blank and carry through the • IDENTIFICATION TESTS FOR CLASSES
following procedure for both the Sample and the blank: Tests
Evaporate the casserole contents on a steam bath to • ABSORBANCE RATIO
the consistency of paste. Place the casserole in a cold Sample solution: 1 mg/mL [NOTE—Any cloudiness
electric muffle furnace, gradually heat to 525°, and appearing can be eliminated by centrifuging the
hold at that temperature until all nitrogen dioxide solution.]
fumes are driven off. Cool the casserole, add 100 mL of Dilute sample solution: 50 µg/mL: made from Sample
water (the sample should dissolve), and neutralize to solution
pH 7 with hydrochloric acid, using short-range pH Analysis: Use a spectrophotometer equipped with a
indicator paper as an external indicator. Add an monochromator to provide a band width of 2 nm or
additional 2 mL of hydrochloric acid, filter the solution less and of such quality that the stray-light
into a suitable beaker, heat to boiling, and while characteristic is 0.5% or less. With water as a
stirring, slowly add 20 mL of barium chloride TS to the reference, measure the absorbance of the Sample
hot solution. Boil the contents of the beaker for 5 min, solution solution in a 1-cm cell at 560 nm and that of
and allow it to stand overnight. Filter the contents of the Dilute sample solution at 280 nm. Calculate the
the beaker through a tight, ashless filter paper and Absorbance Ratio of the sample by the formula:
quantitatively transfer the precipitate to the paper.
Thoroughly wash the paper and the precipitate with Result = (A280 × 20)/A560
hot water and transfer the paper to a tared crucible
previously ignited for 1 h at 800° in a muffle furnace.
Dry the paper in the crucible for 1 h at 105°. Then A280 = absorbance at 280 nm for the Dilute sample
carefully char it, with free access to air, at low heat over solution
a burner. Gradually increase the heat to burn away the A560 = absorbance at 560 nm for the Sample
paper and ignite the crucible and contents for 1 h at solution
800°. Cool and weigh it and calculate the percent sulfur 20 = dilution factor
by the formula: • COLOR BOUND BY DEAE CELLULOSE
[NOTE—Color Bound by DEAE Cellulose is defined here as
Result = [(WS – WB)/S] × 13.74 × F/A610 the percent decrease in absorbance of a caramel
solution at 560 nm after treatment with DEAE
cellulose.]
WS = weight of the ignited residue of barium DEAE cellulose: Use material with a capacity of 1.0
sulfate from the sample determination (g) mEq/g. DEAE cellulose of higher or lower capacities
WB = weight of the ignited residue from the blank may be used in proportionately higher or lower
determination (g) quantities.
Sample solution: Prepare a Sample solution of 2,4-Dinitrophenylhydrazine-hydrochloride (DNPH-

approximately 0.5 absorbance unit at 560 nm by HCl): Add 5 g of reagent-grade 2,4-
transferring an appropriate amount of sample into a dinitrophenylhydrazine (DNPH) to 10 mL of
100-mL volumetric flask with the aid of 0.025 N hydrochloric acid contained in a 100-mL Erlenmeyer
hydrochloric acid. Dilute to volume with 0.025 N flask, and gently shake the latter until the free base
hydrochloric acid, and centrifuge or filter if the (red) is converted to the hydrochloride (yellow). Add
solution is cloudy. 100 mL of ethanol and heat the mixture on a steam
Supernatant: To a 20-mL aliquot of the Sample bath until all of the solids have dissolved. Cool to
solution, add 140 mg of DEAE cellulose, mix thoroughly room temperature and, after the solution has
for several minutes, centrifuge or filter, and collect the crystallized, filter off the hydrochloride. Wash the
Monographs
clear supernatant liquid. hydrochloride with ether, dry it at room temperature,
Analysis: Using a suitable spectrophotometer previously and store it in a desiccator. Upon storage, the
standardized with 0.025 N hydrochloric acid, measure hydrochloride slowly converts to the free base. The
the absorbance of the Sample solution and of the latter can be removed by washing with purified
Supernatant in a 1-cm cell at 560 nm. Calculate the (peroxide-free) dimethoxyethane.
percent of color bound by DEAE cellulose by the DNPH-HCl reagent: Mix 0.5 g of DNPH–HCl with 15
formula: mL of 5% methanol in dimethoxyethane for 30 min.
Store in a refrigerator at 4°. When properly prepared
Result = 100[(X1 – X2)/X1] and stored, this reagent is stable for at least 3 months.
THI-DNPH standard: Add 0.5 g of DNPH–HCl to 1 mL
of hydrochloric acid, followed by 10 mL of ethyl
X1 = absorbance of the Sample solution at 560
alcohol, and heat on a steam bath until the
nm
hydrochloride dissolves. Add 100 mg of 2-acetyl-4(5)-
X2 = absorbance of the Supernatant at 560 nm
tetrahydroxybutylimidazole (THI) to the hot solution.
• COLOR BOUND BY PHOSPHORYL CELLULOSE
Crystallization begins in a few minutes. Filter off the
[NOTE—Color Bound by Phosphoryl Cellulose is defined
THI-DNPH when the suspension reaches room
here as the percent decrease in absorbance of a
temperature. Recrystallize the THI-DNPH with a
caramel solution at 560 nm after treatment with
hydrochloric acid-ethyl alcohol mixture (1 drop of
phosphoryl cellulose.]
hydrochloric acid per 5 mL of ethyl alcohol). The yield
Phosphoryl cellulose: Use material with a capacity of
is 70% to 80% based on the THI used. When stored in
1.2 mEq/g. Phosphoryl cellulose (cellulose phosphate)
the refrigerator, the THI–DNPH standard is stable for at
of higher or lower capacities may be used in
least 1 year.
proportionately higher or lower quantities.
Stock THI-DNPH solution: Dissolve 10 mg of
Sample solution: Transfer 200 to 300 mg of sample
THI–DNPH standard in a 100-mL volumetric flask and
into a 100-mL volumetric flask, dilute to volume with
dilute to volume with absolute Carbonyl-free methanol
0.025 N hydrochloric acid; centrifuge or filter if the
(see below). Dilute a portion of this solution tenfold
solution is cloudy.
with the methanol. The THI concentration (mg/L) of
Supernatant: To a 40-mL aliquot of the Sample
the Stock THI–DNPH solution is 0.47 times that of
solution, add 1.42 g of Phosphoryl cellulose, mix
THI–DNPH standard. When stored in the refrigerator,
thoroughly for several minutes, centrifuge or filter, and
the Stock THI–DNPH solution is stable for at least 20
collect the clear supernatant liquid.
weeks.
Analysis: Using a suitable spectrophotometer previously
Cation-exchange resin (Strong): Dowex 50 AG × 8,
standardized with 0.025 N hydrochloric acid, measure
proton form, 100- to 200-mesh
the absorbance of the Sample solution and of the
Cation-exchange resin (Weak): Amberlite CG AG 50 I,
Supernatant in a 1-cm cell at 560 nm. Calculate the
proton form, 100- to 200-mesh [NOTE—Sediment two
percent of color bound by Phosphoryl cellulose by the
or three times before use.]
formula:
Carbonyl-free methanol: Add 5 g of Girard’s Reagent
Result = 100[(X1 – X2)/X1] P (Aldrich, or equivalent) and 0.2 mL of hydrochloric
acid to 500 mL of methanol and reflux for 2 h. Distill
the refluxed methanol through a short Vigreux
X1 = absorbance of the Sample solution at 560 column, and store in tightly closed bottles.
nm Purified dimethoxyethane: Distill dimethoxyethane
X2 = absorbance of the Supernatant at 560 nm from DNPH in the presence of acid and redistill it from
• 2-ACETYL-4(5)-TETRAHYDROXYBUTYLIMIDAZOLE (THI) sodium hydroxide. Immediately before use, pass it
[NOTE—Class III (Ammonia Caramel) is the only class of through a column of neutral alumina to remove
caramel color found to contain THI. Because some peroxides.
countries have a THI limit of 25 mg/kg on an Combination column
equivalent color basis (on the basis of a product with Dropping funnel: 100-mL, equipped with a Teflon
Color intensity standardized to 0.1 absorbance units), stopcock and fitted with a 14.5-mm standard
the following method for determining THI is provided.] ground-glass joint, as the solvent reservoir
Upper column: Glass, 150 × 12.5 mm (id), filling sample from the standard curve. [NOTE—For THI limits
height: max 9 cm and bed height: 50 to 60 mm; or greater than 25 mg/kg, prepare a series of THI–DNPH
200 × 10 mm (id), filling height: max 14 cm and bed standard solutions in a range encompassing the
height 80 to 90 mm, equipped with a 1-mm (id) expected THI concentration in the sample.]
capillary outlet and fitted with 14.5-mm standard
ground-glass joints
Lower column: Glass, 175 mm × 10 mm (id), bed
Caraway Oil
.
height 60 mm, equipped with a 1-mm (id) capillary

outlet and a Teflon stopcock and fitted with a 14.5-
mm standard ground-glass joint
Monographs
Assembly: Fill the Upper column with Cation-exchange

CAS: [8000-42-8]
resin (Weak). Fill the Lower column with Cation-
UNII: C2J9B08Q3I [caraway oil]
exchange resin (Strong). Connect the Dropping funnel
and the two columns, one fitted above the other. DESCRIPTION
Sample solution: Dissolve 200 to 250 mg of sample in Caraway Oil occurs as a colorless to pale yellow liquid with
3 mL of water. Quantitatively transfer the solution to the characteristic odor and taste of caraway. It is a volatile
the upper part of the Combination column. Elute with oil distilled from the dried, ripe fruit of Carum carvi L.
water until a total of about 100 mL of water has (Fam. Umbelliferae).
passed through the column. Disconnect the Upper Function: Flavoring agent
column and elute the Lower column with 0.5 N Packaging and Storage: Store in full, tight containers in
hydrochloric acid. Discard the first 10.0 mL of eluate a cool place protected from light.
and subsequently collect a volume of 35 mL.
Concentrate the solution to dryness at 40° (15 mm IDENTIFICATION
Hg). Then, dissolve the syrupy residue in 250 µL of • INFRARED SPECTRA, Spectrophotometric Identification Tests,
Carbonyl-free methanol and add 250 µL of DNPH–HCl Appendix IIIC
reagent. Transfer the reaction mixture (Sample solution) Acceptance criteria: The spectrum of the sample
to a septum-capped vial, and store the vial for 5 h at exhibits relative maxima at the same wavelengths as
room temperature. those of the spectrum below.
Mobile phase: 50:50 (v/v) methanol:0.1 M phosphoric
acid ASSAY
THI–DNPH standard solutions: Pipet 1, 2, and 5 mL • KETONES, Aldehydes and Ketones, Neutral Sulfite Method,
of the Stock THI–DNPH solution into separate 10-mL Appendix VI
volumetric flasks and dilute to volume with absolute Acceptance criteria: NLT 50.0%, by volume, of ketones
Carbonyl-free methanol. as carvone
SPECIFIC TESTS
Detector: UV 385 nm
IIB
Column: 250-mm × 4-mm (id), 10-1m LiChrosorb RP-
Acceptance criteria: Between +70° and +80°
8 HPLC column (Alltech Associates, Inc., or
equivalent)
greater accuracy.]
Flow rate: 2 mL/min
Acceptance criteria: Between 1.484 and 1.488 at 20°
Analysis: Prepare a standard curve by injecting the
Stock THI–DNPH solution, and the serially diluted
Acceptance criteria: One mL of the sample dissolves in
THI–DNPH standard solutions, recording the
8 mL of 80% alcohol.
chromatograms, and measuring the peak areas for
• SPECIFIC GRAVITY: Determine by any reliable method (see
THI–DNPH. Inject the Sample solution and measure the
General Provisions).
peak response. [NOTE—THI-DNPH elutes at about
Acceptance criteria: Between 0.900 and 0.910
6.3±0.1 min.] Calculate the amount of THI in the
FCC 9 Monographs / Carbon, Activated / 235
Monographs
Caraway Oil
Analysis: Place the Sample in a glass-stoppered

Carbon, Activated
.
Erlenmeyer flask containing 10 mL of dilute

First Published: Prior to FCC 6 hydrochloric acid (5%), boil for 30 s, and cool to room
Last Revision: First Supplement, FCC 7 temperature. Add 100 mL of iodine TS, stopper, and
shake vigorously for 30 s. Filter through Whatman No.
2 filter paper, or equivalent, discarding the first portion
UNII: 2P3VWU3H10 [activated charcoal]
of filtrate. Compare 50 mL of the subsequent filtrate
DESCRIPTION with the Control.
Carbon, Activated occurs as a black substance, varying in Acceptance criteria: The color of the carbon-treated
particle size from coarse granules to a fine powder. It is a iodine solution is no darker than that of the Control,
solid, porous, carbonaceous material prepared by indicating the adsorptivity of the sample.
carbonizing and activating organic substances. The raw • B. PROCEDURE
materials, which include sawdust, peat, lignite, coal, Analysis: Ignite a sample in air.
cellulose residues, coconut shells, and petroleum coke, may Acceptance criteria: Carbon monoxide and carbon
be carbonized and activated at a high temperature with or dioxide are produced, and an ash remains.
without the addition of inorganic salts in a stream of IMPURITIES
activating gases such as steam or carbon dioxide. Inorganic Impurities
Alternatively, carbonaceous matter may be treated with a • ARSENIC, Arsenic Limit Test, Appendix IIIB
chemical activating agent such as phosphoric acid or zinc Sample solution: Use a 20-mL portion of the filtrate
chloride, and the mixture carbonized at an elevated obtained in the test for Water Extractables, diluted to
temperature, followed by removal of the chemical 35 mL.
activating agent by water washing. Activated Carbon is Acceptance criteria: NMT 3 mg/kg
insoluble in water and in organic solvents. • LEAD, Lead Limit Test, Appendix IIIB
Function: Decolorizing agent; taste- and odor-removing Sample solution: Use a 20-mL portion of the filtrate
agent; purification agent in food processing obtained in the test for Water Extractables.
Packaging and Storage: Store in well-closed containers. Control: 10 µg Pb (10 mL of Diluted Standard Lead
IDENTIFICATION Solution)
• A. PROCEDURE Acceptance criteria: NMT 10 mg/kg
Sample: 3 g, powdered • HEAVY METALS (AS PB)
Control: Dilute 10 mL of iodine TS with water to 50 Method I
mL. [NOTE—This test is designed to limit the content of
common metallic impurities colored by sulfide ion
236 / Carbon, Activated / Monographs FCC 9
(Ag, As, Bi, Cd, Cu, Hg, Pb, Sb, Sn) by comparing used for Solution A, adjust the pH to between 3.0 and
the color with a standard containing lead (Pb) ion 4.0 (using short-range pH indicator paper) by adding
under the specified test conditions. It demonstrates 1 N acetic acid or 6 N ammonia, dilute with water to
that the test substance is not grossly contaminated 40 mL, and mix.
by such heavy metals, and within the precision of the Solution C: Transfer 10 mL of the Sample solution into
test, that it does not exceed the Heavy Metals limit a third color-comparison tube that matches those
given as determined by concomitant visual used for Solutions A and B, and add 2.0 mL of
comparison with a control solution. In the specified Standard lead solution. Adjust the pH to between 3.0
pH range, the optimum concentration of lead (Pb) and 4.0 (using short-range pH indicator paper) by
ion for matching purposes by this method is 20 µg in adding 1 N acetic acid or 6 N ammonia, dilute with
Monographs
50 mL of solution. water to 40 mL, and mix.

