50.1.23 C.

Reagents
AOAC Official Method 995.05 (a) Sol vents (HPLC grade).—n-Hexane, dichloromethane,
Vitamin D in Infant Formulas acetonitrile, isopropanol, methanol, ethyl acetate.
and Enteral Products (b) Ethanol.—Absolute, pharmaceutical grade.
Liquid Chromatographic Method (c) Phenolphthalein solution.—1%. Dissolve 1 g
First Action 1995 phenolphthalein in 100 mL absolute ethanol.
[Applicable to determination of 8–2600 IU (International Unit; (d) Dichloromethane–isopropanol (IPA) solutions.—(1) 99.8 +
1 mg vitamin D = 40 IU) vitamin D/qt (1 qt = 0.946 L) in infant 0.2 (v/v).—Transfer 2 mL isopropanol into 1 L volumetric flask.
formulas and enteral products.] Dilute to volume with dichloromethane and mix. (2) 80 + 20
See Table 995.05A for the results of the interlaboratory study (v/v).—Transfer 200 mL isopropanol into 1 L volumetric flask,
supporting acceptance of the method. dilute to volume with dichloromethane, and mix.
(e) Acetic acid solution.—10%. Transfer 10 mL glacial acetic
A. Principle acid (AR grade) into 100 mL volumetric flask, dilute to volume with
Test por tion is saponified, ex tracted, and evap o rated to H2O, and mix well.
concentrate nutrient. Vitamin D is determined by reversed-phase LC (f) Ethanolic potassium hydroxide (KOH) solution.—Dissolve
equipped with UV detector at 265 nm. 140 g KOH pellets (AR grade) in 310 mL absolute ethanol and add
50 mL H2O. Prepare on day of use.
B. Apparatus
(g) Mobile phase.—Gradient mixture of acetonitrile, methanol,
(a) Liquid chromatograph.—With UV detector, meeting system and ethyl acetate. See Table 995.05B for concentrations of mobile
suitability requirements. Operate at 27° ± 1°C; higher temperature phase components.
results in loss of resolution. (h) Vitamin D2 standard solutions.—USP reference standard or
(b) LC column.—250 ´ 4.6 mm id, C18, 5 mm particle size. traceable secondary standard (NIST, USP, etc). (1) Stock standard
Op er ating con di tions: in jec tion vol ume, 250 mL; col umn solution.—180 mg/mL. Accurately weigh 45 mg vitamin D2 and
tem per a ture, 27°C; wave length, 265 nm; flow rates, see quantitatively transfer to 250 mL amber volumetric flask. Dilute to
Table 995.05B; stop time, 35 min; retention times: vitamin D2, vol ume with ab so lute eth a nol. (2) Working stan dard
19.5 min; vitamin D3, 23 min. (Note: The column must not be solution.—2.88 mg/mL. Transfer 4.0 mL stock standard solution
end-capped.) into 250 mL amber volumetric flask, and dilute to volume with
(c) Solid-phase extraction (SPE) column.—Silica; absolute ethanol. Store in refrigera tor £7 days. (3) Internal
500 mg/2.8 mL. standard solution.—46 ng/mL. Use to quantitate vitamin D3.
(d) Vacuum manifold.—For SPE column, (c). Transfer 4.0 mL working standard solution into 250 mL amber
volumetric flask and dilute to volume with absolute ethanol. Store in
(e) Evaporator.—With N flow.
refrigerator £7 days.
(f) Rotary evaporator.
(i) Vitamin D3 standard solutions.—USP reference standard or
(g) Water bath shaker.—Maintaining 60°C. traceable secondary standard. Prepare and store in refrigerator
(h) Separatory funnel.—250 mL. £7 days. (1) Stock standard solution.—180 mg/mL. (2) Working
(i) Amber volumetric flask.—250 mL. stan dard so lu tion.—2.88 mg/mL. (3) In ter nal stan dard
(j) Reflux apparatus. solution.—46 ng/mL. Use to quantitate vitamin D2.