The most common limitation of the Heavy Metals test is Analysis: Add 10 mL of freshly prepared hydrogen
that the color the sulfide ion produces in the Sample sulfide TS to each tube, mix, allow to stand for 5
solution depends on the metals present and may not min, and view downward over a white surface.
match the color in the dilution of the Standard lead [NOTE—If the color of Solution C is lighter than that of
solution used for matching purposes. Lead sulfide is Solution A, the sample is interfering with the test
brown, as are Ag, Bi, Cu, Hg, and Sn sulfides. While it procedure and Method II must be used.]
is possible that ions not mentioned here may also yield Acceptance criteria: The color of Solution B is not
nonmatching colors, among the nine common metallic darker than that of Solution A, and the intensity of
impurities listed above, the sulfides with different colors the color of Solution C is equal to or greater than that
are those of As and Cd, which are yellow, and that of of Solution A (NMT 0.004%).
Sb, which is orange. If these criteria are met, Cd may Method II
be a contributor to the yellow color, so the Cd content Solution A: Prepare as directed in Method I.
should be determined. If an orange color is observed, Solution B: Place 0.5 g of sample into a suitable
the Sb content should be determined. These additional crucible, add sufficient sulfuric acid to wet the
tests are in accord with the section on Trace Impurities sample, and carefully ignite at a low temperature
in the General Provisions, as follows: “if other possible until thoroughly charred, covering the crucible
impurities may be present, additional tests may be loosely with a suitable lid during the ignition. After
required, and should be applied, as necessary, by the the sample is thoroughly carbonized, add 2 mL of
manufacturer, vendor, or user to demonstrate that the nitric acid and 5 drops of sulfuric acid, cautiously
substance is suitable for its intended application.” heat until white fumes no longer evolve, then ignite,
Determine the amount of heavy metals by Method I for preferably in a muffle furnace, at 500°– 600° until all
substances that yield clear, colorless solutions before of the carbon is burned off. Cool, add 4 mL of 1:2
adding sulfide ion. Use Method II for those substances hydrochloric acid, cover, and digest on a steam bath
that do not yield clear, colorless solutions under the for 10–15 min. Uncover, and slowly evaporate on a
test conditions specified for Method I or for substances steam bath to dryness. Moisten the residue with 1
that by virtue of their complex nature, interfere with drop of hydrochloric acid, add 10 mL of hot water,
the precipitation of metals by sulfide ion.] and digest for 2 min. Add 6 N ammonia dropwise
[NOTE—In the following tests, failure to accurately adjust until the solution is just alkaline to litmus paper,
the pH of the solution within the specified limits may dilute with water to 25 mL, and adjust the pH to
result in a significant loss of test sensitivity.] between 3.0 and 4.0 (using short-range pH indicator
Lead nitrate stock solution: Dissolve 159.8 mg of paper) by adding 1 N acetic acid. Filter if necessary,
Reagent-Grade ACS Lead Nitrate [Pb(NO3)2] in 100 rinse the crucible and the filter with 10 mL of water,
mL of water containing 1 mL of nitric acid, dilute to transfer the solution and rinsings into a 50-mL color-
1000.0 mL, and mix. [NOTE—Prepare and store this comparison tube, dilute with water to 40 mL, and
solution in glass containers that are free from lead mix.
salts.] Analysis: Add 10 mL of freshly prepared hydrogen
Standard lead solution: Dilute 10.0 mL of Lead sulfide TS to each tube, mix, allow to stand for 5
nitrate stock solution with water to 100.0 mL. Each min, and view downward over a white surface.
mL is equivalent to 10 µg of lead (Pb) ion. [NOTE— Acceptance criteria: The color of Solution B is not
Prepare on the day of use.] darker than that of Solution A (NMT 0.004%).
Sample solution: Use the filtrate obtained in the test Organic Impurities
for Water Extractables. • CYANOGEN COMPOUNDS
Solution A: Pipet 2.0 mL of Standard lead solution Sample: 5 g
(20 µg of Pb) into a 50-mL color-comparison tube, Analysis: Mix the Sample with 50 mL of water and 2 g
and add water to make 25 mL. Adjust the pH to of tartaric acid and distill the mixture, collecting 25 mL
between 3.0 and 4.0 (using short-range pH indicator of distillate below the surface of a mixture of 2 mL of
paper) by adding 1 N acetic acid or 6 N ammonia, 1 N sodium hydroxide and 10 mL of water contained
dilute with water to 40 mL, and mix. in a small flask placed in an ice bath. Dilute the
Solution B: Transfer 10 mL of the Sample solution into distillate with water to 50 mL and mix. Add 12 drops
a 50-mL color-comparison tube that matches the one of ferrous sulfate TS to 25 mL of the diluted distillate,
FCC 9 Monographs / Carbon, Activated / 237
heat almost to boiling, cool, and add 1 mL of R = normality of the 0.1000 N Potassium iodate
hydrochloric acid. solution
Acceptance criteria: No blue color is produced. S = volume of Sodium thiosulfate solution (mL)
• HIGHER AROMATIC HYDROCARBONS Average the three normality results. Repeat the test if the
Sample solution: Extract 1 g of sample with 12 mL of range of values exceeds 0.003 N.
cyclohexane in a continuous-extraction apparatus for 2 Iodine solution (0.100 ± 0.001 N): Transfer 12.700 g of
h. Place the extract in a Nessler tube. iodine and 19.100 g of potassium iodide (KI),
Control solution: Dissolve 100 µg of quinine sulfate in accurately weighed, into a beaker, and mix. Add 2–5
1000 mL of 0.1 N sulfuric, and transfer into a mL of water, and stir well. While stirring, continue to
matching Nessler tube. add small increments, approximately 5 mL each, of
Monographs
Acceptance criteria: The Sample solution shows no water until the total volume is 50–60 mL. Allow the
more color or fluorescence than the Control solution solution to stand a minimum of 4 h to ensure crystal
when observed under ultraviolet light. dissolution, stirring occasionally. Quantitatively transfer
the solution to a 1-L volumetric flask, and dilute with
SPECIFIC TESTS water to volume. The iodide-to-iodine weight ratio
• IODINE NUMBER1 must be 1.5:1. Store the solution in an amber bottle.
Hydrochloric acid solution (5% by weight): Add 70 mL [NOTE—Standardize this solution just before use.]
of concentrated hydrochloric acid to 550 mL of water, To standardize this solution, perform the following in
and mix well. triplicate. Pipet 25.0 mL into a 250-mL wide-mouthed
Potassium iodate solution (0.1000 N): Dry 4 or more g Erlenmeyer flask. Titrate with the standardized Sodium
of primary standard-grade potassium iodate (KIO3) at thiosulfate solution until a light yellow color develops.
110° ± 5° for 2 h, and cool to room temperature in a Add a few drops of Starch solution, and continue the
desiccator. Dissolve 3.5667 g ± 0.1 mg of the dry titration until 1 drop produces a colorless solution.
potassium iodate in about 100 mL of water. Determine the Iodine solution normality using the
Quantitatively transfer to a 1-L volumetric flask, dilute following formula:
with water to volume, and mix thoroughly. Store in a
glass-stoppered bottle. Result = (S × N1)/I
Starch solution: Mix 1.0 ± 0.5 g of starch with 5–10 mL
of cold water to make a paste. Continue to stir while
adding an additional 25 ± 5 mL of water to the starch S = volume of the standardized Sodium
paste. Pour the mixture, while stirring, into 1 L of thiosulfate solution (mL)
boiling water, and boil for 4–5 min. [NOTE—Make this N1 = normality of the standardized Sodium
solution fresh daily.] thiosulfate solution, determined above
Sodium thiosulfate solution (0.100 N): Dissolve 24.820 I = volume of Iodine solution (mL)
g of sodium thiosulfate in approximately 75 ± 25 mL of Average the three normality results. Repeat the test if the
freshly boiled water, and add 0.10 ± 0.01 g of sodium range of values exceeds 0.003 N. The standardized
carbonate. Quantitatively transfer the mixture to a 1-L Iodine solution concentration must be 0.100 ± 0.001 N.
volumetric flask, and dilute with water to volume. Allow Sample preparation: Grind a representative sample
the solution to stand for a minimum of 4 days before until 60 wt % (or more) passes through a 325-mesh
standardizing. Store the solution in an amber bottle. To screen and 95 wt % (or more) passes through a 100-
standardize the solution, perform the following in mesh screen. Dry the ground sample, and cool to room
triplicate: Pipet 25.0 mL of 0.1000 N Potassium iodate temperature in a desiccator.
solution into a wide-mouthed Erlenmeyer flask. Add Analysis: Three dosages of Sample preparation must be
2.00 ± 0.01 g of potassium iodide, and shake the flask estimated to determine the iodine number. Weigh the
to dissolve the potassium iodide crystals. Pipet 5.0 mL three dosages (M) of dry carbon to the nearest mg.
of concentrated hydrochloric acid into the flask, and Transfer each to one of three clean, dry 250-mL
titrate the free iodine with Sodium thiosulfate solution to Erlenmeyer flasks equipped with ground-glass stoppers.
a light yellow color. Add a few drops of Starch solution, Pipet 10.0 mL of Hydrochloric acid solution into each
and continue the titration until 1 drop produces a flask, stopper each flask, and swirl gently until the
colorless solution. Determine the Sodium thiosulfate carbon is completely wetted. Loosen the stoppers to
solution normality using the following formula: vent the flasks, place on a hot plate in a fume hood,
and bring the contents to a boil. Allow to boil gently
Result = (P × R)/S for 30 ± 2 s to remove any sulfur (which may interfere
with the test results). Remove the flasks from the hot
plate and cool to room temperature. Standardize and
P = volume of 0.1000 N Potassium iodate then pipet 100.0 mL of the standardized Iodine solution
solution (mL) into each flask. [NOTE—Stagger the addition of
standardized Iodine solution to the three flasks so that
1Portions of this test are adapted from “ASTM D 4607-94(1999)—Standard no delays are encountered in handling.]
Test Method for Determination of Iodine Number of Activated Carbon.” The
original ASTM method is available in its entirety from ASTM, 100 Barr Harbor
Immediately stopper the flasks, and shake the contents
Drive, West Conshohocken, PA 19428; phone: 610-832-9585; fax: 610-832- vigorously for 30 ± 1 s. Quickly filter each mixture by
9555; email: service@astm.org; website: <www.astm.org>. gravity through one sheet of folded filter paper
238 / Carbon, Activated / Monographs FCC 9
(Whatman No. 2V, or equivalent) into one of three greater than 0.995. Carbon dosages may be estimated
beakers. [NOTE—Prepare the filtration equipment in initially by using three values of C (usually 0.01, 0.02,
advance to avoid delays in filtering the samples.] and 0.03):
For each filtrate, use the first 20–30 mL to rinse a pipet,
and discard the rinse portions. Use clean beakers to M = [A − (DF) × (C) × (126.93) × (50)]/E
collect the remaining filtrates. Mix each filtrate by
swirling the beaker, and pipet 50.0 mL of each filtrate
M = weight of the carbon dosage (g)
into one of three clean 250-mL Erlenmeyer flasks.
E = nominal iodine number of the Sample
Titrate each filtrate with standardized Sodium thiosulfate
If new carbon dosages have been determined, repeat the
solution until a pale yellow color develops. Add 2 mL of
Analysis and Calculations.
Monographs
Starch solution, and continue the titration with

Acceptance criteria: NLT 400
standardized Sodium thiosulfate solution until 1 drop
produces a colorless solution. Record the volume (S) of
Acceptance criteria: Results conform to the
standardized Sodium thiosulfate solution used.
Calculations: The capacity of a carbon for any adsorbate
• RESIDUE ON IGNITION (SULFATED ASH), Appendix IIC
depends on the concentration of the adsorbate. The
Sample: 500 mg
concentrations of the standardized Iodine solution and
Acceptance criteria: Results conform to the
filtrate must be known to determine an appropriate
carbon weight to produce final concentrations agreeing
• WATER EXTRACTABLES
with the definition of iodine number. The amount of
Sample: 5 g
sample to be used in the determination is governed by
Analysis: Transfer the Sample into a 250-mL flask
the activity of the sample. If filtrate normalities (C) are
provided with a reflux condenser and a Bunsen valve.
not within the range of 0.008–0.040 N, repeat the
Add 100 mL of water and several glass beads, and
procedure using different sample weights. Once filtrate
reflux for 1 h. Cool slightly, and filter through
normalities are set within the specified range, perform
Whatman No. 2, or equivalent, filter paper, discarding
the following calculations for each carbon dosage:
the first 10 mL of filtrate. Cool the subsequent filtrate
A = N2 × 12693.0 to room temperature, and pipet 25.0 mL into a tared
crystallization dish. [NOTE—Retain the remainder of the
filtrate for the Arsenic and Lead tests.] Evaporate the
N2 = normality of the standardized Iodine solution filtrate in the dish to incipient dryness on a hot plate,
never allowing the solution to boil. Dry for 1 h at 100°
B = N1 × 126.93 in a vacuum oven, cool, and weigh.
N1 = normality of the standardized Sodium
thiosulfate solution
Calculate the dilution factor (DF) using the equation:
Carbon Dioxide
.
DF = (I + H)/F First Published: Prior to FCC 6

I = volume of standardized Iodine solution used
in the standardization procedure (mL) CO2 Formula wt 44.01
H = volume of Hydrochloric acid solution used INS: 290 CAS: [124-38-9]
(mL) UNII: 142M471B3J [carbon dioxide]
F = volume of filtrate (mL)
Calculate the weight, in mg, of iodine adsorbed per g of
DESCRIPTION
Carbon Dioxide occurs as a colorless gas. One L of Carbon
sample (X/M) by the equation:
Dioxide weighs about 1.98 g at 0° and a pressure of 760
X/M = [A − (DF) × (B) × (S)]/M mm Hg. Under a pressure of about 59 atmospheres, it may
be condensed to a liquid, a portion of which forms a white
S = volume of standardized Sodium thiosulfate solid (“Dry Ice”) upon rapid vaporization. Solid Carbon
solution (mL) Dioxide evaporates without melting upon exposure to air.
M = weight of the Sample taken (g) One volume of the gas dissolves in about 1 volume of
Calculate the normality of the residual filtrate (C): water, forming a solution that is acid to litmus.
Function: Propellant and aerating agent; carbonating
C = (N1 × S)/F agent; direct-contact freezing agent
Using logarithmic paper, plot X/M (as the ordinate) Packaging and Storage: Store in metal cylinders.
versus C (as the abscissa) for each of the three carbon [NOTE—The following tests are designed to reflect the
dosages. Calculate the least squares fit for the three quality of Carbon Dioxide in both its vapor and its liquid
points, and plot. The iodine number is the X/M value at phases, which are present in previously unopened
a residual iodine concentration (C) of 0.02 N. The cylinders. Reduce the container pressure by means of a
regression coefficient for the least squares fit should be regulator. Withdraw the samples for the tests with the least
FCC 9 Monographs / Carbon Dioxide / 239
possible release of gas, consistent with proper purging of IMPURITIES

the sampling apparatus. Measure the gases with a gas Inorganic Impurities
volume meter downstream from the detector tubes to • CARBONYL SULFIDE
minimize contamination of or changes to the samples. The Standard: Gas mixture of 50 ppm carbonyl sulfide in
various detector tubes called for in the respective tests are helium (obtain from a specialty gas supplier)
listed under Detector Tubes, Solutions and Indicators.] Chromatographic system, Appendix IIA
Mode: Gas chromatography
IDENTIFICATION Detector: Sievers 350 (or equivalent)1
• A. CARBON DIOXIDE Chemiluminescence Detector (SCD) [NOTE—Operate
Sample: 100 ± 5 mL (Released from the vapor phase of the SCD with 190 mL/min of hydrogen and 396 mL/
Monographs
the contents of the container) min of air, and optimize the gas flows and probe
Analysis: Pass the Sample through a carbon dioxide position of the SCD for maximum sensitivity.]
detector tube (see Detector Tubes, Solutions and Column: 30-m × 0.53-mm (id), 5 mm DB-5 capillary
Indicators) at the rate specified for the tube. Note the column (J&W Scientific Company, or equivalent)
indicator change. Column temperature: 30°
Acceptance criteria: The indicator change extends Injection port temperature: 100°
throughout the entire indicating range of the tube. Carrier gas: Helium, 5 psig head-pressure
• B. PROCEDURE Split ratio: 1:1
Analysis: Pass a sample through barium hydroxide TS. Injection volume: 5.00 mL
Acceptance criteria: A precipitate is formed that System suitability
dissolves with effervescence in acetic acid. [NOTE—The retention time for carbonyl sulfide is
[NOTE—Perform the Assay and the tests under Inorganic approximately 3 min.]
Impurities and Organic Impurities in the following order: Suitability requirement: The peak areas resulting
Assay, Carbonyl sulfide, Hydrogen sulfide, Nitric Oxide from triplicate injections of the Standard give a
(NO) and Nitrogen Dioxide (NO2), Nonvolatile relative standard deviation of NMT 5.0%.
hydrocarbons, Sulfur dioxide, Volatile hydrocarbons, and Analysis: Inject the sample, in triplicate, average the
Water.] peak area responses, and calculate the concentration, C
ASSAY (ppm), of carbonyl sulfide in the sample by the
• PROCEDURE equation:
[NOTE—Sampling for this Assay may be done from the C (ppm) = S(AU / AS)
vapor phase for convenience, but this results in more
residual volume than sampling from the liquid phase. If
the specification of 0.5 mL is exceeded from a vapor S = the calculated concentration (ppm) of
phase sample, a liquid sample may be taken.] carbonyl sulfide in the Standard
Sample: 100.0 mL taken from the liquid phase as (approximately 50 ppm)
directed in the test for Nitric Oxide and Nitrogen Dioxide AU = average of the peak area responses for the
(below) sample
Analysis: Assemble a 100-mL gas buret provided with a AS = average of the peak area responses for the
leveling bulb and a two-way stopcock to a gas Standard
absorption pipet of suitable capacity by connecting the Acceptance criteria: NMT 0.5 ppm, by volume
pipet to one of the buret outlets. Fill the buret with • HYDROGEN SULFIDE
slightly acidified water (turned pink with methyl Sample: 50 mL, released from the vapor phase
orange), and fill the pipet with potassium hydroxide Analysis: Pass the Sample through a hydrogen sulfide
solution (1:2). By manipulating the leveling bulb and detector tube (Dräger #672804, 0.5 to 15 ppm, or
leveling water, draw the potassium hydroxide solution equivalent) at the rate specified for the tube. Note the
to fill the pipet and capillary connection up to the indicator change.
stopcock, and then fill the buret with the leveling Acceptance criteria: NMT 0.5 ppm, by volume, for the
water, and draw it through the other stopcock, opening volume of Sample specified in this test
it in such a manner that all gas bubbles are eliminated • NITRIC OXIDE (NO) AND NITROGEN DIOXIDE (NO2)
from the system. Draw the Sample into the buret. By Sample: 500 mL of liquid sample
raising the leveling bottle, force the measured Sample Analysis: Position the sample container so that when its
into the pipet. The absorption may be facilitated by valve is opened, the liquid phase can be sampled;
rocking the pipet or by flowing the Sample between generally this requires that the cylinder be inverted.
pipet and buret. Draw any residual gas into the buret, Attach a section of tubing long enough to act as a
and measure its volume. vaporizer for the small quantity of liquid to be
Acceptance criteria: NMT 0.5 mL of gas remains (NLT sampled. Connect one end of a nitric oxide–nitrogen
99.5% of CO2, by volume) dioxide detector tube (Detector Tubes, Solutions and
1 Any sulfur-selective detector may be used; e.g., electrolytic conductivity,
flame photometric, or sulfur chemiluminescence. The detector must be

capable of detecting less than 0.1 ppm v/v of carbonyl sulfide with a signal-
to-noise ratio of 10:1.
240 / Carbon Dioxide / Monographs FCC 9
Indicators) to the tubing and the other end to a gas Sample: Standard [NOTE—The typical retention time
flow meter. Pass the Sample through the tube at a for methane is 0.4 min.]
suitable rate. No frost should reach the tube inlet from Suitability requirement: The peak areas resulting
the expanding sample. Note the indicator change. from triplicate injections give a relative standard
Acceptance criteria: NMT 5 ppm total, by volume deviation of NMT 5.0%
• SULFUR DIOXIDE Analysis: Inject the sample, in triplicate. [NOTE—The
Sample: 1050 ± 50 mL from the liquid phase (see Nitric composition of hydrocarbons present will vary from
Oxide and Nitrogen Dioxide, above) sample to sample.] Sum the averages of the individual
Analysis: Pass the Sample through a sulfur dioxide peak areas for the individual hydrocarbons (Do not
detector tube (see Detector Tubes, Solutions and include the peak areas for carbon dioxide.). [NOTE—
Monographs
Indicators) at the rate specified for the tube. Note the The typical retention times for methane, carbon
indicator change. dioxide, and hexane are 0.4, 0.8, and 14.4 min,
Acceptance criteria: NMT 5 ppm, by volume respectively.] Calculate the total concentration, C
• WATER (ppm), of Volatile Hydrocarbons in the sample by the
Sample: 24,000 mL of gas equation:
Analysis: Pass the Sample through a suitable water-
absorption tube (see Detector Tubes, Solutions and C (ppm) = S(AU/AS)
Indicators), NLT 100 mm long, which previously has
been flushed with about 500 mL of sample and
S = calculated concentration (ppm) of methane
weighed. Regulate the flow so that about 60 min will
in the Standard (approximately 50 ppm)
be required for passage of the Sample. Then weigh the
AU = sum of the averages of the individual peak
absorption tube and calculate the weight gain.
area responses in the sample
Acceptance criteria: NMT 1.0 mg
AS = average of the peak area responses for the
Organic Impurities
Standard
• NONVOLATILE HYDROCARBONS
Acceptance criteria: NMT 0.005%, by volume (as
Sample: 500 g solid, prepared by passing liquid sample
methane)
from a storage container or sample cylinder through a
commercial carbon dioxide snow horn directly into an
open, clean container.
Analysis: Weigh the specified amount of Sample
Cardamom Oil
.
collected and transfer it into a clean beaker. Allow the

solid sample to sublime completely; place a watch First Published: Prior to FCC 6
glass placed over the beaker to prevent ambient
contamination. Wash the beaker with a residue-free CAS: [8000-66-6]
solvent, and transfer the solvent from the beaker to a UNII: JM0KJ091HZ [cardamom oil]
clean, tared watch glass or petri dish with two
additional rinses of the beaker with the solvent. Allow DESCRIPTION
the solvent to evaporate, by heating to 104°, until the Cardamom Oil occurs as a colorless or very pale yellow
watch glass or petri dish is at a constant weight. liquid with the aromatic, penetrating, and somewhat
Determine the weight of the residue by difference. camphoraceous odor of cardamom and a pungent,
Acceptance criteria: The weight of the residue is NMT strongly aromatic taste. It is the volatile oil distilled from
5 mg. (NMT 10 mg/kg) the seed of Elettaria cardamomum (L.) Maton (Fam.
• VOLATILE HYDROCARBONS (AS METHANE) Zingiberaceae). It is affected by light. It is miscible with
Standard: Standard gas mixture of 50 ppm methane in alcohol.
helium (obtain from a specialty gas supplier) Function: Flavoring agent
Chromatographic system, Appendix IIA Packaging and Storage: Store in full, tight containers in
Mode: Gas chromatography a cool place protected from light.
Detector: Flame ionization with a sensitivity range of
10−12 A/mV and an attenuation of 32
IDENTIFICATION
Column: 1.8-m × 3-mm (od) metal column, or
Appendix IIIC
equivalent, packed with 80- to 100-mesh HayeSep Q
(or equivalent)
Column temperature: 70° for 1 min, increase to 200°
those of the spectrum below.
at a rate of 20°/min, hold at 200° for 10 min
Injection port temperature: 230° SPECIFIC TESTS
Detector: 230° • ANGULAR ROTATION, Optical (Specific) Rotation, Appendix
Carrier gas: Helium IIB: Use a 100 mm tube.
Flow rate: 30 mL/min Acceptance criteria: Between +22° and +44°
Injection volume: 1.00 mL • REFRACTIVE INDEX, Appendix IIB
System suitability [NOTE—Use an Abbé or other refractometer of equal or
greater accuracy.]
FCC 9 Monographs / Carmine / 241
Acceptance criteria: Between 1.462 and 1.466 at 20° • SPECIFIC GRAVITY: Determine by any reliable method (see
• SOLUBILITY IN ALCOHOL, Appendix VI General Provisions).
Acceptance criteria: One mL of the sample dissolves in Acceptance criteria: Between 0.917 and 0.947
5 mL of 70% alcohol. The solution can be clear or
hazy.
Monographs
Cardamom Oil
soluble in hot water, and practically insoluble in cold water

Carmine
.
and in dilute acids.

First Published: Prior to FCC 6 Carminic acid crystallizes from water as bright red crystals
that darken at 130° and decompose at 250°; it is freely
soluble in water, in alcohol, in ether, in concentrated
Carminic Acid
sulfuric acid, and in solutions of alkali hydroxides; it is
insoluble in petroleum ether and in chloroform. Its aqueous
solutions at pH 4.8 are red-orange to yellow, and at 6.2
are dark red to violet.
[NOTE—Before use in food, Carmine must be pasteurized or
otherwise treated to destroy all viable Salmonella
microorganisms. According to the pertinent U.S. color
additive regulation (21 CFR 73.100(b)(2)), “…
C22H20O13 Formula wt 492.39 pasteurization or such other treatment is deemed to permit
INS: 120 CAS: [1390-65-4] the adding of safe and suitable substances (other than
UNII: TZ8Z31B35M [cochineal] chemical preservatives) that are essential to the method of
pasteurization or other treatment used.”]
DESCRIPTION [NOTE—The specifications and tests in this monograph refer
Carmine occurs as bright red, friable pieces or as a dark red to Carmine without any added substances for
powder. It is the aluminum or the calcium-aluminum lake, pasteurization or any other such treatment.]
on an aluminum hydroxide substrate, of the coloring Function: Color
principles obtained by an aqueous extraction of cochineal. Packaging and Storage: Store in well-closed containers
Cochineal consists of the dried female insects Dactylopius in a cool, dry place.
coccus costa (Coccus cacti L.), enclosing young larvae; the
coloring principles thus derived consist mainly of carminic IDENTIFICATION
acid (C22H20O13). It is soluble in alkali solutions, slightly • PROCEDURE
Sample solution: Mix 333 mg of sample with 44 mL of
water, 0.15 mL of a 1:10 sodium hydroxide solution,
242 / Carmine / Monographs FCC 9
and 0.2 mL of ammonium hydroxide in a 500-mL Sample solution: Prepare as directed for organic
volumetric flask. Warm the mixture to dissolve the compounds.
sample, allow the solution to cool, dilute to volume Control: 2 µg Pb (2 mL of Diluted Standard Lead
with water, and mix. Pipet 10.0 mL of this solution into Solution)
a 250-mL volumetric flask, dilute to volume with water, Acceptance criteria: NMT 2 mg/kg
and mix.
Analysis: Using a suitable spectrophotometer and a 1- SPECIFIC TESTS
cm cell, measure the absorbance of the Sample solution; • ASH
use water as a blank. Sample: 1 g
Acceptance criteria: The Sample solution exhibits Analysis: Transfer the Sample into a tared, previously
Monographs
absorption maxima at 520 nm and 550 nm and the ignited and cooled porcelain crucible, and ignite red-
absorbance at 520 nm is not less than 0.30. hot with a Meker burner to constant weight.
ASSAY • LOSS ON DRYING, Appendix IIC: 135° for 3 h
• PROCEDURE Sample: 1 g
Sample: 0.100 g (∼52% carminic acid content) Acceptance criteria: NMT 20.0%
Sample solution: Dissolve the Sample in 30 mL of 2 N • MICROBIAL LIMITS
hydrochloric acid and heat to a boil for 30 s. After [NOTE—Current methods for the following tests may be
cooling, dilute to a volume of one liter. If a black or found by accessing the US Food and Drug
brown precipitate forms, filter the solution. Administration’s Bacteriological Analytical Manual
Analysis: Using a suitable spectrophotometer and a 1- (BAM) online at www.fda.gov/Food/default.htm.]
cm cell, measure the absorbance of the Sample solution Acceptance criteria
at the wavelength maximum of about 494 nm; use a Salmonella: Negative in 25 g
1:3 aqueous dilution of 2 N hydrochloric acid as the • PROTEIN, Nitrogen Determination, Method II, Appendix IIIC
blank. [NOTE—To obtain accurate results, the Analysis: Calculate the protein content, in percent, by
absorbance must be in the range of 0.650 to 0.750. the formula:
Adjust the starting weight as necessary to achieve this
absorbance.] Calculate the percent carminic acid in the Result = (F × N/W) × 100
Result = 100A/13.9W F = conversion factor from nitrogen to protein,

6.25
N = weight of nitrogen (mg)
A = absorbance of the Sample solution W = weight of sample taken (mg)
W = weight of the sample taken (g) Acceptance criteria: NMT 25%
Acceptance criteria: NLT 50.0% of carminic acid
(C22H20O13), calculated on the dried basis
IMPURITIES Carnauba Wax

.
• ARSENIC, Arsenic Limit Test, Appendix IIIB First Published: Prior to FCC 6
Sample: 3.0 g
Sample solution: Transfer the Sample into a 500-mL INS: 903 CAS: [8015-86-9]
Kjeldahl flask equipped with a steam trap, add 5 g of UNII: R12CBM0EIZ [carnauba wax]
ferrous sulfate and 75 mL of hydrochloric acid, and
mix. Connect the flask with the steam trap and with a DESCRIPTION
condenser, the delivery tube of which consists of a Carnauba Wax occurs as a hard, brittle substance with a
large-sized straight adapter and extends to slightly resinous fracture and a color ranging from light brown to
above the bottom of a 500-mL Erlenmeyer flask pale yellow. It is a purified wax obtained from the leaf
containing 100 mL of water. Begin heating the Kjeldahl buds and leaves of the Brazilian wax palm Copernicia
flask and collect about 40 mL of distillate in the cereferia (Arruda) Mart. [synonym C. prunifera (Muell.)]. Its
Erlenmeyer flask. Pour the distillate mixture into a 600- specific gravity is about 0.997. It is partially soluble in
mL beaker, and add 20 mL of bromine TS (bromine boiling alcohol, is soluble in chloroform and in ether, but is
water), and heat on a hot plate until the volume is insoluble in water.
reduced to about 2 mL. Transfer the residual liquid into Function: Anticaking agent; surface-finishing (glazing)
a 125-mL arsine generator flask (see Figure 11) with agent; release agent; carrier for flavors
the aid of 35 mL of water, and continue as directed in Packaging and Storage: Store in well-closed containers.
the Procedure beginning with “Add 20 mL of 1:5
IMPURITIES
sulfuric acid…”
• LEAD, Appendix IIIB
• LEAD, Lead Limit Test, Appendix IIIB
Sample solution: Prepare as directed for organic
compounds.
FCC 9 Monographs / L-Carnitine / 243
Control: 5 µg Pb (5 mL of Diluted Standard Lead • B. INFRARED ABSORPTION, Spectrophotometric Identification

Solution) Tests, Appendix IIIC
Acceptance criteria: NMT 5 mg/kg Reference standard: USP Levocarnitine RS
Sample and standard preparation: K (previously dried
SPECIFIC TESTS in vacuum at 60° for 5 h)
• ACID VALUE [FATS AND RELATED SUBSTANCES], Method I, Acceptance criteria: The spectrum of the sample
Appendix VII exhibits maxima at the same wavelengths as those in
Acceptance criteria: Between 2 and 7 the spectrum of the Reference standard.
• ESTER VALUE
Analysis: Subtract the Acid Value (above) from the ASSAY
Monographs
Saponification Value (below) to obtain this value. • PROCEDURE
Acceptance criteria: Between 71 and 88 Sample: 1.0 g [NOTE—Avoid atmospheric moisture
• MELTING RANGE OR TEMPERATURE DETERMINATION, Procedure uptake during weighing.]
for Class II, Appendix IIB Analysis: Dissolve the Sample in water contained in a
Acceptance criteria: Between 80° and 86° 250-mL flask. Titrate with 1.0 N hydrochloric acid to a
• RESIDUE ON IGNITION (SULFATED ASH), Appendix IIC potentiometric endpoint. Perform a blank determination
Sample: 2 g (see General Provisions), and make any necessary
Analysis: Heat the Sample in a tared, open, porcelain or correction. Each mL of 1.0 N hydrochloric acid is
platinum dish over an open flame. It volatilizes without equivalent to 161.2 mg of C7H15NO3.
emitting an acrid odor. Ignite as directed. Acceptance criteria: NLT 97.0% and NMT 103.0% of
Acceptance criteria: NMT 0.25% C7H15NO3, calculated on the anhydrous basis
• SAPONIFICATION VALUE, Appendix VII
Sample: 5 g IMPURITIES
Acceptance criteria: Between 78 and 95 Inorganic Impurities
• UNSAPONIFIABLE MATTER, Appendix VII • CHLORIDE
Acceptance criteria: Between 50.0% and 55.0% Sample solution: Add 100 mg of sample in 30 to 40
mL of water in a 50-mL flask, and mix. Add 10% nitric
acid dropwise until the solution is neutral to litmus.
Add an additional 1 mL of 10% nitric acid and dilute
with water to a total volume of 50 mL.
L-Carnitine
.
Control: Add 0.56 mL of 0.02 N hydrochloric acid

First Published: Prior to FCC 6 solution to 30 to 40 mL of water in a 50-mL flask. Add
1 mL of 10% nitric acid and dilute to volume with
4-Amino-3-hydroxybutyric Acid Trimethylbetaine water.
Levocarnitine Analysis: Add 1 mL of 0.1 N silver nitrate to both the
4-Trimethylamino-3-hydroxybutyrate Sample solution and the Control. Mix, allow to stand for
(R)-3-Carboxy-2-hydroxy-N,N,N-trimethyl-1-propanaminium 5 min protected from direct sunlight, and visually
Hydroxide, Inner Salt compare the two solutions.
Sample solution does not exceed that shown in the
Control (NMT 0.4%).
C7H15NO3 Formula wt 161.20 Spectrophotometric Method, Appendix IIIB
CAS: [541-15-1] Sample: 10 g
UNII: 0G389FZZ9M [levocarnitine] Acceptance criteria: NMT 1 mg/kg
• POTASSIUM
DESCRIPTION [NOTE—The Standard solution and the Sample solutions
L-Carnitine occurs as white crystals or as a white, crystalline, may be modified, if necessary, to obtain solutions of
hygroscopic powder. It is freely soluble in water, in alcohol, suitable concentrations adaptable to the linear or
in alkaline solutions, and in dilute mineral acids. It is working range of the spectrophotometer.]
practically insoluble in acetone and in ethyl acetate. It Standard stock solution: 12.5 mg/mL potassium, made
decomposes without melting at about 185° to 195°. by transferring 5.959 g of potassium chloride,
Function: Nutrient previously dried at 105° for 2 h, into a 250-mL
Packaging and Storage: Store in tight containers. volumetric flask, dilute to volume with water, and mix.
Standard solution: 31.25 µg/mL potassium: from
IDENTIFICATION Standard stock solution
• A. PROCEDURE
Sample: 62.5 mg
Analysis: Dissolve 1 g of sample in 10 mL of water and
Sample stock solution: Transfer the Sample into a 100-
10 mL of 1 N hydrochloric acid, and add 5 mL of
mL volumetric flask, dissolve in and dilute to volume
sodium tetraphenylborate TS.
with water, and mix.
Sample solutions: Add 0, 2.0, and 4.0 mL of the
Standard solution to three separate 25-mL volumetric
244 / L-Carnitine / Monographs FCC 9
flasks. Add 20.0 mL of the Sample stock solution to each Acceptance criteria: NMT 0.1%
flask, dilute to volume with water, and mix. These
solutions contain 0 (Sample solution A), 2.5 (Sample SPECIFIC TESTS
solution B), and 5.0 (Sample solution C) µg/mL of • OPTICAL (SPECIFIC) ROTATION, Appendix IIB
potassium. Sample solution: 100 mg/mL (using a previously dried
Analysis: Using a suitable atomic absorption sample)
spectrophotometer equipped with an air-acetylene Acceptance criteria: [α]D20 between −29.0° and
flame and using water as the blank, concomitantly −32.0°, calculated on the anhydrous basis
determine the absorbance values of the Sample • PH, pH Determination, Appendix IIB
solutions at the potassium emission line at 766.7 nm. Sample solution: 50 mg/mL
Monographs
Plot the absorbance values of the Sample solutions Acceptance criteria: Between 5.5 and 9.5
versus their contents of potassium, in µg/mL; draw the • RESIDUE ON IGNITION (SULFATED ASH), Appendix IIC
straight line best fitting the three points and Sample: 2 g
extrapolate the line until it intersects with the Acceptance criteria: NMT 0.5%
concentration axis. From the intercept, determine the • WATER, Water Determination, Appendix IIB
amount, in µg, of potassium in each mL of Sample Acceptance criteria: NMT 4.0%
solution A. Calculate the percent potassium in the
portion of Sample taken by multiplying the
concentration, in µg/mL, of potassium found in Sample
β-Carotene
.
solution A by 0.2.
Acceptance criteria: NMT 0.2% First Published: Prior to FCC 6
• SODIUM Last Revision: FCC 9
[NOTE—The Standard solution and the Sample solutions
may be modified, if necessary, to obtain solutions of Carotene
suitable concentrations adaptable to the linear or Beta-Carotene
working range of the spectrophotometer.]
Standard stock solution: 10.0 mg/mL sodium, made
by transferring 6.355 g of sodium chloride, previously
dried at 105° for 2 h, into a 250-mL volumetric flask,
dilute to volume with water, and mix.
Standard solution: 250 µg/mL sodium: from Standard
stock solution C40H56 Formula wt 536.88
Sample: 4 g INS: 160a(i) CAS: [7235-40-7]
Sample stock solution: Transfer the Sample into a 100- UNII: 01YAE03M7J [β-carotene]
mL volumetric flask, dissolve in and dilute to volume
with water, and mix. DESCRIPTION
Sample solutions: Add 0, 2.0, and 4.0 mL of the
Standard solution to three separate 25-mL volumetric Change to read:
flasks. Add 20.0 mL of the Sample stock solution to each β-Carotene occurs as red crystals or as crystalline powder.
▲ It consists predominantly of all-trans-β-carotene, but may
flask, dilute to volume with water, and mix. These
solutions contain 0 (Sample solution A), 20.0 (Sample also contain minor amounts of cis-isomers and other
solution B), and 40.0 (Sample solution C) µg/mL of carotenoids (see specifications).▲ FCC9 It is soluble in carbon
sodium. disulfide and in chloroform; sparingly soluble in ether, in
Analysis: Using a suitable atomic absorption solvent hexane, and in vegetable oils; practically insoluble
spectrophotometer equipped with an air-acetylene in methanol and in ethanol; insoluble in water and in acids
flame and using water as the blank, concomitantly and alkalies. It melts between 176° and 182°, with
determine the absorbance values of the Sample decomposition. ▲ Commercial preparations of β-Carotene
solutions at the sodium emission line at 589.0 nm. Plot intended for use in foods are prepared from β-Carotene
the absorbance values of the Sample solutions versus meeting these specifications and are formulated as
their contents of sodium, in µg/mL; draw the straight suspensions in edible oils or water-dispersible (edible)
line best fitting the three points and extrapolate the emulsions and powders. These preparations may have a
line until it intersects with the concentration axis. From different ratio of trans/cis isomers.▲ FCC9
the intercept, determine the amount, in µg, of sodium Function: Nutrient; color
in each mL of Sample solution A. Calculate the percent Packaging and Storage: Store in a cool place in tight,
sodium in the portion of Sample taken by multiplying light-resistant containers under inert gas.
the concentration, in µg/mL, of sodium found in [NOTE—Carry out all work in low-actinic glassware and in
Sample solution A by 0.003125. subdued light.]
FCC 9 Monographs / β-Carotene / 245
IDENTIFICATION Acceptance criteria: 96.0%–101.0% of total

• A. UV-VIS ABSORPTION SPECTRUM carotenoids, calculated as β-carotene (C40H56)
Sample solution: Prepare as directed for the Sample ▲
▲ FCC9
solution in the test for Total Carotenoids.

Analysis: Record the UV-Vis spectrum from 300–600 IMPURITIES
nm. Change to read:
Acceptance criteria: The Sample solution shows a
shoulder at about 427 nm, an absorption maximum at
• LEAD, Lead Limit Test, ▲ Flame Atomic Absorption
about 455 nm, and another maximum at about 483
Spectrophotometric Method,▲ FCC9 Appendix IIIB
nm. The absorbance ratio A455/A483 is 1.14–1.18.
Monographs
Sample: 5 g
Add the following: Acceptance criteria: NMT 5 mg/kg
• ▲ B. ABSORBANCE RATIO SPECIFIC TESTS
Sample solution A: Transfer 50 mg of sample into a
100-mL volumetric flask, dissolve it in 10 mL of acid- Change to read:
free chloroform, immediately dilute with cyclohexane to • ALPHA-CAROTENE AND OTHER RELATED COMPOUNDS
volume, and mix. Transfer 5.0 mL of this solution into a ▲ [NOTE—Use low-actinic glassware.]
100-mL volumetric flask, and dilute with cyclohexane to Mobile phase: Transfer 50 mg of butylated
volume. hydroxytoluene into a 1-L volumetric flask, and dissolve
Sample solution B: Dilute 5.0 mL of Sample solution A with 20 mL of 2-propanol. Add 0.2 mL of N-
to 50 mL with cyclohexane. ethyldiisopropylamine, 25 mL of 0.2% ammonium
Analysis: Using a suitable spectrophotometer, determine acetate solution, 455 mL of acetonitrile, and about 450
the absorbance of Sample solution B at 455 nm and that mL of methanol. Allow the solution to reach room
of Sample solution A at 340 nm. temperature, and dilute with methanol to volume.
Acceptance criteria: The ratio of absorbance values Diluent: 50 µg/mL of butylated hydroxytoluene in
obtained, A455/A340, is NLT 1.5.▲ FCC9 alcohol
Change to read: System suitability solution: Transfer 20 mg of USP β-
Carotene System Suitability RS to a 50-mL volumetric
• ▲ C.▲ FCC9 PROCEDURE flask. Add 1 mL of water, 4 mL of tetrahydrofuran, and
Acceptance Criteria: The retention time of the major sonicate for 5 min. Dilute with Diluent to volume, and
peak of the Sample solution corresponds to that of the sonicate for 5 min. Cool to room temperature, pass the
Standard solution, as obtained in the ▲ test for Alpha- suspension through a membrane filter of 0.45-µm pore
Carotene and Other Related Compounds.▲ FCC9 size, and use the clear filtrate.
ASSAY Standard solution: 10 µg/mL of USP β-Carotene RS in
tetrahydrofuran and Diluent (1:9). Dissolve an
Change to read: appropriate amount of USP β-Carotene RS in a
• TOTAL CAROTENOIDS volumetric flask first with tetrahydrofuran, using 10% of
Sample stock solution: 0.1 mg/mL of the sample in the volume of the flask, then dilute with Diluent to
tetrahydrofuran volume.
Sample solution: Transfer 3.0 mL of the Sample stock Sample solution: 10 µg/mL. Prepare by diluting the
solution into a 100-mL volumetric flask, and dilute with freshly prepared Sample stock solution as prepared in the
cyclohexane to volume. test for Total Carotenoids (1 in 10, v/v) with Diluent.
Analysis: Determine the absorbance of the Sample Chromatographic system, Appendix IIA
solution using a suitable spectrophotometer with a 1-cm Mode: HPLC
cell, set to ▲ 455▲ FCC9 nm, using cyclohexane as the Detector: UV 448 nm
blank. Column: 4.6-mm × 25-cm; 5-µm packing of spherical,
Calculate the percentage of total carotenoids as β- porous silica that has been covalently modified with
carotene: alkyl amide groups and not endcapped.1
Result = A/(F × C) × 100 Flow rate: 0.6 mL/min
A = absorbance of the Sample solution System suitability
F = coefficient of extinction (E1%) of pure all- Samples: System suitability solution and Standard
trans-β-carotene in cyclohexane, 2505 (100 solution
mL · g−1 · cm−1)
C = concentration of the Sample solution (g/mL)
1 Supelco Suplex pKb-100, available from http://www.sigmaaldrich.com/
Supelco, or equivalent.
246 / β-Carotene / Monographs FCC 9
[NOTE—The approximate relative retention times of the Total related compounds (including alpha-
components in the System suitability solution are listed carotene): NMT 5.0% (determined as percentage of
in Table 1.] the total carotenoids content)
• LOSS ON DRYING, Appendix IIC: In a vacuum over
Table 1 phosphorus pentoxide at 40° for 4 h
Relative Relative Acceptance criteria: NMT 0.2%
Retention Response • RESIDUE ON IGNITION (SULFATED ASH), Appendix IIC
Analyte Time Factor Sample: 2 g
All-trans-alpha-carotene 0.93 1.1 Acceptance criteria: NMT 0.2%
▲
All-trans-beta-carotene 1.00 1.0 ▲ FCC9
Monographs
9-cis-beta-carotene 1.07 1.0