Table 995.05A. Interlaboratory study results for determination of vitamin D in infant formulas and enteral products by liquid
chromatography
a b
Sample x mg/L sr sR RSDr, % RSDR, % r R HorRat
IF liquid 1086.70 28.7 112.40 142.13 10.34 13.08 314.72 397.96 0.48
IF liquid 983.50 26.0 76.89 135.98 7.82 13.83 214.73 380.74 0.47
IF liquid 585.00 15.5 35.59 42.61 6.08 7.28 99.65 119.31 0.25
IF liquid 494.70 13.1 46.27 62.69 9.35 12.67 129.56 175.53 0.42
IF liquid 488.10 12.9 36.08 57.81 7.39 11.84 101.02 161.87 0.39
IF liquid 472.50 12.5 20.99 46.46 4.44 9.83 58.77 130.09 0.32
E liquid 357.60 9.45 31.03 58.19 8.68 16.27 86.88 162.93 0.51
E liquid 346.10 9.15 27.49 33.55 7.94 9.69 76.97 93.94 0.30
IF powder 5.69 0.15 0.22 0.60 3.87 10.46 0.62 1.68 0.18
IF powder 3.96 0.10 0.54 0.77 13.71 19.44 1.51 2.16 0.31
E powder 1.09 0.029 0.25 0.25 23.11 23.11 0.70 0.70 0.30
a
IF= infant formula; E = enteral product.
b
Mean concentration of vitamin D in IU/QND for liquid formulas (IU = International Unit [1 mg vitamin D = 40 IU]; QND = quart normal dilution [1 qt = 0.946 L]),
and in IU/g for powders.

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Table 995.05B. Flow rates and concentrations of mobile F. Evaporation and Solid-Phase Extraction
phase components for determination of vitamin D in infant Place round-bottom flask on rotary evaporator and evaporate
formulas and enteral products by liquid chromatography hexane to dryness at 40°C. Immediately after evaporation, add
Time, Flow rate, Acetonitrile, Methanol, Ethyl acetate, 2.0 mL dichloromethane–IPA solution, C(d)(1), to flask.
min mL/min % % % Wash SPE column with 4.0 mL dichloromethane–IPA solution,
0.0 0.7 91.0 9.0 0.0 C(d)(2), and then with 5.0 mL dichloromethane–IPA solution,
28.0 0.7 91.0 9.0 0.0 C(d)(1). Transfer solution from round-bottom flask to SPE column.
28.5 2.5 0.0 0.0 100.0
Rinse empty flask with 1.0 mL dichloromethane–IPA solution,
C(d)(1), and transfer rinse to SPE column. Wash SPE column with
31.0 2.5 0.0 0.0 100.0
2.0 mL dichloromethane–IPA solution, C(d)(1), and discard this
31.5 2.5 91.0 9.0 0.0 fraction. Elute vitamins D2 and D3 with 7.0 mL dichloromethane–IPA
33.0 2.5 91.0 9.0 0.0 solution, C(d)(1), into 16 ´ 100 mm disposable culture tube.
34.0 0.7 91.0 9.0 0.0 Place culture tube in water bath and evaporate
dichloromethane–IPA solution at 40°C using nitrogen. When dry,
immediately add 1.0 mL acetonitrile to tube and swirl to rinse down
sides of tube. Transfer solution to LC vial using disposable dropper.
(j) System suitability standard solution.—Dissolve 125 mg
certified vitamin D2 standard and 125 mg certified vitamin D3 G. Chromatographic Determination
standard in 10 mL acetonitrile. Heat solution 45 min at 90°C under Inject standards mixture, D(1), onto LC column at beginning,
reflux, then cool. Heating forms previtamin D2 and previtamin D3 middle, and end of each run of test solutions.
for use in System Suitability. Transfer 1.0 mL refluxed solution to To calculate content of vitamins D2 and D3, use internal standard
100 mL amber vol umetric flask and di lute to vol ume with procedure and peak heights.
acetonitrile.
H. Calculations
(Note: Protect vitamins D2 and D3 standard solutions from high
Perform following calculations:
temperature, oxygen, and light, to minimize degradation.)
(1) Calculate concentration of vitamin D2 in standards mixture
D. Preparation of Standards Mixture and Test Solutions (CSD2) as follows:
(Note: Perform the assay in subdued lighting to minimize vitamin
W ´ 4 ´ 4 ´ 4 ´ 40000
degradation.) CSD2(IU/mL) = ´ 105
.
250 ´ 250 ´ 250
(1) Standards mixture.—Transfer 4.0 mL vitamin D2 internal
standard solution, C(h), and 4.0 mL vitamin D3 internal standard
where W = weight of vitamin D2 certified reference standard, mg;
solution, C(i), into 125 mL Erlenmeyer flask, and add 15.0 mL H2O.