13-cis-beta-carotene 1.17 1.2
15-cis-beta-carotenef 1.21 1.4
Carrageenan
.
[NOTE—The chromatogram from the System suitability First Published: First Supplement, FCC 6
solution is similar to the Reference Chromatogram
provided with the USP β-Carotene System Suitability Irish moss (from Chondrus spp.)
RS being used.] Eucheuman (from Eucheuma spp.)
Suitability requirement 1: The resolution between all- Iridophycan (from Iridaea spp.)
trans-beta-carotene and all-trans-alpha-carotene and Hypnean (from Hypnea spp.)
between all-trans-beta-carotene and 9-cis-beta- Processed Eucheuma Seaweed, PES, PNG-carrageenan, and
carotene is NLT 1.5 for the System suitability solution. Semi-refined carrageenan (from E. spinosum or E. cottonii)
Suitability requirement 2: The tailing factor for the INS: 407 CAS: [9000-07-1]
all-trans-beta-carotene peak is NMT 2.0 for the UNII: 5C69YCD2YJ [carrageenan]
Standard solution.
Suitability requirement 3: The relative standard DESCRIPTION
deviation is NMT 2.0% for the all-trans-beta-carotene Carrageenan occurs as a yellow or tan to white, coarse to
peak from replicate injections of the Standard fine powder. It is obtained from certain members of the
solution. class Rhodophyceae (red seaweeds). The principal
Analysis: Separately inject equal volumes of the Sample commercial sources of carrageenans are the following
solution and the System suitability solution into the families and genera of the class Rhodophyceae1:
chromatograph, record the chromatograms, and Furcellariaceae such as Furcellaria;
identify the peaks of the relevant analytes in the Gigartinaceae such as Chondrus, Gigartina, Iridaea;
chromatogram of the Sample solution by comparing Hypnaeceae such as Hypnea;
with those in the chromatogram of the System Phyllophoraceae such as Phyllophora, Gynmogongrus,
suitability solution. Measure the peak area responses. Ahnfeltia;
Calculate the percentage of alpha-carotene and other Solieriaceae such as Eucheuma, Anatheca, Meristotheca.
individual related compounds (relative to total Carrageenan is a hydrocolloid consisting mainly of the
carotenoids) in the sample taken: ammonium, calcium, magnesium, potassium, and sodium
sulfate esters of galactose and 3,6-anhydrogalactose
Result = rA/rT × 100 polysaccharides. These hexoses are alternately linked
α-(1→3) and β-(1→4) in the copolymer. The relative
rA = peak area of the analyte of interest
proportions of cations existing in carrageenan may be
multiplied by the appropriate relative
changed during processing to the extent that one may
response factor (see Table 1) from the
become predominant.
chromatogram of the Sample solution
The prevalent polysaccharides in carrageenan are designated
rT = sum of (peak area × relative response factor)
as kappa-, iota-, and lambda-carrageenan. Kappa-
for all-trans-alpha-carotene, all-trans-beta-
carrageenan is mostly the alternating polymer of D-
carotene, 9-cis-beta-carotene, 13-cis-beta-
galactose-4-sulfate and 3,6-anhydro-D-galactose; iota-
carotene, 15-cis-beta-carotene, and other
carrageenan is similar except that the 3,6-anhydrogalactose
cis-isomers of β-carotene from the
is sulfated at carbon 2. Between kappa-carrageenan and
chromatogram of the Sample solution. The
iota-carrageenan, there is a continuum of intermediate
relative response factors are obtained from
compositions differing in degree of sulfation at carbon 2.
Table 1. Use 1.0 as the relative response
In lambda-carrageenan, the alternating monomeric units
factor for the other cis-isomers of β-
carotene not identified in Table 1. 1In the United States, only the following seaweed species from the families
Gigartinaceae and Solieriaceae are authorized as sources of carrageenan
▲ FCC9
intended for use in foods (Title 21 US Code of Federal Regulations Part 172,
Acceptance criteria section 620 (21 CFR 172.620)): Chondrus crispus, C. ocellatus, Eucheuma
Alpha-carotene: NMT 1.0% (determined as percentage cottonii, E. spinosum, Gigartina acicularis, G. pistillata, G. radula, and G.
of the total carotenoids content) stellata.
FCC 9 Monographs / Carrageenan / 247
are mostly D-galactose-2-sulfate (1→3-linked) and D- crystalline or syrupy residue is obtained. Dissolve this
galactose-2,6-disulfate (1→4-linked). hydrolysate in 10 mL of 40% methanol.
Carrageenan may be obtained from any of the cited Standard solution: Separately, dissolve each of the
seaweeds by extraction into water or aqueous dilute alkali. Standards in 10 mL of 40% methanol.
It may be recovered by alcohol precipitation, by drum Spray reagent: Dissolve 1.23 g of anisidine and 1.66 g
drying, or by precipitation in aqueous potassium chloride of phthalic acid in 100 mL ethanol.
and subsequent freezing. Additionally, carrageenan may be Application volumes: 1-5 µL Sample solution and 1-10
obtained by extracting the cleaned seaweed with alkali for µL of the Standard solutions
a short time at elevated temperatures. The material is then Analysis: Apply the Sample solution on the starting lines
thoroughly washed with water to remove residual salts of two Silica Gel G plates. On the same plates apply the
Monographs
followed by purification, drying and milling to a powder. Standard solutions. Develop one plate in Developing
Carrageenan obtained by this method contains a higher solvent system A and the other in Developing solvent
percentage of algal cellulose. The alcohols used during system B. After developing the two plates, remove the
recovery and purification of carrageenan are restricted to plates from the chambers and dry them at 110° for 20
methanol, ethanol, and isopropanol. min. Allow the plates to cool and spray with Spray
Carrageenan is insoluble in ethanol but it is soluble in water reagent. After spraying, heat the plates at 100° for 10
at 80°, forming a viscous clear or cloudy and slightly min. [NOTE—A greenish yellow color is produced with
opalescent solution that flows readily. Some samples form hexoses, a red color with pentoses and a brown color
a cloudy viscous suspension in water. Carrageenan with uronic acids.]
disperses in water more readily if first moistened with Acceptance criteria: Spots corresponding in color and
alcohol, glycerol, or a saturated solution of glucose or RF with galactose and 3,6-anhydrogalactose from the
sucrose in water. Standard solutions are obtained from the Sample
Articles of commerce may include sugars for standardization solution.
purposes, salts to obtain specific gelling or thickening • INFRARED SPECTRA, Spectrophotometric Identification Tests,
characteristics, or emulsifiers carried over from drum-drying Appendix IIIC
processes. Sample preparation
Function: Thickener, gelling agent, stabilizer, emulsifier [NOTE—If the carrageenan sample does not contain
Packaging and Storage: Store in well-closed containers. standardizing salts or sugars, the following purification
[NOTE—Carrageenan must be well dispersed in water in step is not necessary.] Disperse 1 g of carrageenan in
many of the following tests so dispersion technique must 250 mL of cold water, heat the mixture at 90° for 10
be kept in mind throughout this monograph. Carrageenan min, cool it to 60°, and dissolve 1 g of potassium
is best dispersed by slowly sprinkling the powder into cold chloride into the solution. Coagulate the mixture with
water with continuous vigorous stirring. This allows the 2 volumes of isopropyl alcohol, then recover, wash,
carrageenan particles to wet and hydrate effectively prior and dry the purified carrageenan. Disperse 0.5 g of
to dissolving. Adding carrageenan directly to hot water, or the purified carrageenan sample in 250 mL of cold
too rapidly to cold water, or not stirring vigorously will water, heat the mixture at 90° for 10 min, and cool it
cause the carrageenan particles to form lumps which are to 60°. Cast films 0.5 mm thick (when dry) on a
very difficult to break down and solubilize. If appropriate, suitable non-stick surface such as Teflon or a plastic
carrageenan disperses more readily in cold water if first petri dish. Alternatively, use films cast on a potassium
moistened with alcohol, glycerol, or saturated solutions of bromide plate, care being taken to avoid moisture.
glucose/sucrose/salt. Alternatively, for improved dispersion, Acceptance criteria
carrageenan powder may be pre-blended with other water All types: The spectrum of the sample exhibits strong,
soluble solids before adding to cold water.] broad absorption bands, typical of all polysaccharides,
in the 1000 cm–1 to 1100 cm–1 region. Maxima are at
IDENTIFICATION 1065 cm–1 and 1020 cm–1 for gelling and non-gelling
• GUM CONSTITUENTS, Thin-Layer Chromatography, Appendix types, respectively.
IIA Kappa-type (see appropriate spectrum below):
Absorbent: Silica Gel G Low ester sulfate absorbance at 1220–1260 cm–1
Standards: Galactose, rhamnose, galacturonic acid, 3,6- Strong 3,6-AG absorbance at 930–935 cm–1
anhydrogalactose, mannose, arabinose, and xylose Strong galactose-4-sulfate absorbance at 840–850
Developing solvent systems cm–1
A: Formic acid, methyl ethyl ketone, tertiary butanol No 3,6-AG-2-sulfate absorbance at 800–805 cm–1
and water (3:6:8:3) Iota-type (see appropriate spectrum below):
B: Glacial acetic acid, chloroform, water (74:65:11) Same as kappa-type except strong ester sulfate
Sample solution: Boil a mixture of 200 mg of sample absorbance at 1220–1260 cm–1 and strong 3,6-AG-2-
and 20 mL of 10% sulfuric acid for 3 h. Allow the sulfate absorbance at 800–805 cm–1
mixture to cool and add excess barium carbonate, Lambda-type (see appropriate spectrum below):
mixing with a magnetic stirrer until the solution reaches Strong ester sulfate absorbance 1220–1260 cm–1
pH 7; filter the solution. Evaporate the filtrate in a Weak to no 3,6AG absorbance at 930–935 cm–1
rotary evaporator at 30 - 50° under vacuum until a Strong galactose-2-sulfate absorbance at 825–830
cm–1
248 / Carrageenan / Monographs FCC 9
Strong galactose-6-sulfate absorbance at 810–820 Analysis: Use any suitable atomic absorption
cm–1 spectrophotometer equipped with a Boling-type
• PREDOMINANT POLYSACCHARIDES burner, an air–acetylene flame, and a hollow-cathode
Sample: 4 g cadmium lamp. Optimize the instrument according to
Analysis: Transfer the Sample to a flask containing 200 the manufacturer’s instructions. Determine the
mL of water and heat the mixture in a water bath at absorbance of each of the Standard solutions, of the
80°, with constant stirring, until dissolved. Replace any Sample solution, and of the Reagent blank at 228.8 nm.
water lost by evaporation and allow the solution to cool Determine the corrected absorbance value for the
to room temperature. [NOTE—The solution becomes Sample solution by subtracting the Reagent blank
viscous and may form a gel.] To 50 mL of the solution absorbance from the Sample solution absorbance.
Monographs
or gel, add 200 mg of potassium chloride; then reheat, Prepare a standard curve by plotting the corrected
mix well, and cool. absorbance of the Standard solutions versus
Acceptance criteria concentration of lead (µg/mL). Calculate the
kappa type: A short-textured “brittle” gel forms. concentration (mg/kg) of cadmium in the Sample using
iota type: A compliant “elastic” gel forms. the following formula:
lambda type: The solution does not gel.
Result = C/W × 50
IMPURITIES
Inorganic Impurities C = the concentration (µg/mL) of cadmium in
• ARSENIC, Arsenic Limit Test, Appendix IIIB the Sample solution determined from the
Sample solution: Prepare as directed for organic standard curve
compounds. W = the weight of Sample taken (g)
Acceptance criteria: NMT 3 mg/kg 50 = the sample dilution factor
• CADMIUM Acceptance criteria: NMT 2 mg/kg
[NOTE—Throughout this test, use distilled, deionized • LEAD
water.] [NOTE—Throughout this test, use distilled, deionized
Standard stock solution: 10 µg/mL cadmium prepared water.]
by diluting a commercially available standard solution Standard stock solution: 100 µg/mL of lead prepared
Standard solutions: 0.05, 0.1, 0.2, 0.4, and 0.6 µg/mL by diluting a commercially available standard solution
of cadmium: from Standard stock solution Standard solutions: 0.1, 0.2, 0.4, 0.8, and 1.6 µg/mL
Sample: 7.5 g [NOTE—The sample should be powdered of lead: from Standard stock solution
and dry.] Sample: 7.5 g [NOTE—The sample should be powdered
Sample solution: [CAUTION—Handle perchloric acid in and dry.]
an appropriate fume hood.] Transfer the Sample to a Sample solution: [CAUTION—Handle perchloric acid in
250-mL Erlenmeyer flask and wet it with 10 mL of an appropriate fume hood.] Transfer the Sample to a
water; add 25 mL of nitric acid. As soon as any initial 250-mL Erlenmeyer flask and wet it with 10 mL of
reaction subsides, heat gently on a hot plate set at water; add 25 mL of nitric acid. As soon as any initial
100–150° for about 1 h or until most of the dark reaction subsides, heat gently on a hot plate set at
fumes that form are evolved. Swirl the flask 100–150° for about 1 h or until most of the dark
occasionally. Cool the flask and add 5 mL of perchloric fumes that form are evolved. Swirl the flask
acid. Salt-like particles are visible at this stage. Resume occasionally. Cool the flask and add 5 mL of perchloric
heating the flask on the hot plate at 100–150° until the acid. Salt-like particles are visible at this stage. Resume
digest is yellow or colorless; this takes about 1 h. Do heating the flask on the hot plate at 100–150° until the
not allow the solution to dry; if necessary add 2–3 mL digest is yellowish or colorless; this takes about 1 h. Do
of nitric acid. Cool the digest and wash the insides of not allow the solution to dry; if necessary add 2–3 mL
the flask with 5 mL of water and swirl the flask. Add 2 of nitric acid. Cool the digest and wash the insides of
mL of hydrochloric acid to complete the digestion. the flask with 5 mL of water and swirl the flask. Add 2
Resume heating the solution on the hot plate at mL of hydrochloric acid to complete the digestion.
100–150° until brown fumes are no longer visible and Resume heating the solution on the hot plate at
the solution is white to yellowish in color. Again, do 100–150° until brown fumes are no longer visible and
not allow the solution to dry; if necessary add 2–3 mL the solution is white to yellowish in color. Again, do
of nitric acid. Cool the solution. It will become slightly not allow the solution to dry; if necessary add 2–3 mL
viscous and salt-like particles will be visible. Add 10 mL of nitric acid. Cool the solution. It will become slightly
of water to the flask, while washing the sides. Transfer viscous and salt-like particles will be visible. Add 10 mL
the viscous solution to a 50-mL volumetric flask, dilute of water to the flask, while washing the sides. Transfer
to volume with water, and mix. Filter the salt-like the viscous solution to a 50-mL volumetric flask, dilute
particles from the solution using two layers of to volume with water, and mix. Filter the salt-like
Whatman no. 5 filter paper (or equivalent). particles from the solution using two layers of
Reagent blank: Use the same quantities of reagents as Whatman no. 5 filter paper (or equivalent).
used to prepare the Sample solution, but omitting the Reagent blank: Use the same quantities of reagents as
Sample. used to prepare the Sample solution, but omitting the
Sample.
Analysis: Use any suitable atomic absorption Erlenmeyer flask and, in an ultrasonic bath, sonicate
spectrophotometer equipped with a lead electrodeless the flask for 10 min or until bubbles no longer form on
discharge lamp (EDL) and an air–acetylene flame. the surface; this indicates that all dissolved gas has
Optimize the instrument according to the been removed.
manufacturer’s instructions. Determine the absorbance Reagent blank: Use the same quantities of reagents as
of each of the Standard solutions, of the Sample used to prepare the Sample solution.
solution, and of the Reagent blank at 283.3 nm. Cold-Vapor Atomic Absorption Method
Determine the corrected absorbance value for the Instrument: Use any suitable atomic absorption
Sample solution by subtracting the Reagent blank spectrophotometer equipped with a hydride vapor
absorbance from the Sample solution absorbance. generator (e.g., Shimadzu Model 6601F or
Monographs
Prepare a standard curve by plotting the corrected equivalent) or atomic vapor assembly. [NOTE—
absorbance of the Standard solutions versus Integral to the hydride generator is a reactor tube or
concentration of lead (µg/mL). Calculate the coil and a peristaltic pump with dual tubing channels:
concentration (mg/kg) of lead in the Sample using the one channel for the Sample solution and one for the
following formula: two Reagent solutions. Flow control is determined by
tubing size and tubing clamps. Flow rates are
Result = C/W × 50 measured at the exit of the hydride generator. The
hydride generator manifold is where the three
C = the concentration (µg/mL) of lead in the
solutions are mixed and pass into the reactor coil to
Sample solution determined from the
generate atomic mercury, which is carried into the
standard curve.
absorbance cell of the instrument.]
W = the weight of Sample taken (g)
Lamp: Mercury at 253.7 nm
50 = the sample dilution factor
Purge gas: Argon
Pump calibration: Calibrate (using water) the
• MERCURY
peristaltic pump so that it will provide a flow rate of
[NOTE—Throughout this test, use distilled, deionized
the Sample solution of 8 mL/min and a combined
water.]
flow rate for the two Reagent solutions of 2 mL/min.
[CAUTION—Handle perchloric acid in an appropriate fume
[NOTE—The combined flow rate is achieved with a
hood.]
single pump setting.]
Reagent solutions
Analysis: Set the spectrophotometer to previously
Sodium borohydride solution: 0.4% solution
established optimum conditions at 253.7 nm. Transfer
prepared by first dissolving 2.5 g of sodium
suitable quantities of the two Reagent solutions into
hydroxide in water and then adding and dissolving
separate graduated cylinders. Insert separate aspirator
2.0 g of sodium borohydride (>98%) followed by
tubing leading from the peristaltic pump into each of
dilution with water to 500 mL. [NOTE—Prepare
the Reagent solutions and into the Sample solution
immediately before use.]
contained in the 100-mL Erlenmeyer flask. Start the
5 M Hydrochloric acid: Dilute 417 mL of hydrochloric
flow of argon gas (tank outlet pressure: 3.2 ± 0.2 kg/
acid to 1 L.
cm2) through the hydride vapor generator of the
Standard stock solution: 1 mg/mL mercury prepared
spectrophotometer. Start the pump to initiate flow of
by diluting a commercially available standard solution.
the three solutions into the hydride generator
Standard solutions: 10, 25, 50, 100, and 200 ng/mL
manifold. Measure the absorbance for the Sample
of mercury, from Standard stock solution
solution. Repeat for the Reagent blank and for each of
Sample: 2 g [NOTE—The sample should be powdered
the Standard solutions.
and dry.]
Determine the corrected absorbance value for the
Sample solution: Transfer the Sample to a 250-mL
Sample solution by subtracting the Reagent blank
Erlenmeyer flask and wet it with 5 mL of water; add 10
absorbance from the Sample solution absorbance.
mL of nitric acid–perchloric acid (1:1) solution. As soon
Prepare a standard curve by plotting the corrected
as any initial reaction subsides, heat gently on a hot
absorbance of the Standard solutions versus
plate set at 100–150° for about 1 h until all of the dark
concentration of mercury (ng/mL). Calculate the
fumes that form are evolved and the solution turns
concentration (mg/kg) of mercury in the Sample using
yellowish or colorless. Swirl the flask occasionally. Salt-
the following formula:
like particles are visible at this stage. Do not allow the
solution to dry. Cool the solution. It will become Result = (C/W) × (50/1000)
slightly viscous and salt-like particles will be visible.
Rinse down the sides of the flask with 5 mL of water C = the concentration (ng/mL) of mercury in the
and allow the solution to stand overnight to facilitate Sample solution determined from the
elimination of dissolved gas. Transfer the viscous standard curve
solution to a 200-mL volumetric flask, dilute to volume W = the weight of Sample taken (g)
with water, and mix. Filter the salt-like particles from 50 = the sample dilution factor
the solution using two layers of Whatman no. 5 filter 1000 = ng/g to mg/kg conversion factor
paper (or equivalent). Transfer the filtrate to a 500-mL Acceptance criteria: NMT 1 mg/kg
Organic Impurities Calculations: For each solvent being analyzed,

• RESIDUAL SOLVENTS determine the calibration factor, C, from the following
Internal standard solution: Add 50.0 mL of water to a equation:
50 mL injection vial and seal. Weigh and inject 15 µL
of 3-methyl-2-pentanone through the septum and C = 50D/(E(F − G))
reweigh to within 0.01 mg.
D = the weight (mg) of solvent in the Standard
Blank: A sample with very low solvent content [NOTE—
solution
Depending on the solvent or solvents used in the
E = the weight (mg) of internal standard in the
purification and recovery of the Sample, more than one
Standard solution
solvent may be present.]
F = the relative peak area for the solvent in the
Monographs
Blank solution: Weigh 0.20 g of the Blank into an

Standard solution
injection vial. Add 5.0 mL of water and 1.0 mL of the
G = the relative peak area for the same solvent in
Internal standard solution. Heat the vial at 60° for 10
the Blank solution
min and shake it vigorously for 10 s.
For each solvent being analyzed, its weight (mg) in the
Standard solution: Weigh 0.20 g of the Blank into an
Sample solution is given by the formula:
injection vial. Add 5.0 mL of water and 1.0 mL of the
Internal standard solution. Weigh the vial to within 0.01 Result = ABC/50
mg. Inject 4 µL each of ethanol, isopropanol, and
methanol through the septum, reweighing the vial A = the relative peak area of the solvent
between the addition of each solvent. Heat the vial at B = the weight (mg) of internal standard
60° for 10 min and shake it vigorously for 10 s. C = the calibration factor for the solvent
Sample: 5 g For each solvent being analyzed, its percentage in the
Sample solution: Disperse 1 mL of a suitable antifoam Sample is given by the formula:
emulsion, such as Dow-Corning G-10 or equivalent, in
200 mL of water contained in a 1000-mL 24/40 round- Result = 0.1 w/W
bottom distilling flask. Add the Sample and shake the w = the weight (mg) of the solvent in the Sample
flask for 1 h on a wrist-action mechanical shaker. solution
Connect the flask to a fractionating column and distill W = the weight (g) of the sample taken
about 100 mL, adjusting the heat so that the foam Acceptance criteria: NMT 0.1% of ethanol,
does not enter the column. Quantitatively transfer the isopropanol, or methanol, singly or in combination
distillate to a 200-mL volumetric flask, fill to the mark
with water, and shake the flask to mix. Weigh 8.0 g of SPECIFIC TESTS
this solution into an injection vial. Add 1.0 mL of the • ASH (Total)
Internal standard solution. Heat the vial at 60° for 10 Sample: 2 g of Sample 1 from the procedure for
min and shake it vigorously for 10 s. determination of Sulfate (below).
Chromatographic system, Appendix IIA Analysis: Transfer the Sample to a previously ignited,
Mode: Head space gas chromatography tared silica or platinum crucible. Heat the Sample with a
Detector: Flame-ionization detector suitable infrared lamp, increasing the intensity
Column: Fused silica, (0.8 m × 0.53 mm id) coated gradually, until the Sample is completely charred;
with DB-wax (1-µm thickness) coupled with fused continue heating for an additional 30 min. Transfer the
silica column, (30 m × 0.53 mm id) coated with DB-1 crucible with the charred Sample into a muffle furnace
(5-µm thickness) and ignite at about 550° for 1 h. Cool in a desiccator
Temperature and weigh. Repeat the ignition in the muffle furnace
Oven: 35° for 5 min; 5°/min to 90°; 6 min at 90° until a constant weight is obtained. If a carbon-free ash
Injection port: 140° is not obtained after the first ignition, moisten the
Detector: 300° charred spot with a 10% solution of ammonium nitrate
Headspace sampling conditions and dry under an infrared lamp. Repeat the ignition
Sample heating temperature: 60° step. Calculate the percentage of total ash of the
Sample heating time: 10 min sample using the formula:
Syringe temperature: 70°
Transfer temperature: 80° Result = W2/W1 × 100%
Carrier gas: Helium
Flow rate: 5 mL/min (208 kPa)
W1 = the weight of Sample (g)
Injection volume: 1.0 mL
W2 = the weight of ash determined (g)
Analysis: Inject equal volumes from the Sample solution,
[NOTE—Retain the ash for the Acid-Insoluble Ash test.]
Blank solution, and Standard solution into the
Acceptance criteria: NLT 15% and NMT 40%, on the
dried basis
determine the peak areas. [NOTE—The approximate
• ACID-INSOLUBLE ASH, Appendix IIC
retention times for ethanol, methanol, isopropanol, and
Sample: Use the ash from the test for Ash (Total)
3-methyl-2-pentanone are 2.81, 2.93, 5.23, and 16.90
Analysis: Ignite to constant weight at 800° ± 25°
min, respectively.]
Acceptance criteria: NMT 1% sulfate, %ES, in the sample taken using the following
• ACID-INSOLUBLE MATTER equation:
Sample: 2 g of Sample 1 obtained from the procedure
for determination of Sulfate (below). %ES = (W2/W1) × 0.4116 × 100%
Analysis: Transfer the Sample into a 250-mL beaker
containing 150 mL of water and 1.5 mL of sulfuric acid
W1 = the weight (g) of Sample 2 taken
TS. Cover the beaker with a watch glass and heat the
W2 = the weight (g) of the ash (barium sulfate)
mixture on a steam bath for 6 h, rubbing down the
Calculate the acid-insoluble matter corrected percentage
wall of the beaker frequently with a rubber-tipped
of ester sulfate, %ESC, in the sample taken using the
stirring rod and replacing any water lost by
following equation:
Monographs
evaporation. Weigh 500 mg of a suitable acid-washed
filter aid, pre-dried at 105° for 1 h, to the nearest 0.1 %ESC = %ES/[1 − (%AIM/100)]
mg, add the filter aid to the sample solution, and filter
it through a tared sintered-glass filter crucible. Wash the
residue several times with hot water, dry the crucible %AIM = the percent Acid-Insoluble Matter
and its contents at 105° for 3 h, cool in a desiccator, (determined above).
and weigh. The difference between the total weight Acceptance criteria: NLT 20% and NMT 40% (as ester
and the weight of the filter aid plus crucible is the sulfate), on the washed, dried, and acid-insoluble
weight of the Acid-Insoluble Matter. Calculate as a matter corrected basis
percentage. • VISCOSITY OF A 1.5% SOLUTION
Acceptance criteria: NMT 15% Sample: 7.5 g of Sample 1 obtained from the procedure
• LOSS ON DRYING, Appendix IIC: 105° to constant weight for determination of Sulfate (above)
Acceptance criteria: NMT 12% Sample solution: Transfer the Sample into a tared, 600-
• PH, pH Determination, Appendix IIB mL tall-form (Berzelius) beaker, and disperse it with
Sample solution: 1:100 suspension agitation for 10 to 20 min in 450 mL of deionized
Acceptance criteria: Between 8 and 11 water. Add sufficient water to bring the final weight to
• SULFATE 500 g and heat in a water bath with continuous
Sample 1: Disperse 15 g of a sample of product in 500 agitation, until a temperature of 80° is reached (20–30
mL of 60% w/w isopropanol/water at room min). Add water to adjust for loss by evaporation, cool
temperature. Stir gently for 4 h. Filter through ash-free to 76–77°, and heat in a constant temperature bath at
filter paper and discard the filtrate. Wash the material 75°.
remaining on the filter paper with two 15-mL portions Analysis: Pre-heat the bob and guard of a Brookfield
of 60% isopropanol/water. Dry the material at 105° to LVF or LVT viscometer to approximately 75° in water.
constant weight. Dry the bob and guard and attach them to the
Sample 2: 1 g of Sample 1 [NOTE—Retain the remainder viscometer, which should be equipped with a No.1
of Sample 1 for determination of Ash (Total), Acid- spindle (19 mm in diameter, approximately 65 mm in
insoluble matter, and Viscosity of a 1.5% solution.] length) and capable of rotating at 30 rpm. Adjust the
Sample solution: Transfer Sample 2 to a 100-mL long- height of the bob in the Sample solution, start the
necked round-bottom flask. Add 50 mL of 0.2 N viscometer rotating at 30 rpm and, after six complete
hydrochloric acid. Fit a condenser, preferably one with revolutions of the viscometer, take the viscometer
at least five condensing bulbs, to the flask and reflux for reading on the 0–100 scale. Record the result in
1 h. Add 25 mL of a 10% (by volume) hydrogen centipoises, obtained by multiplying the reading on the
peroxide solution and resume refluxing for about 5 h or scale by the factor given by the Brookfield
until the solution becomes completely clear. manufacturer. [NOTE—If the viscosity is very low,
Analysis: Transfer the Sample solution to a 600-mL increased precision may be obtained by using the
beaker, bring to a boil, and add dropwise 10 mL of a Brookfield UL (ultra low) adapter or equivalent, in
10% barium chloride solution. Heat the reaction which case the viscometer reading on the 0–100 scale
mixture for 2 h on a boiling water bath. Filter the should be multiplied by 0.2 to obtain the viscosity in
mixture through ash-free slow-filtration filter paper. centipoises. On the other hand, samples of some types
Wash with boiling distilled water until the filtrate is free of carrageenan may be too viscous to read when a No.
from chloride. Dry the filter paper and contents in a 1 spindle is used. Such samples obviously pass the
drying oven. Gently burn and ash the paper at 800° in specification, but if a viscosity reading is desired for
a tared porcelain or silica crucible until the ash is white. other reasons, use a No. 2 spindle and take the reading
Cool in a desiccator. Weigh the cooled crucible on the 0–100 scale or on the 0–500 scale.]
containing the ash. Calculate the percentage ester Acceptance criteria: NLT 5 cP at 75°
Monographs
Kappa-Carrageenan
Monographs
Kappa-PES-Carrageenan
Monographs
Iota-Carrageenan
Monographs
Iota-PES-Carrageenan
Lambda-Carrageenan
256 / Carrot Seed Oil / Monographs FCC 9

Carrot Seed Oil
.

First Published: Prior to FCC 6 those of the spectrum below.
SPECIFIC TESTS
FEMA: 2244 • ACID VALUE (ESSENTIAL OILS AND FLAVORS), Appendix VI
CAS: [8015-88-1]
Acceptance criteria: NMT 5.0
UNII: 595AO13F11 [carrot seed oil] • ANGULAR ROTATION, Optical (Specific) Rotation, Appendix
IIB: Use a 100-mm tube.
DESCRIPTION
Acceptance criteria: Between –4° and –30°
Carrot Seed Oil occurs as a light yellow to amber liquid
Monographs

having a pleasant, aromatic odor. It is the volatile oil
obtained by steam distillation from the crushed seeds of
greater accuracy.]
Daucus carota L. (Fam. Umbelliferae). It is soluble in most
fixed oils, and is soluble, with opalescence, in mineral oil. It
• SAPONIFICATION VALUE, Appendix VI
is practically insoluble in glycerin and in propylene glycol.
Sample: 5 g
Packaging and Storage: Store in a cool place protected
from light in full, tight containers that are made from glass
or aluminum or that are lined with tin.
0.5 mL of 90% alcohol. The solution can become
IDENTIFICATION opalescent upon further dilution up to 10 mL.
• INFRARED SPECTRA, Spectrophotometric Identification Tests, • SPECIFIC GRAVITY: Determine by any reliable method (see
Appendix IIIC General Provisions).
Carrot Seed Oil

FCC 9 Monographs / (−)-Carveol / 257

Carvacrol
.
IDENTIFICATION
Appendix IIIC
ASSAY
• PROCEDURE: Proceed as directed under M-1b, Appendix
Monographs
C10H14O Formula wt 150.22
FEMA: 2245 XI.
Acceptance criteria: NLT 98.0% of C10H14O
UNII: 9B1J4V995Q [carvacrol]
SPECIFIC TESTS
DESCRIPTION • REFRACTIVE INDEX, Appendix II: At 20°
Carvacrol occurs as a colorless to pale yellow liquid.
Odor: Pungent, spicy, thymol
• SPECIFIC GRAVITY: Determine at 25° by any reliable
Solubility: Soluble in alcohol, ether; insoluble or practically
method (see General Provisions).
insoluble in water
Boiling Point: ∼238°
mL of 60% alcohol to give a clear solution.
Carvacrol
(–)-Carveol
.

l-Carveol
p-Mentha-6,8-dien-2-ol C10H16O Formula wt 152.24
FEMA: 2247
UNII: 1L9KXT85R9 [carveol, (-)-]
258 / (−)-Carveol / Monographs FCC 9
DESCRIPTION ASSAY
(–)-Carveol occurs as a colorless to pale yellow liquid. • PROCEDURE: Proceed as directed under M-1b, Appendix
Odor: Spearminty XI.
Solubility: Soluble in propylene glycol, vegetable oils; Acceptance criteria: NLT 96.0% of C10H16O
insoluble or practically insoluble in water
Boiling Point: ∼226° to 227° (751 mm Hg) SPECIFIC TESTS
Solubility in Alcohol, Appendix VI: One mL dissolves in 1 • REFRACTIVE INDEX, Appendix II: At 20°
mL of 95% alcohol. Acceptance criteria: Between 1.493 and 1.497
Function: Flavoring agent • SPECIFIC GRAVITY: Determine at 25° by any reliable
Monographs
IDENTIFICATION Acceptance criteria: Between 0.947 and 0.953

Appendix IIIC OTHER REQUIREMENTS
Acceptance criteria: The spectrum of the sample • ANGULAR ROTATION, Optical (Specific) Rotation, Appendix
exhibits relative maxima at the same wavelengths as IIB
those of the spectrum below. Acceptance criteria: Between −117° and −130°
(–)-Carveol
UNII: 4RWC1CMS3X [carvone, (+)-]

(+)-Carvone
.

DESCRIPTION
(+)-Carvone occurs as a colorless to light yellow liquid.
Last Revision: Second Supplement, FCC 8
Odor: Caraway
Solubility: Soluble in propylene glycol, most fixed oils;
d-Carvone miscible in alcohol; insoluble or practically insoluble in
dextro-Carvone glycerin
d-1-Methyl-4-isopropenyl-6-cyclohexen-2-one Boiling Point: ∼230°
mL of 60% alcohol.
IDENTIFICATION
Appendix IIIC
C10H14O Formula wt 150.22
FEMA: 2249
FCC 9 Monographs / (−)-Carvone / 259
Acceptance criteria: The spectrum of the sample • SPECIFIC GRAVITY: Determine at 25° by any reliable
exhibits relative maxima at the same wavelengths as method (see General Provisions).
those of the spectrum below. Acceptance criteria: Between 0.956 and 0.961
ASSAY OTHER REQUIREMENTS

• PROCEDURE: Proceed as directed under M-1b, Appendix • ANGULAR ROTATION, Optical (Specific) Rotation, Appendix
XI. IIB: Use a 100-mm tube.
Acceptance criteria: NLT 95.0% of C10H14O Acceptance criteria: Between +50° and +60°
SPECIFIC TESTS
• REFRACTIVE INDEX, Appendix IIB: At 20°
Monographs
(+)-Carvone
DESCRIPTION
(–)-Carvone
.
(−)-Carvone occurs as a colorless to pale strawberry colored

First Published: Prior to FCC 6 liquid.
Last Revision: Second Supplement, FCC 8 Odor: Spearminty
Solubility: Soluble in propylene glycol, most fixed oils;
l-Carvone miscible in alcohol; insoluble or practically insoluble in
levo-Carvone glycerin
l-1-Methyl-4-isopropenyl-6-cyclohexen-2-one Boiling Point: ∼231°
mL of 70% alcohol.
IDENTIFICATION
Appendix IIIC
C10H14O Formula wt 150.22 Acceptance criteria: The spectrum of the sample
FEMA: 2249 exhibits relative maxima at the same wavelengths as
UNII: 5TO7X34D3D [carvone, (-)-] those of the spectrum below.
260 / (−)-Carvone / Monographs FCC 9
ASSAY • SPECIFIC GRAVITY: Determine at 25° by any reliable

• PROCEDURE: Proceed as directed under M-1b, Appendix method (see General Provisions).
XI. Acceptance criteria: Between 0.956 and 0.961
Acceptance criteria: NLT 97.0% of C10H14O
OTHER REQUIREMENTS
SPECIFIC TESTS • ANGULAR ROTATION, Optical (Specific) Rotation, Appendix
• REFRACTIVE INDEX, Appendix IIB: At 20° IIB: Use a 100-mm tube.
Acceptance criteria: Between 1.496 and 1.502 Acceptance criteria: Between −57° and −62°
Monographs
(−)-Carvone
IDENTIFICATION
(–)-Carvyl Acetate
.

First Published: Prior to FCC 6 Appendix IIIC
Last Revision: First Supplement, FCC 7 Acceptance criteria: The spectrum of the sample
l-Carvyl Acetate those of the spectrum below.
p-Mentha-6,8-dien-2-yl Acetate ASSAY
• PROCEDURE: Proceed as directed under M-1b, Appendix
XI.
Acceptance criteria: NLT 98.0% of C12H18O2
SPECIFIC TESTS
• ACID VALUE, FLAVOR CHEMICALS (OTHER THAN ESSENTIAL
OILS), M-15, Appendix XI
C12H18O2 Formula wt 194.27
Acceptance criteria: NMT 1.0
FEMA: 2250
• REFRACTIVE INDEX, Appendix II: At 20°
UNII: IJM42H65AV [carvyl acetate, (-)-] Acceptance criteria: Between 1.473 and 1.479
• SPECIFIC GRAVITY: Determine at 25° by any reliable
DESCRIPTION
(–)-Carvyl Acetate occurs as a colorless to pale yellow liquid.
Odor: Spearminty
Solubility: Soluble in alcohol OTHER REQUIREMENTS
Boiling Point: ∼77° to 79° (0.1 mm Hg) • ANGULAR ROTATION, Optical (Specific) Rotation, Appendix
Function: Flavoring agent IIB
FCC 9 Monographs / β-Caryophyllene / 261
Monographs
(–)-Carvyl Acetate

β-Caryophyllene
.
IDENTIFICATION
Appendix IIIC
ASSAY
• PROCEDURE: Proceed as directed under M-1a, Appendix
XI.
C15H24 Formula wt 204.36 Acceptance criteria: NLT 80.0% of C15H24
FEMA: 2252
UNII: BHW853AU9H [caryophyllene] SPECIFIC TESTS
• REFRACTIVE INDEX, Appendix II: At 20°
DESCRIPTION Acceptance criteria: Between 1.498 and 1.504
β-Caryophyllene occurs as a colorless to slightly yellow, oily • SPECIFIC GRAVITY: Determine at 25° by any reliable
liquid. It may contain a suitable antioxidant. method (see General Provisions).
Odor: Woody, spicy Acceptance criteria: Between 0.897 and 0.910
Solubility: Soluble in alcohol, ether; insoluble or practically
insoluble in water
OTHER REQUIREMENTS
Boiling Point: ∼256°
mL of 95% alcohol to give a clear solution.
262 / β-Caryophyllene / Monographs FCC 9
• PHENOLS, M-1b, Appendix XI

Monographs
β-Caryophyllene

Cascarilla Oil
.