4 = dilution factor; 40 000 = IU vitamin D/mg; 250 = volumes of
(2) Test portion.—Prepare formula or enteral product according
subsequent dilutions of vitamin D2 standard solutions; 1.05 =
to label directions and use as test sample. Target vitamin D
correction factor for pre-vitamin D.
concentration will be 0.5 IU/mL. Transfer 15.0 mL test portion into
(2) Calculate concentration of vitamin D3 in standards mixture
125 mL Erlenmeyer flask, using pipet or syringe (depending on
(CSD3) as above, using weight of vitamin D3 certified reference
viscosity). Add 4.0 mL internal standard solution to flask. Use
standard.
vitamin D2 standard solution, C(h)(3), to quantitate vitamin D3 and
(3) Calculate response ratio of vitamin D2 in standards mixture
vitamin D3 standard solution, C(i)(3), to quantitate vitamin D2.
(RSD2) as follows:
E. Saponification and Extraction
PS D 2
Add 15.0 mL aliquots of ethanolic KOH solution, C(f), to RSD2 =
standards mixture, D(1), and to test portion from D(2). Close flasks PS D 3
with stoppers and place them in water bath shaker for 30 min at
60°C. Remove flasks from shaker and let cool to room temperature. where PSD2 = peak height of vitamin D2 in standards mixture; and PSD3
Transfer contents of flask to 250 mL separatory funnels. To empty = peak height of vitamin D3 in standards mixture.
flask add 15.0 mL H2O, close with stopper, and shake vigorously. (4) Calculate response ratio of vitamin D3 in standards mixture
Transfer rinse to corresponding funnel, rinse empty flask again with (RSD3) as above, using peak heights of vitamins D3 and D2, respectively.
60 mL hexane, and transfer rinse to funnel. Close funnel with (5) Calculate response ratio of vitamin D2 in test portion (RTD2) as
stopper and shake vigorously 90 s. Let layers separate ca 10 min. follows:
Drain and discard aqueous layer. Add 15.0 mL H2O to hexane layer
re main ing in fun nel, close fun nel with stop per, and shake PTD 2
RTD2 =
vigorously. Let layers separate; drain and discard aqueous layer. PTD 3
Add 1 drop phenolphthalein solution, C(c), and 15.0 mL H2O to
funnel. Add 10% acetic acid solution, C(e), to funnel, dropwise with where PTD2 = peak height of vitamin D2 in test portion; and PTD3 =
shaking, until washing is neutral to phenolphthalein (colorless). peak height of vitamin D3 in test sample.
Drain and discard aqueous layer. Drain hexane layer through (6) Calculate response ratio of vitamin D3 in test portion (RTD3)
Na2SO4, supported by small cotton plug, into 100 mL round-bottom as above, using peak heights of vitamins D3 and D2, respectively.
flask. Rinse funnel and Na2SO4 with few mL hexane, collecting (7) Calculate concentration of vitamin D2 (CTD2) or D3 (CTD3) in
hexane in same round-bottom flask. test portion as follows:
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 RTD2 CS D2 measured as the time interval between the points where the baseline
CTD2 (IU/mL) = ´ ´U ´ D
RS D2 Vt intersects the 2 lines produced by extending the relatively straight
sides of the peak.
RTD3 CS D3 Separation between these peaks should be sufficient to allow
CTD3 (IU/mL) = ´ ´U ´ D
RS D3 Vt additional peak to be resolved between vitamin D2 and D3, namely,
pre-vitamin D3.
where Vt = volume of test portion, mL (usually 15); U = conversion
factor to appropriate units (if necessary); and D = dilution factor for To verify that pre-vitamin D3 is resolved, inject system suitability
diluted powders or liquids. standard solution, C(j). The system suitability standard solution
con tains, in or der of elu tion, pre-vitamin D 2 , vi ta min D 2 ,
I. System Suitability
pre-vitamin D3, and vitamin D3.
For system suitability use standards mixture, D(1), during routine
operation. Resolution factor between vitamin D2 and vitamin D3 Optimize chromatography by adjusting amount of methanol in
should be 2.0. Calculate resolution factor as follows: mobile phase; decreasing methanol content increases retention
times. Maintain column temperature at 27° ± 1°C; increased
2(t2 - t1 ) temperature decreases retention, and vice versa. Six-replicate
R=
W1 + W 2 injections of standard should have <2% relative standard deviation
(RSD). RSD values for standards throughout run should be <4%.
where t1 and t2 = elution times for vitamins D2 and D3, respectively;
and w1 and w2 = peak widths of vitamins D2 and D3. Peak widths are References: J. AOAC Int. 75, 566(1992); 79, 73(1996).

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