First Published: Prior to FCC 6 Acceptance criteria: Between −1° and +8°
• ESTER VALUE AFTER ACETYLATION, Total Alcohols, Appendix
VI
Sweetwood Bark Oil
CAS: [8007-06-5] Sample: 2 g of the dried, acetylated sample oil
UNII: N81EA2A6NP [cascarilla oil] Analysis: Calculate the Ester Value after Acetylation by
the formula:
DESCRIPTION
Result = A × 28.05 / B
Cascarilla Oil occurs as a light yellow to brown amber liquid
with a pleasant, spicy odor. It is the volatile oil obtained by
steam distillation of the dried bark of Croton cascarilla A = amount (mL) of 0.5 N alcoholic potassium
Benn. and of Croton eluteria Benn. (Fam. Euphorbiaceae). It hydroxide consumed in the saponification
is soluble in most fixed oils and in mineral oil, but it is B = weight (g) of acetylated sample oil taken
practically insoluble in glycerin and in propylene glycol. Acceptance criteria: Between 62 and 88
Function: Flavoring agent • REFRACTIVE INDEX, Appendix IIB
Packaging and Storage: Store in a cool place protected [NOTE—Use an Abbé or other refractometer of equal or
from light in full, tight containers that are made from steel greater accuracy.]
or aluminum and that are suitably lined. Acceptance criteria: Between 1.488 and 1.494 at 20°
• SAPONIFICATION VALUE, Appendix VI
IDENTIFICATION
Sample: 5 g
Appendix IIIC
0.5 mL of 90% alcohol and remains in solution on
dilution to 10 mL.
SPECIFIC TESTS • SPECIFIC GRAVITY: Determine by any reliable method (see
• ACID VALUE (ESSENTIAL OILS AND FLAVORS), Appendix VI General Provisions).
Acceptance criteria: Between 3 and 10 Acceptance criteria: Between 0.892 and 0.914
FCC 9 Monographs / Casein and Caseinate Salts / 263
Monographs
Cascarilla Oil
ASSAY
Casein and Caseinate Salts
.
• PROTEIN, Nitrogen Determination, Appendix IIIC

First Published: Prior to FCC 6 Analysis: Calculate the percent protein (P) by the
equation:
CAS: [9000-71-9]
P = N × 6.38
UNII: 48268V50D5 [casein]
DESCRIPTION N = percent nitrogen

Casein occurs as an off-white to cream-colored, granular or Acceptance criteria
fine powder. It is derived from the coagulum formed by Acid casein: NLT 90.0% protein, calculated on the
treating skim milk with a food-grade acid (Acid Casein), dried basis
enzyme (Rennet Casein), or other food-grade precipitating Rennet casein: NLT 86.0% protein, calculated on the
agent. After the precipitation, Casein is separated from the dried basis
soluble milk fraction, washed, and dried. Chemically, Caseinate salts: NLT 84.0% protein, calculated on the
Casein is a mixture of at least 20 electrophoretically distinct dried basis
phosphoproteins. The main fractions—designated α-casein,
β-casein, and κ-casein—are known to be mixtures, rather IMPURITIES
than single proteins. Casein contains all the amino acids Inorganic Impurities
known to be essential for human nutrition. It is insoluble in • LEAD, Lead Limit Test, Flame Atomic Absorption
water and alcohol, but it can be dissolved by aqueous Spectrophotometric Method, Appendix IIIB
alkalies to form Caseinate Salts. Sample: 10 g
Caseinate Salts occur as white to cream-colored granules or Acceptance criteria: NMT 1 mg/kg
powders. They are soluble or dispersible in water. They are
prepared by treatment of Casein with food-grade alkalies, SPECIFIC TESTS
neutralizing agents, enzymes, buffers, or sequestrants. • FAT
Common counter-ions are NH4+, Ca++, Mg++, K+, and Na+. Sample: 1 g
Function: Binder; extender; clarifying agent; emulsifier; Analysis: Transfer the Sample to a fat-extraction flask,
stabilizer add 10 mL of water, and shake until homogeneous
Packaging and Storage: Store in well-closed containers. (warm if necessary). Add approximately 1 mL of
ammonium hydroxide and heat in a water bath for 15
min at 60° to 70°, shaking occasionally. Add 10 mL of
alcohol, and mix well. Add 25 mL of peroxide-free
264 / Casein and Caseinate Salts / Monographs FCC 9
ether, stopper, and shake vigorously for 1 min. Allow to Standard solutions: Into each of five test tubes add, in
cool if necessary, add 25 mL of petroleum ether and sequence, 2 mL of water and, respectively, 3 mL of
shake vigorously. Allow the layers to separate and each of the Standard stock solutions. To each tube, add
clarify, or centrifuge to expedite the process. Decant 0.2 mL of Phenol reagent, and mix well. Add 5 mL of
the organic layer into a suitable flask or dish, and sulfuric acid to each tube using an automatic dispenser
repeat the extraction twice with 15 mL each of ether or by other means that permit mixing within 1 to 2 s.
and petroleum ether for each extraction. Evaporate the Ensure that the solutions have been thoroughly mixed,
combined ether extractions on a steam bath, and dry and allow them to stand for 15 min, then cool to 20°
the residue to a constant weight at 102°, or 70° to 75° in a water bath for 5 min.
at less than 50 mm Hg. Calculate the percent of fat in Analysis: Using a suitable spectrophotometer, determine
Monographs
the sample taken with the equation: the absorbance of each of the Standard solutions in a 1-
cm pathlength cell at 490 nm against the water blank.
%Fat = R/S × 100% Calculate the slope of the curve obtained by plotting
absorbances versus µg/mL of lactose. The slope of the
resulting curve is the absorptivity (a) of the lactose-
R = weight (g) of the residue
reagent product.
S = weight (g) of the sample taken
Determine the absorbance of the Sample solution against
a blank prepared using identical reagents. Calculate the
• FREE ACID
percent lactose (L) in the sample by the equation:
[NOTE—This test is required only for casein.]
Sample preparation: Transfer 10 g of finely-ground L = (A × F) / (a × m)
sample into a 500-mL conical flask. Add 200 mL of
freshly boiled water maintained at 60°, swirl, and
stopper. Place the flask in an 80° water bath for 30 A = absorbance of the Sample solution at 490
min, shaking at 10-min intervals. Cool to room nm
temperature, and filter. F = dilution factor and conversion to percent
Analysis: Transfer a 100.0-mL portion of the clear filtrate from µg/mL, 0.00475
from the Sample preparation into a 250-mL conical flask, a = absorptivity of the lactose-reagent product
add 0.5 mL of phenolphthalein TS, and titrate with 0.1 calculated above
N sodium hydroxide to a pink endpoint that persists for m = weight of the sample (g) taken
30 s. Acceptance criteria: NMT 2.0%
Acceptance criteria • LOSS ON DRYING, Appendix IIC: 102° for 3 h
Casein: NMT 2.7 mL of 0.1 N sodium hydroxide is Acceptance criteria: NMT 12.0%
consumed
• LACTOSE
Phenol reagent: Heat a mixture of 8 g of phenol and
Cassia Oil
.
2 g of water until the crystals dissolve.

Sample solution: Transfer 1 g of sample into a 150-mL
beaker. If the sample is Acid Casein, add 0.10 g of
Last Revision: FCC 8
sodium hydrogen carbonate to the beaker. If the
sample is Rennet Casein, add 0.10 g of sodium
tripolyphosphate. Add 25 mL of water, and dissolve the Cinnamon Oil
sample by gently swirling while warming to 60° to 70° FEMA: 2258
CAS: [8007-80-5]
on a hot plate. Cool the solution to ambient
UNII: A4WO0626T5 [chinese cinnamon oil]
temperature, and add 15 mL of water, 8 mL of 0.1 N
hydrochloric acid, and 1 mL of a 10% solution of acetic DESCRIPTION
acid. Mix well by swirling and, after 5 min, add 1 mL of Cassia Oil occurs as a yellow or brown liquid having the
1 M sodium acetate, and mix well. characteristic odor and taste of cassia cinnamon. It is the
After the precipitate has settled, filter and discard the volatile oil obtained by steam distillation from the leaves
first 5 mL of filtrate. Pipet 2 mL of the remaining filtrate and twigs of Cinnamomum cassia Blume (Fam. Lauraceae),
into a test tube, add 0.2 mL of Phenol reagent, and mix rectified by distillation, and consisting mainly of cinnamic
well. Add 5 mL of sulfuric acid using an automatic aldehyde. Upon aging or exposure to air it darkens and
dispenser or by other means that permit mixing within thickens. It is soluble in glacial acetic acid and in alcohol.
1 to 2 s. Ensure that the solution has been thoroughly Function: Flavoring agent
mixed, and allow it to stand for 15 min, then cool to Packaging and Storage: Store in full, tight, light-resistant
20° in a water bath for 5 min. containers. Avoid exposure to excessive heat.
Lactose solution: 2 mg/mL lactose monohydrate
Standard stock solutions: Transfer, respectively, 1, 2, 3, IDENTIFICATION
and 4 mL of Lactose solution into four separate 500-mL • INFRARED SPECTRA, Spectrophotometric Identification Tests,
volumetric flasks and dilute to volume with water. Appendix IIIC
These dilutions contain 20, 40, 60, and 80
µg/mL of lactose, respectively.
FCC 9 Monographs / Castor Oil / 265
Acceptance criteria: The spectrum of the sample Acceptance criteria: 1.602–1.614 at 20°
exhibits relative maxima at the same wavelengths as • ROSIN OR ROSIN OILS
those of the spectrum below. Sample: 2 mL
Analysis: Shake the Sample in a test tube with 5–10 mL
ASSAY of solvent hexane, and allow the liquids to separate.
• ALDEHYDES, Aldehydes and Ketones, Neutral Sulfite Method, Decant the hexane layer, which is just slightly colored,
Appendix VI into another test tube, shake it with an equal volume of
Acceptance criteria: NLT 80.0%, by volume, of total 1:1000 cupric acetate solution, and allow the phases to
aldehydes separate.
Acceptance criteria: The hexane layer does not turn
SPECIFIC TESTS
Monographs
green.
Acceptance criteria: One mL of sample dissolves in 2
Acceptance criteria: Between −1° and +1°
mL of 70% alcohol.
• CHLORINATED COMPOUNDS, Appendix VI
• SPECIFIC GRAVITY: Determine by any reliable method (see
Acceptance criteria: Passes test
General Provisions).
Acceptance criteria: 1.045–1.063
greater accuracy.]
Cassia Oil
soluble in alcohol, and is miscible with absolute alcohol,

Castor Oil
.
with glacial acetic acid, with chloroform, and with ether.

First Published: Prior to FCC 6 Function: Antisticking agent; release agent; component of
protective coatings
Packaging and Storage: Store in tight containers, and
Ricinus Oil
INS: 1503 CAS: [8001-79-4] avoid exposure to excessive heat.
UNII: D5340Y2I9G [castor oil] IDENTIFICATION
• A. PROCEDURE
DESCRIPTION Acceptance criteria: A sample is only partly soluble in
Castor Oil occurs as a pale yellow or almost colorless,
solvent hexane (distinction from most other fixed oils),
transparent, viscous liquid. It is the fixed oil obtained from
but it yields a clear liquid with an equal volume of
the seed of Ricinus communis L. (Fam. Euphorbiaceae) and
alcohol (foreign fixed oils).
consists mainly of the triglyceride of ricinoleic acid. It is
266 / Castor Oil / Monographs FCC 9
• B. FATTY ACID COMPOSITION, Appendix VII DESCRIPTION

Acceptance criteria: Castor oil exhibits the following Cedar Leaf Oil occurs as a colorless to yellow liquid having a
composition profile of fatty acids: strong camphoraceous and sage odor. It is the volatile oil
obtained by steam distillation from the fresh leaves and
Fatty Acid Weight % (Range) branch ends of the eastern arborvitae, Thuja occidentalis L.
16:0 0.9-1.6 (Fam. Cupressaceae). It is soluble in most fixed oils, in
18:0 1.0-1.8 mineral oil, and in propylene glycol. It is practically
18:1 3.7-6.7
insoluble in glycerin.
18:3 0.2-0.6
Monographs
18:0 di-OH 0.4-1.3 from light in full, tight containers that are made from glass
18:1 -OH 83.6-89.0 or that are lined with tin.
20:0 0.2-0.5
IDENTIFICATION
IMPURITIES Appendix IIIC
Inorganic Impurities Acceptance criteria: The spectrum of the sample
• LEAD, Lead Limit Test, Atomic Absorption Spectrophotometric exhibits relative maxima at the same wavelengths as
Graphite Furnace Method II, Appendix IIIB those of the spectrum below.
Acceptance criteria: NMT 0.1 mg/kg
ASSAY
SPECIFIC TESTS • KETONES, Aldehydes and Ketones, Hydroxylamine Method,
• FREE FATTY ACIDS Appendix VI
Sample: 10 g Sample: 1 g
Analysis: Neutralize a mixture of alcohol and ether Analysis: Use 76.10 as the equivalence factor (e) in the
(50:50) to phenolphthalein with 0.1 N sodium calculation.
hydroxide. Add 50 mL of this mixture to a flask and Acceptance criteria: NLT 60.0% of ketones, calculated
dissolve the Sample in the mixture. Add 1 mL of as thujone (C10H16O)
phenolphthalein TS, and titrate with 0.1 N sodium
hydroxide until the solution remains pink after shaking SPECIFIC TESTS
the flask for 30 s. • ANGULAR ROTATION, Optical (Specific) Rotation Appendix
Acceptance criteria: Not more than 7 mL of 0.1 N IIB: Use a 100-mm tube.
sodium hydroxide is required for a 10.0-g sample. Acceptance criteria: Between −7° and −14°
greater accuracy.]
Cedar Leaf Oil
.

First Published: Prior to FCC 6 Acceptance criteria: One mL of the sample dissolves in
3 mL of 70% alcohol, occasionally becoming cloudy on
Thuja Oil dilution to 10 mL.
White Cedar Leaf Oil • SPECIFIC GRAVITY: Determine by any reliable method (see
FEMA: 2267 General Provisions).
CAS: [8007-20-3] Acceptance criteria: Between 0.906 and 0.916
UNII: BJ169U4NLG [cedar leaf oil]
FCC 9 Monographs / Celery Seed Oil / 267
Monographs
Cedar Leaf Oil
SPECIFIC TESTS
Celery Seed Oil
.
• ACID VALUE (ESSENTIAL OILS AND FLAVORS), Appendix VI

First Published: Prior to FCC 6 Acceptance criteria: NMT 4.5
UNII: 8MBL58728K [celery seed oil]
Acceptance criteria: Between +48° and +78°
DESCRIPTION • REFRACTIVE INDEX, Appendix IIB
Celery Seed Oil occurs as a yellow to green-brown liquid [NOTE—Use an Abbé or other refractometer of equal or
with a pleasant, aromatic odor. It is the volatile oil greater accuracy.]
obtained by steam distillation of the fruit or seed of Apium Acceptance criteria: Between 1.480 and 1.490 at 20°
graveolens L. It is soluble in most fixed oils with the • SAPONIFICATION VALUE, Appendix VI
formation of a flocculent precipitate, and in mineral oil Sample: 5 g
with turbidity. It is partly soluble in propylene glycol, but it Acceptance criteria: Between 25 and 65
is insoluble in glycerin. • SOLUBILITY IN ALCOHOL, Appendix VI
Function: Flavoring agent Acceptance criteria: One mL of sample dissolves in 8
Packaging and Storage: Store in a cool place protected mL of 90% alcohol, usually with turbidity.
from light in full, tight containers that are made from steel • SPECIFIC GRAVITY: Determine by any reliable method (see
or aluminum and that are suitably lined. General Provisions).
IDENTIFICATION
Appendix IIIC
268 / Celery Seed Oil / Monographs FCC 9
Monographs
Celery Seed Oil
clover-shaped jar design. The jar and blades meet the

Cellulose Gel
.
following specifications: the jar has a 7.0-cm id at the

First Published: Prior to FCC 6 bottom and a 9.2-cm id at the top and an overall
height of 21.9 cm, and the four blades are arranged so
that two of the blades are pointed up and two are
Cellulose, Microcrystalline
INS: 460 CAS: [9004-34-6] pointed down.
Analysis: Mix the Sample preparation for 5 min with the
UNII: OP1R32D61U [cellulose, microcrystalline]
Blender setup. Transfer 100 mL of the dispersion into a
DESCRIPTION 100-mL graduated cylinder, and allow it to stand for 3
Cellulose Gel occurs as a fine, white or almost white h.
powder. It is purified, partially depolymerized cellulose Acceptance criteria: A white, opaque, bubble-free
prepared by treating alpha-cellulose, obtained as a pulp dispersion that does not form a supernatant liquid at
from fibrous plant material, with mineral acids. It consists the surface is obtained. [NOTE—Save this dispersion for
of free-flowing, nonfibrous particles that may be use in Identification test B (below).]
compressed into self-binding tablets that disintegrate • B. PROCEDURE
rapidly in water. It is insoluble in water, in dilute acids, in Sample: Use the dispersion obtained from Identification
dilute sodium hydroxide solutions, and in most organic test A (above).
solvents. Analysis: Add a few drops of iodine TS to a 20-mL
Function: Anticaking agent; binding agent; dispersing aliquot of the Sample and mix.
agent Acceptance criteria: No purple to blue or blue color
Packaging and Storage: Store in well-closed containers. appears.
IDENTIFICATION ASSAY
• A. PROCEDURE • PROCEDURE
Sample preparation: Sift 20 g of sample for 5 min on Sample: 125 mg
an air-jet sieve equipped with a screen having 38-µm Analysis: With the aid of about 25 mL of water, transfer
openings. If more than 5% is retained on the screen, the Sample into a 300-mL Erlenmeyer flask. Add 50.0
mix 30 g of sample with 270 mL of water; otherwise, mL of 0.5 N potassium dichromate, mix, then carefully
mix 45 g of sample with 255 mL of water. add 100 mL of sulfuric acid, and heat to boiling.
Blender setup: Use a single-speed, high-speed Remove the mixture from the heat, allow it to stand at
(equal to or greater than 18,000 rpm) power blender room temperature for 15 min, cool it in a water bath,
(Waring Blender, Model 700G, or equivalent) that has a and transfer it into a 250-mL volumetric flask. Dilute
almost to volume with water, cool to 25°, then dilute
FCC 9 Monographs / Cellulose Gum / 269
to volume with water, and mix. Titrate a 50.0-mL DESCRIPTION

aliquot with 0.1 N ferrous ammonium sulfate, using 2 Cellulose Gum occurs as a white to cream colored powder
or 3 drops of orthophenanthroline TS as the indicator, or as granules. The powder is hygroscopic. It readily
and record the volume required (S), in mL. Perform a disperses in water to form colloidal solutions. It is insoluble
blank determination (see General Provisions), and record in most solvents. A 1:100 aqueous suspension has a pH
the volume of 0.1 N ferrous ammonium sulfate required between 6.5 and 8.5.
(B), in mL. Calculate the percent cellulose in the sample Function: Thickener; stabilizer
by the formula: Packaging and Storage: Store in well-closed containers.
Result = (B − S) × 338/W IDENTIFICATION
Monographs
• A. PROCEDURE
Sample: 1 g, powdered
W = weight of sample (mg), on the dried basis Sample dispersion: Add the Sample to 50 mL of warm
Acceptance criteria: NLT 97.0% and NMT 102.0% of water while stirring to produce a uniform dispersion.
carbohydrate, calculated as cellulose on the dried basis Continue stirring until a colloidal solution is produced,
IMPURITIES then cool to room temperature.
Inorganic Impurities Analysis: Add 10 mL of cupric sulfate TS to 10 mL of
• LEAD, Lead Limit Test, Flame Atomic Absorption Sample dispersion. [NOTE—Save the remainder of the
Spectrophotometric Method, Appendix IIIB Sample dispersion for Identification test B (below).]
Sample: 10 g Acceptance criteria: A fluffy, blue-white precipitate
Acceptance criteria: NMT 2 mg/kg forms.
• B. SODIUM, Appendix IIIA
SPECIFIC TESTS Sample: Use a portion of the Sample dispersion prepared
• LOSS ON DRYING, Appendix IIC: 105° to constant weight in Identification test A (above).
Acceptance criteria: NMT 7.0% Acceptance criteria: Passes tests
• PH, pH Determination, Appendix IIB
Sample mixture: Shake 5 g of sample with 40 mL of ASSAY
water for 20 min and centrifuge. • PROCEDURE
Analysis: Determine the pH of the supernatant liquid Calculation: Calculate the Percent Cellulose Gum by
from the Sample mixture. subtracting from 100 the Percent Sodium Chloride and
Acceptance criteria: Between 5.0 and 7.5 Percent Sodium Glycolate determined using the
• RESIDUE ON IGNITION (SULFATED ASH), Appendix IIC procedures below.
Sample: 2 g Acceptance criteria: NLT 99.5% and NMT 100.5% of
Acceptance criteria: NMT 0.05% cellulose gum, calculated on the dried basis
• WATER-SOLUBLE SUBSTANCES • PERCENT SODIUM CHLORIDE
Sample mixture: 5 g of sample with 80 mL of water Sample: 5 g
Analysis: Shake the Sample mixture for 10 min. Filter the Analysis: Transfer the Sample into a 250-mL beaker, add
mixture through Whatman No. 42, or equivalent, filter 50 mL of water and 5 mL of 30% hydrogen peroxide,
paper into a tared beaker, evaporate the filtrate to and heat on a steam bath for 20 min, stirring
dryness on a steam bath, dry at 105° for occasionally to ensure complete dissolution. Cool, add
1 h, cool, and weigh. 100 mL of water and 10 mL of nitric acid, and titrate
Acceptance criteria: NMT 0.24% with 0.05 N silver nitrate to a potentiometric endpoint,
using a silver/calomel (AgCl) electrode set, and stirring
constantly. Calculate the percent sodium chloride in the
sample by the formula:
Cellulose Gum
.
Result = (V × N × FE)/[(100 − b) × W]
V = volume of the silver nitrate (mL)
N = normality of the silver nitrate
Sodium Carboxymethylcellulose
FE = equivalence factor for sodium chloride,
CMC
584.4
Modified Cellulose
b = percent Loss on Drying (determined
separately, below)
• PERCENT SODIUM GLYCOLATE
Standard stock solution: 1 mg/mL of glycolic acid
(using glycolic acid previously dried in a desiccator at
room temperature overnight) [NOTE—Use this solution
INS: 466 CAS: [9004-32-4] within 30 days.]
UNII: K679OBS311 [carboxymethylcellulose sodium] Standard solutions: Transfer 1.0, 2.0, 3.0, and 4.0 mL,
respectively, of the Standard solution into separate 100-
270 / Cellulose Gum / Monographs FCC 9
mL volumetric flasks, add sufficient water to each flask Acceptance criteria: NMT 3 mg/kg
to make 5 mL, then add 5 mL of glacial acetic acid,
and dilute to volume with acetone. SPECIFIC TESTS
Sample solution: Transfer 500 mg of sample into a 100- • DEGREE OF SUBSTITUTION
mL beaker, moisten thoroughly with 5 mL glacial acetic Electrode system: Use a standard glass electrode and a
acid followed by 5 mL of water, and stir with a glass calomel electrode modified as follows: Discard the
rod until dissolution is complete (usually about 15 min). aqueous potassium chloride solution contained in the
While stirring, slowly add 50 mL of acetone, then add electrode, rinse and fill with the supernatant liquid
1 g of sodium chloride, and stir for several minutes to obtained by shaking thoroughly 2 g each of potassium
ensure complete precipitation of the cellulose gum. chloride and silver chloride (or silver oxide) with 100
Monographs
Filter through a soft, open-textured paper, previously mL of methanol, then add a few crystals of potassium
wetted with a small amount of acetone, and collect the chloride and silver chloride (or silver oxide) to the
filtrate in a 100-mL volumetric flask. Use an additional electrode.
30 mL of acetone to facilitate transfer of the solids and Sample: 200 mg, previously dried
to wash the filter cake, then dilute to volume with Analysis: Add 75 mL of glacial acetic acid to the Sample
acetone, and mix. contained in a 250-mL glass stoppered Erlenmeyer flask,
Blank solution: 5% each of glacial acetic acid and water connect the flask with a water-cooled condenser, and
in acetone reflux gently on a hot plate for 2 h. Cool, transfer the
Analysis: Transfer 2.0 mL of the Sample solution, 2.0 mL solution to a 250-mL beaker with the aid of 50 mL of
of each of the Standard solutions, and 2.0 mL of the glacial acetic acid, and titrate with 0.1 N perchloric acid
Blank solution into separate 25-mL volumetric flasks. in dioxane while stirring with a magnetic stirrer.
Place the uncovered flasks in a boiling water bath for [CAUTION—Handle perchloric acid in an appropriate
exactly 20 min to remove the acetone, remove from fume hood.] Determine the endpoint potentiometrically
the bath, and cool. Add to each flask 5.0 mL of 2,7- with a pH meter equipped with the Electrode system
dihydroxynaphthalene TS, mix thoroughly, add an described. Record the mL of 0.1 N perchloric acid
additional 15 mL, and again mix thoroughly. Cover the versus mV (0- to 700-mV range), and continue the
mouth of each flask with a small piece of aluminum titration to a few mL beyond the endpoint. Plot the
foil. Place the flasks upright in a boiling water bath for titration curve and read the volume (A), in mL, of 0.1 N
20 min, then remove from the bath, cool, dilute to perchloric acid at the inflection point. Calculate the
volume with sulfuric acid, and mix. Using a suitable degree of substitution by the formula:
spectrophotometer, determine the absorbance of the
Result = [F1V/W]/[1.000 − (F2V/W)]
solutions prepared from the Sample solution and from
the Standard solutions at 540 nm against the solution
prepared from the Blank solution. Prepare a standard F1 = 1/10th of the molecular weight of 1
curve using the absorbance obtained from each of the anhydroglucose unit, 16.2
Standard solutions. From the standard curve and the V = volume of 0.1 N perchloric acid (mL)
absorbance of the Sample solution, determine the W = weight of the sample taken (mg)
weight (w), in mg, of glycolic acid in the sample taken, F2 = 1/10th of the molecular weight of 1 sodium
and calculate the percent sodium glycolate in the carboxymethyl group, 8.0
sample by the formula: Acceptance criteria: NLT 0.2 and NMT 1.50
carboxymethyl groups (–CH2COOH) per
Result = (w × F)/[(100 − b) × W]
anhydroglucose unit on the dried basis
• LOSS ON DRYING, Appendix IIC: 105° to constant weight
w = weight of glycolic acid in the Sample, as Acceptance criteria: NMT 10.0%
determined from the standard curve • SODIUM
F = factor converting glycolic acid to sodium Analysis: From the weight of the sample taken and the
glycolate, 12.9 number of mL of 0.1 N perchloric acid consumed in
b = percent Loss on Drying, determined the determination of Degree of Substitution (above),
separately (below) calculate the percent sodium. Each mL of 0.1 N
W = weight of the sample taken (g) perchloric acid is equivalent to 2.299 mg of sodium
(Na).
IMPURITIES Acceptance criteria: NMT 12.4%, on the dried basis
Inorganic Impurities • VISCOSITY DETERMINATION, Viscosity of Cellulose Gum,
• LEAD, Lead Limit Test, Appendix IIIB Appendix IIB
Sample solution: Prepare as directed for organic Sample solution: 2% (w/w)
compounds using 2 g of sample. Acceptance criteria: NLT 25 cP
Solution)
FCC 9 Monographs / Cellulose, Powdered / 271
Acceptance criteria: The solution turns blue-green

Cellulose, Powdered
.
within 5 min.
First Published: Prior to FCC 6 • D. MICROSCOPY
Sample: Use the mixture obtained from Identification
test A.
Analysis: Place a few drops of the stirred Sample on a
microscope slide, and insert a cover glass. Observe at
100 magnifications with a microscope.
Acceptance criteria: Fibers and fiber fragments are
visible, regardless of the degree of fineness of the
Monographs
sample.
INS: 460(ii) CAS: [9004-34-6] • E. PROCEDURE
UNII: SMD1X3XO9M [powdered cellulose] Sample: Use the mixture obtained from Identification
test A.
DESCRIPTION Analysis: Dilute 10 mL of the stirred Sample to 1000 mL
Cellulose, Powdered, occurs as a white substance and with water, and filter 125 mL of the dilution through a
consists of fibrous particles that may be compressed into Büchner funnel using Whatman No. 4 filter paper, or
self-binding tablets that disintegrate rapidly in water. It equivalent. Rinse the pad with 25 mL of acetone, and
exists in various grades, exhibiting degrees of fineness dry (paper included) at 105°. Transfer the powder to a
ranging from a dense, free-flowing powder to a coarse, tared weighing bottle, weigh, then transfer to a 50-mL
fluffy, nonflowing material. It is purified, mechanically Erlenmeyer flask, and seal with a rubber stopper.
disintegrated cellulose prepared by processing bleached Record the weight of the sample (w), in mg, of the
cellulose obtained as a pulp from such fibrous materials as sample. Prepare 0.167 M and 1.0 M solutions of
wood or cotton. It is insoluble in water, in dilute acids, and cupriethylenediamine (CED), determining the necessary
in nearly all organic solvents. It is slightly soluble in 1 N volumes of each as follows: 0.12 × w equals the mL of
sodium hydroxide. 0.167 M CED to use, and 0.08 × w equals the mL of
Function: Anticaking agent; binding agent; bulking agent; 1.0 M CED to use. Add a few 3-mm glass beads and
dispersing agent; filter aid; texturizing agent; thickening the calculated volume of 0.167 M CED, blow nitrogen
agent over the surface of the solution, and shake for 2 min.
Packaging and Storage: Store in well-closed containers. Add the calculated volume of 1.0 M CED, again
introduce the nitrogen, and shake vigorously for at least
IDENTIFICATION 3 min.
• A. PROCEDURE Acceptance criteria: A dark blue solution, clear under
Sample mixture: Mix the 30 g of sample with 270 mL microscopic examination, appears.
of water in a high-speed (approximately 12,000 rpm)
power blender for 5 min. ASSAY
Analysis: If the Sample mixture is a free-flowing • PROCEDURE
suspension, transfer 100 mL of it (saving the remainder Sample: 125 mg
for Identification tests D and E (below)), into a 100-mL Analysis: With the aid of about 25 mL of water, transfer
graduated cylinder, and allow it to settle for 1 h. the Sample into a 300-mL Erlenmeyer flask. Add 50.0
Acceptance criteria: The Sample mixture will be either a mL of 0.5 N potassium dichromate, mix, then carefully
free-flowing suspension or a heavy, lumpy suspension add 100 mL of sulfuric acid and heat to boiling.
that flows poorly (if at all), settles only slightly, and Remove the flask from the heat, allow the solution to
contains many trapped air bubbles. The mixture is not stand at room temperature for 15 min, cool it in a
slimy. If the Analysis is carried out, a supernatant liquid water bath, and transfer the solution to a 250-mL
appears above the layer of sample. volumetric flask. Dilute with water almost to volume,
• B. PROCEDURE cool to 25°, dilute to volume with water, and mix.
Sample mixture: 10 g in 90 mL of water Titrate a 50-mL aliquot with 0.1 N ferrous ammonium
Analysis: Boil the Sample mixture for 5 min, filter while sulfate, using 2 or 3 drops of orthophenanthroline TS.
hot through ashless, fine quantitative paper Perform a blank determination (see General Provisions),
(S & S 589 Blue Ribbon, or equivalent), and add 2 and make any necessary correction. Calculate the
drops of iodine TS to the filtrate. normality, N, of the ferrous ammonium sulfate solution
Acceptance criteria: The color does not change from by the formula:
yellow-red.
• C. PROCEDURE Result = (0.1 × 50)/B
Sample: 2 to 5 mg
Analysis: Add the Sample to 20 mL of a 0.1% solution
B = volume of ferrous ammonium sulfate
of anthrone in 75% sulfuric acid, and heat on a steam
solution required in the blank titration (mL)
bath.
272 / Cellulose, Powdered / Monographs FCC 9
Calculate the percent cellulose in the Sample by the 105°, and ignite at 800° ± 25° for 1 h. Perform a blank
formula: determination (see General Provisions) to obtain the
corrected weight of the sample precipitate, each mg of
Result = 6.75(B − S) × N/2W which is equivalent to 0.137 mg of sulfur.
S = volume (mL) of ferrous ammonium sulfate SPECIFIC TESTS
solution used in the sample titration • ASH (TOTAL)
W = weight (g) of the sample taken, on the Sample: 3 g
dried basis Analysis: Heat the Sample at 550° ± 50° until completely
Acceptance criteria: NLT 97.0% and not more than
Monographs
charred, then ignite at 800° ± 25° until free from

102.0% of carbohydrate, calculated as cellulose carbon, cool in a desiccator, and weigh.
IMPURITIES
• LOSS ON DRYING, Appendix IIC: 105° to constant weight
• CHLORIDE
• PH, pH Determination, Appendix IIB
Sample: 5 g
Sample mixture: Mix 10 g of sample in 90 mL of water
Analysis: Add 250 mL of water to the Sample contained
and allow to stand with occasional stirring for 1 h.
in a 500-mL conical flask and reflux the mixture for 1
Analysis: Determine the pH of the supernatant liquid
h. Filter through Whatman No. 4 filter paper, or
from the Sample mixture.
equivalent, and reflux the sample with 200 mL of
Acceptance criteria: Between 5.0 and 7.5.
water for 30 min. Filter as before, and combine the
• WATER-SOLUBLE SUBSTANCES
filtrates and hot water rinses. Add 1 mL of nitric acid,
Sample: 6 g
heat to boiling, and slowly add 5 mL of a 5% solution
Analysis: Mix the Sample with 90 mL of recently boiled
of silver nitrate. After the precipitate has coagulated,
and cooled water and allow to stand with occasional
cool and filter through a sintered-glass filtering funnel.
stirring for 10 min. Filter through Whatman No. 2 filter
Wash with a 1:100 nitric acid solution until free from
paper, or equivalent, discard the first 10 mL of filtrate,
silver nitrate, rinse with water, dry at 130°, and weigh.
and pass the filtrate through the same filter a second
Perform a blank determination (see General Provisions)
time, if necessary, to obtain a clear filtrate. Evaporate a
to obtain the corrected weight of the sample
15-mL portion of the filtrate to dryness in a tared
precipitate, each mg of which is equivalent to 0.247
evaporating dish on a steam bath, dry at 105° for 1 h,
mg of chloride.
cool in a desiccator, and weigh.
Sample: 3 g
Chamomile Oil, English Type
.
• SULFUR (TOTAL)
Sample: 5 g, previously dried First Published: Prior to FCC 6
Analysis: Transfer the Sample into a 300-mL conical
flask, and add 50 mL of 2:3 perchloric acid: nitric acid CAS: [8015-92-7]
(v/v). [CAUTION—Handle perchloric acid in an UNII: UB27587839 [chamaemelum nobile flower oil]
appropriate fume hood.] Heat on a hot plate in a
hood, and boil until all organic matter has been DESCRIPTION
destroyed and copious fumes of perchloric acid evolve. Chamomile Oil, English Type occurs as a light blue or light
If the organic matter chars and cannot be destroyed green-blue liquid with a strong, aromatic odor,
quickly by further heating for a short time, add 10 to characteristic of the flowers. The color may change with
20 mL of the acid mixture, and continue the treatment age to green-yellow or yellow-brown. It is the oil obtained
until a clear, syrupy residue is obtained. [NOTE—All of by steam distillation of the dried flowers of the so-called
the nitric acid must be driven from the flask, because it English or Roman Chamomile, Anthemis nobilis L. (Fam.
will otherwise form a double salt with the barium Asteraceae). It is soluble in most fixed oils, and it is almost
sulfate formed later.] Allow the mixture to cool for a completely soluble in mineral oil. It is soluble, with slight
few min, then add 200 mL of hot water, and heat haziness, in propylene glycol, but it is insoluble in glycerin.
again to boiling. (If the mixture is cloudy, filter, and Function: Flavoring agent
rinse the filter with a small amount of hot water before Packaging and Storage: Store in a cool place protected
boiling.) As soon as the mixture is boiling gently, from light in full, tight containers that are made from steel
carefully run in 20 mL of barium chloride TS, boil for a or aluminum and that are suitably lined.
few minutes longer, and allow to stand for at least 12
h on a steam bath. Filter any barium sulfate onto an IDENTIFICATION
ashless filter paper, and rinse with five portions of • INFRARED SPECTRA, Spectrophotometric Identification Tests,
boiling water to remove traces of perchloric acid. Place Appendix IIIC
the paper in a tared platinum dish, dry in an oven at
FCC 9 Monographs / Chamomile Oil, German Type / 273
Acceptance criteria: The spectrum of the sample • REFRACTIVE INDEX, Appendix IIB
exhibits relative maxima at the same wavelengths as [NOTE—Use an Abbé or other refractometer of equal or
those of the spectrum below. greater accuracy.]
SPECIFIC TESTS • SOLUBILITY IN ALCOHOL, Appendix VI
• ACID VALUE (ESSENTIAL OILS AND FLAVORS), Appendix VI Acceptance criteria: One mL of sample dissolves in 2
Acceptance criteria: NMT 15.0 mL of 80% alcohol, sometimes with a slight precipitate.
• ESTER VALUE, Esters, Appendix VI • SPECIFIC GRAVITY: Determine by any reliable method (see
Sample: 1 g General Provisions).
Acceptance criteria: Between 250 and 310 Acceptance criteria: Between 0.892 and 0.910
Monographs
Chamomile Oil, English Type
IDENTIFICATION
Chamomile Oil, German Type
.

First Published: Prior to FCC 6 Appendix IIIC
Chamomile Oil, Hungarian Type exhibits relative maxima at the same wavelengths as
UNII: 60F80Z61A9 [chamomile flower oil]
SPECIFIC TESTS
DESCRIPTION • ACID VALUE (ESSENTIAL OILS AND FLAVORS), Appendix VI
Chamomile Oil, German Type occurs as a deep blue or
blue-green liquid with a strong, characteristic odor and a
• ESTER VALUE, Esters, Appendix VI
bitter, aromatic taste. When the oil is exposed to light or
Sample: 5 g
air, the blue color changes to green and finally to brown.
Acceptance criteria: NMT 40
It is the oil obtained by steam distillation of the flowers
• ESTER VALUE AFTER ACETYLATION
and stalks of Matricaria chamomilla L. (Fam. Asteraceae).
Sample: 10 mL
Upon cooling, the oil may become viscous. It is soluble in
Acetylated sample: Using the Sample, prepare 1.5 g of
most fixed oils and in propylene glycol. It is insoluble in
dried, acetylated oil as directed under Total Alcohols,
glycerin and in mineral oil.
Appendix VI.
Analysis: Using the Acetylated sample, proceed as
directed in Esters, Ester Value, Appendix VI.
from light in full, tight containers that are made from steel
or aluminum and that are suitably lined.
274 / Chamomile Oil, German Type / Monographs FCC 9
• SOLUBILITY IN ALCOHOL, Appendix VI • SPECIFIC GRAVITY: Determine by any reliable method (see
Acceptance criteria: The oil does not usually dissolve General Provisions).
clearly in 95% alcohol. Acceptance criteria: Between 0.910 and 0.950
Monographs
Chamomile Oil, German Type
IDENTIFICATION
Chitosan
.
• A. INFRARED ABSORPTION, Spectrophotometric Identification

First Published: Third Supplement, FCC 8 Tests, Appendix IIIC
Reference standard: USP Chitosan RS
Poly-β-(1,4)-2-amino-2-deoxy-D-glucose Sample and standard preparation: A
the spectrum of the Reference standard.
• B. PROCEDURE
Solution A: 0.1 g of glycolic acid in 20 g of water
Sample preparation: Add 0.2 g of sample to 80 g of
water, and stir briefly to obtain a dispersion.
Analysis: To the Sample preparation, add the entire
volume of Solution A in one step. Stir the mixture gently
at room temperature until it becomes clear. [NOTE—It
CAS: [9012-76-4] takes approximately 30–60 min of stirring to obtain a
clear solution.] To the clear solution, add 5 g of a 0.5%
DESCRIPTION aqueous solution of sodium lauryl sulfate.
Chitosan occurs as a white to light yellow powder. It is an Acceptance criteria: A gelatinous mass is formed.
unbranched binary polysaccharide consisting of N-acetyl-D-
glucosamine and D-glucosamine units linked in a β(1→4) IMPURITIES
manner. Chitosan is obtained by partial deacetylation of Inorganic Impurities
chitin, which is extracted from the shells of edible shrimps [NOTE—In the tests for Arsenic, Cadmium, Chromium, Iron,
and crabs suitable for human use. Lead, Mercury, and Nickel: When water is specified as a
Function: Antimicrobial; stabilizer; acidity regulator diluent, use deionized ultra-filtered water. When nitric acid
Packaging and Storage: Store in a tight, light-resistant is specified, use nitric acid of a grade suitable for trace
container. Store in a dry place below 30°. element analysis with as low a content of metals as
practical. ACS reagent-grade ultratrace nitric acid with a
purity of 65%–70% HNO3, or equivalent, is
recommended.]
FCC 9 Monographs / Chitosan / 275
• ARSENIC flask, and dilute with water to volume. (The final

Internal standard solution: Transfer 0.2 mL of a concentration is 2.5 mg/mL of chitosan.)
commercially available ICP standard solution containing Sample blank solution: Prepare as directed for the
1000 mg/kg of yttrium and 0.2 mL of a commercially Sample solution, omitting the addition of chitosan.
available ICP standard solution containing 1000 mg/kg [NOTE—Retain the remaining digestion solution for use
of lutetium to a 100-mL volumetric flask, add 1 mL of in the test for Iron.]
nitric acid, and dilute with water to volume. Spectrophotometric system, Plasma Spectrochemistry,
Standard blank solution: Transfer 1.0 mL of the Appendix IIC
Internal standard solution to a 100-mL volumetric flask, Mode: Inductively coupled plasma-mass spectrometer
add 1 mL of nitric acid, and dilute with water to (ICP-MS)
Monographs
volume. ICP-MS: Use a system equipped with a quadrupole
Standard stock solution: Use a commercially available mass spectrometer and an ion detector maintained
multi-element ICP standard solution containing 10 mg/ under vacuum; the system may include a suppression
kg each of lead, mercury, chromium, nickel, cadmium, system to mitigate interference from the 40Ar35Cl+ion.
and arsenic. If not, correction for this interference must be
Standard solution 1: Transfer 0.1 mL of the Standard determined by a suitable method, such as that
stock solution to a 100-mL volumetric flask, add 1.0 mL recommended by the instrument manufacturer. The
of the Internal standard solution and 1 mL of nitric acid, isotope ratio of 40Ar35Cl+/40Ar37Cl+ in the Standard
and dilute with water to volume. Standard solution 1 blank solution may be used to correct this
contains 0.01 ppm each of lead, mercury, chromium, interference. The instrument used should read all
nickel, cadmium, and arsenic. isotopes for the elements shown in Table 1, for the
Standard solution 2: Transfer 0.1 mL of the Standard yttrium internal standard (89 amu) and the lutetium
stock solution to a 1000-mL volumetric flask, add 10.0 internal standard (175 and 176 amu), and should
mL of the Internal standard solution and 10 mL of nitric report the total element contents using the most
acid, and dilute with water to volume. Standard naturally abundant isotopes.
solution 2 contains 0.001 ppm each of lead, mercury,
chromium, nickel, cadmium, and arsenic. Table 1
Sample solution: Transfer a 1.0-g sample to a clean, Isotope
dry, 100-mL Kjeldahl flask. [NOTE—A 300-mL flask may Element (amu)
be used if the reaction foams excessively.] Clamp the Lead 208
flask at an angle of 45°, and add a sufficient quantity 201
of a mixture of 8 mL of sulfuric acid and 10 mL of
Mercury 202
nitric acid to moisten the substance thoroughly. Warm
Chromium 53
gently until the reaction commences, allow the
reaction to subside, and add portions of the same 58
sulfuric/nitric acid mixture, heating after each addition, Nickel 60
until a total of 18 mL of the acid mixture has been Cadmium 114
added. Increase the amount of heat, and boil the Arsenic 75
contents of the flask gently until the solution darkens.
Cool, add 2 mL of nitric acid, and heat again until the Analysis: Instrument performance must be verified to
solution darkens. Continue the heating, followed by conform to the manufacturer’s specifications for
addition of nitric acid, until no further darkening resolution and sensitivity. Before analyzing samples, the
occurs, then heat strongly to the production of dense, instrument must pass a suitable performance check.
white fumes. Cool, cautiously add 5 mL of water to the Perform the evaluation using instrument software such
flask, boil gently to the production of dense, white as correction equations for interferences and taking the
fumes, and continue heating until the volume is Internal standard solution into account. Generate the
reduced to a few mL. calibration curve for the element of interest (arsenic)
Cool, cautiously add 5 mL of water, and examine the using the results obtained from analysis of the Standard
color of the solution. If the color is yellow, cautiously blank solution, Standard solution 1, and Standard
add 1 mL of 30% hydrogen peroxide, and again solution 2, comparing the element of interest to the
evaporate to the production of dense, white fumes and appropriate internal standard (determined by nearest
a volume of 2–3 mL. If the solution is still yellow, isotope mass, amu, or defined by the equipment
repeat the addition of 5 mL of water and the peroxide manufacturer). The linear regression coefficient of the
treatment. Cool, dilute cautiously with a few mL of calibration curve should be NLT 0.999.
water, rinse into a 20-mL volumetric flask, and dilute Aspirate the Sample blank solution and the Sample
with water to volume. solution, separately, at least in duplicate, and report the
Transfer 5.0 mL of the digestion solution to a 100-mL average reading of each element content for the
volumetric flask. [NOTE—Retain the remaining digestion Sample solution. Determine the concentration of the
solution for use in the test for Iron.] Add 1.0 mL of the element of interest (arsenic) in the Sample blank
Internal standard solution to the 100-mL volumetric solution and the Sample solution, each in µg/mL, using
the calibration curve.
276 / Chitosan / Monographs FCC 9
Calculate the quantity, in mg/kg, of the element of Mode: Inductively coupled plasma-atomic emission
interest (arsenic) in the portion of the sample taken: spectrometer (ICP-AES)
Wavelength: 238.040 and 239.562 nm; settings
Result = (CS − CB)/W should be optimized as directed by the manufacturer.
Analysis: Instrument performance must be verified to
CS = concentration of the element of interest in
conform to the manufacturer’s specifications for
the Sample solution, as determined from the
resolution and sensitivity. Before analyzing samples, the
calibration curve (µg/mL)
instrument must pass a suitable performance check.
CB = concentration of the element of interest in
Generate the calibration curve using the Standard blank
the Sample blank solution, as determined
solution, Standard solution 1, and Standard solution 2.
from the calibration curve (µg/mL)
Monographs
The linear regression coefficient of the calibration curve

W = final concentration of chitosan in the Sample
should be NLT 0.999.
solution (g/mL; after digestion, addition of
Aspirate the Sample blank solution and the Sample
Internal standard solution, and dilution)
solution, separately, at least in duplicate, and report the
Acceptance criteria: NMT 0.5 mg/kg
average reading as the iron content of the sample.
• CADMIUM
Determine the concentration of iron in the Sample
Internal standard solution, Standard stock solution,
blank solution and the Sample solution, each in µg/mL,
Standard solution 1, Standard solution 2, Standard
using the calibration curve.
blank solution, Sample solution, and Sample blank
Calculate the quantity, in mg/kg, of iron in the portion
solution: Use the solutions prepared in the test for
of the sample taken:
Arsenic.
Spectrophotometric system and Analysis: Proceed as Result = (CS − CB)/W
directed in the test for Arsenic, except consider
cadmium the element of interest. CS = concentration of iron in the Sample solution,
Acceptance criteria: NMT 0.2 mg/kg as determined from the calibration curve
• CHROMIUM (µg/mL)
Internal standard solution, Standard stock solution, CB = concentration of iron in the Sample blank
Standard solution 1, Standard solution 2, Standard solution, as determined from the calibration
blank solution, Sample solution, and Sample blank curve (µg/mL)
solution: Use the solutions prepared in the test for W = final concentration of chitosan in the Sample
Arsenic. solution (g/mL; after digestion and dilution)
Spectrophotometric system and Analysis: Proceed as Acceptance criteria: NMT 10 mg/kg
directed in the test for Arsenic, except consider • LEAD
chromium the element of interest. Internal standard solution, Standard stock solution,
Acceptance criteria: NMT 1.0 mg/kg Standard solution 1, Standard solution 2, Standard
• IRON blank solution, Sample solution, and Sample blank
Diluent: Dilute nitric acid in water (1 in 100). solution: Use the solutions prepared in the test for
Standard blank solution: Use the solution prepared in Arsenic.
the test for Arsenic. Spectrophotometric system and Analysis: Proceed as
Standard stock solution: Immediately before use, dilute directed in the test for Arsenic, except consider lead the
a commercially prepared ICP standard iron solution element of interest.
with Diluent to prepare an acidic solution containing Acceptance criteria: NMT 0.5 mg/kg
the equivalent of 100 µg/mL of iron. • MERCURY
Standard solution 1: Transfer 0.5 mL of the Standard Internal standard solution, Standard stock solution,
stock solution to a 100-mL volumetric flask, and dilute Standard solution 1, Standard solution 2, Standard
with Diluent to volume. This solution contains 0.5 µg/ blank solution, Sample solution, and Sample blank
mL of iron. solution: Use the solutions prepared in the test for
Standard solution 2: Transfer 0.1 mL of the Standard Arsenic.
stock solution to a 100-mL volumetric flask, and dilute Spectrophotometric system and Analysis: Proceed as
with Diluent to volume. This solution contains 0.1 µg/ directed in the test for Arsenic, except consider mercury
mL of iron. the element of interest.
Sample solution: Transfer 10.0 mL of the digestion Acceptance criteria: NMT 0.2 mg/kg
solution obtained when preparing the Sample solution • NICKEL
in the test for Arsenic to a 100-mL volumetric flask, and Internal standard solution, Standard stock solution,
dilute with water to volume. (The final concentration is Standard solution 1, Standard solution 2, Standard
5.0 mg/mL of chitosan.) blank solution, Sample solution, and Sample blank
Sample blank solution: Transfer 10.0 mL of the solution: Use the solutions prepared in the test for
digestion solution obtained when preparing the Sample Arsenic.
blank solution in the test for Arsenic to a 100-mL Spectrophotometric system and Analysis: Proceed as
volumetric flask, and dilute with water to volume. directed in the test for Arsenic, except consider nickel
Spectrophotometric system, Plasma Spectrochemistry, the element of interest.
Appendix IIC Acceptance criteria: NMT 1.0 mg/kg
Next Page
FCC 9 Monographs / Chitosan / 277
SPECIFIC TESTS record the chromatograms obtained. Determine the

• APPARENT AVERAGE MOLECULAR WEIGHT AND MOLECULAR elution peak maxima and corresponding retention
WEIGHT DISTRIBUTION volumes (elution volume, Vp) for the ten PEG standards,
[NOTE—This test is applicable to Chitosan of an average corresponding to the specified molecular weight, Mp, at
molecular weight of NMT 1,000,000 Da. In the the peak maximum of the standards. Use a suitable gel-
following test, an apparent average molecular weight is permeation chromatography or size-exclusion
determined.] chromatography (GPC/SEC) software, or an equivalent
Mobile phase: Prepare an aqueous solution with a final data handling system, to compute the data and
concentration of 0.15 M sodium nitrate and 0.5 M molecular weight calibration. Plot a calibration curve of
formic acid. (log Mp) for each standard in the Standard solutions
Monographs
System suitability solution: 1.0 mg/mL of ethylene versus its retention volume (Vp), in mL, at each standard
glycol in Mobile phase peak maximum. Construct the best polynomial line
Standard solutions: Prepare several sets of mixtures, fitting the ten points.
containing ten polyethylene glycol (PEG) standards of Based on the PEG molecular weight calibration curve,
different known molecular weight, which are used to calculate the molecular weight and molecular weight
cover the molecular weight range from about 200 to distribution of the sample taken using the slice by slice
1,100,000 g/mol.1 [NOTE—These standards could be method, as follows. From the chromatogram of the
mixtures of polyethylene glycols and polyethylene Sample solution, identify the retention volumes Va and
oxides.] Vb corresponding to the beginning and the end of the
Prepare each set of PEG molecular weight standards to chromatogram. The baseline between Va and Vb is
have a known concentration of about 1.0 mg/mL for assumed to be linear; draw a straight line between Va
each standard in Mobile phase. Allow the solutions to and Vb. Data analysis is based on a suitable GPC/SEC
stand at room temperature for at least 8 h before use. computer software or a real-time data acquisition
Do not filter. system with either off-line or on-line data processing
Sample solution: Prepare a solution containing 1.0 mg/ that is able to provide a means of determining
mL of sample in Mobile phase. Cap and mix well. Allow chromatographic peak heights or integrated area
the solution to stand at room temperature for at least segments as prescribed intervals under the SEC
12 h. Pass the solution through a membrane filter of chromatogram and handling and reporting the data.
0.45-µm pore size, discard an appropriate volume of The following describes the data processes which can
the initial filtrate, and use the remaining filtrate for either be computed by the GPC/SEC software or by an
analysis. equivalent data processing system. Upon acquisition,
Chromatographic system, Appendix IIA handle the data under the chitosan elution peak in
Mode: High-performance liquid chromatography discrete segments, Ai, integrated area slices, or as
Detector: Refractive index digitized chromatogram heights, Hi, by recording the
Detector temperature: 35° vertical displacements between the chromatogram trace
Columns: 7.5-mm × 30-cm analytical column and the baseline at elution volume, Vi, over designated
containing 10-µm methacrylate size exclusion packing; intervals. A minimum of 40 area segments or heights
two 7.5-mm × 30-cm analytical columns containing are required. Obtain the corresponding value for Mi for
17-µm methacrylate size exclusion packing chitosan based on its elution volume, Vi, from the
Flow rate: 1.0 mL/min for the System suitability molecular weight calibration curve.
solution; 0.5 mL/min for the Standard solutions and the Calculate the number- and weight-average molecular
Sample solution weights, Mn and Mw, in g/mol, respectively, using the
Injection volume: 20 µL for the System suitability following formulas.
solution; 100 µL for the Standard solutions and the
Sample solution
System suitability
Suitability requirement 1: The plate count for the
ethylene glycol peak in the chromatogram of the
System suitability solution is NLT 80% of the value that
is certified by the column manufacturer for new
columns.
Suitability requirement 2: The resolution between
the PEG standards in the chromatogram of the
Standard solutions is NLT 1.7. [NOTE—The resolution
between the PEG standard with a molecular weight
of 1,000,000 Da and its adjacent PEG standard
should be NLT 1.0 if NLT 1.7 cannot be met.] If the elution volume internal ∆Vi, for instance, (V2−V1 =
Analysis: Separately inject each of the Standard solutions V3−V2), etc., is constant; parameters Ai and Mi are the
and the Sample solution into the chromatograph and chromatographic peak slice and chitosan molecular
1 Suitable polyethylene glycol molecular weight standards are available as weight associated with the elution volume, Vi; and N is
ReadyCal kits from Polymer Standards Service (PSS), www.polymer.de. the number of data points obtained from the

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182 / Caffeine / Monographs FCC 9

Sample solution: 0.2 mg/mL in Mobile phase. Shake,

and sonicate, if necessary, to dissolve.

Calcium Acetate occurs as a fine, white, bulky powder. It is

form with Mixture A, but forms immediately with

of 2.7 N hydrochloric acid. Formula wt, actual (avg) 219.00

SPECIFIC TESTS Analysis: Add 2 drops of glacial acetic acid and 5 mL of

First Published: Prior to FCC 6

First Published: Second Supplement, FCC 7

C12H14CaO12 · 2H2O Formula wt 426.34

DESCRIPTION C14H10CaO4 · xH2O Formula wt, anhydrous 282.31

Salicylic acid stock solution: 1.0 mg/mL of USP Salicylic IMPURITIES

1 Luna C18(2) (Phenomenex, Torrance, CA), or equivalent.

Calcium Bromate Calcium Carbonate

First Published: Prior to FCC 6 First Published: Prior to FCC 6

Ca(BrO3)2 · H2O Formula wt 313.90 CaCO3 Formula wt 100.09

• LEAD, Lead Limit Test, Appendix IIIB • CALCIUM, Appendix IIIA

dilute to 100 mL with water, and let it stand for 4 h or

Analysis: Add phenolphthalein TS to the Sample solution IMPURITIES

Ca3(C6H5O7)2 · 4H2O Formula wt 570.50

UNII: MLM29U2X85 [calcium citrate]

Acceptance criteria: The spectrum of the sample Carrier gas: Helium

• D. PROCEDURE System suitability

Cupric nitrate solution: 10 mg/mL

UNII: SQE6VB453K [calcium gluconate] Acceptance criteria: NMT 2 mg/kg

three-fourths of the length of the plate. Remove the

Acceptance criteria: NMT 0.005%

mixture through a suitable tared, porous-bottom

porcelain crucible, and wash the residue with hot water

C6H10CaO6 · xH2O Formula wt, anhydrous 218.22

and filter, if necessary. Heat the filtrate or clear solution

Calculate the percent sulfonate sulfur by the formula:

CAS: [8061-52-7] R = weight of the residue (g)

by dissolving 0.1479 g of sodium sulfate in 100 mL of equivalent.

Degree of sulfonation calculation: Calculate the degree Injection size: 200 µL

6 Lignosulfonate calibration standards available from Borregaard Industries

Limited, Borregaard LignoTech Research and Development, P.O. Box 162,

arsenic (ng/mL). Calculate the concentration (mg/kg) Organic Impurities

and the Standard solutions into the analyzer. For each

Analysis: Using a suitably calibrated atomic absorption IDENTIFICATION

mL of Solution A. Mix, and add 50 mg of ammonium

C20H23CaN7O6 · xH2O Formula wt, anhydrous 497.52 Table 1

1 Purospher STAR RP 18e (Merck KGaA); or equivalent.

d Report the impurity Mefox as sum of 6S- and 6R-Mefox.

Shaking: Low rS = peak response for the D,L-5-

and 10.1 min.] M1 = molecular weight of L-5-

Result = (rU/rS) × (CS/CU) × F × (M1/M2) × 100 rD = peak response of D-5-methyltetrahydrofolate

rL = peak response of L-5-methyltetrahydrofolate IDENTIFICATION

solution, and mix.

dried, into a 25-mL volumetric flask, dilute to volume

• ORGANIC IMPURITIES Packaging and Storage: Store in tight containers.

with Internal standard solution, and mix.

Calculate the quantity (mg), of C18H32CaN2O10 in the

sample taken by the formula:

C18H32CaN2O10 Formula wt 476.54

solution hygroscopic powder. It is a mixture of the calcium salts of

C = concentration (mg/mL) of USP Calcium First Published: Prior to FCC 6

Sample: 1.0 g equipped with a magnetic stirrer, and cautiously add