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Status: Currently Official on 11-Nov-2024
Official Date: Official as of 01-Nov-2020
Document Type: Dietary Supplement Monographs
DocId: GUID-FB300FEA-1F49-49E9-AEF0-EDB7C7165AC9_2_en-US
DOI: https://doi.org/10.31003/USPNF_M20457_02_01
DOI Ref: rml6h
Printed from: https://online.uspnf.com/uspnf/document/1_GUID-FB300FEA-1F49-49E9-AEF0-EDB7C7165AC9_2_en-US
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Creatine

C4H9N3O2 · H2O 149.15

Glycine, N-(aminoiminomethyl)-N-methyl-, monohydrate;
N-Carbamimidoyl-N-methylglycine monohydrate CAS RN®: 6020-87-7.
C4H9N3O2 131.14
Anhydrous CAS RN®: 57-00-1.

DEFINITION
Creatine contains one molecule of water of hydration. It contains NLT 98.0% and NMT 102.0% of creatine (C4H9N3O2), calculated on the dried

L
basis.

IDENTIFICATION
IA
• A. Spectroscopic Identification Tests 〈197K〉
• B. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.

ASSAY
• Procedure
IC

Mobile phase: Dissolve 23 g of monobasic ammonium phosphate in 1 L of water and adjust with phosphoric acid to a pH of 4.0.
Diluent: Water
System suitability solution: 0.1 mg/mL of USP Creatine RS, 0.1 mg/mL of USP Creatinine RS, and 0.002 mg/mL of dicyandiamide1 in Diluent
Standard solution: 2.5 mg/mL of USP Creatine RS in Diluent
FF

Sample solution: 2.5 mg/mL of Creatine in Diluent

Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
Mode: LC
Detector: UV 225 nm
O

Column: 4.0-mm × 25-cm; 5-µm packing L9

Column temperature: 25°
Flow rate: 1.0 mL/min
Injection volume: 20 µL
System suitability
Samples: System suitability solution and Standard solution
[Note—The relative retention times for dicyandiamide, creatine, and creatinine are about 0.8, 1.0, and 2.0, respectively, System suitability
solution.]
Suitability requirements
Resolution: NLT 2.0 between dicyandiamide and creatine, System suitability solution
Column efficiency: NLT 1000 theoretical plates, Standard solution
Relative standard deviation: NMT 2.0%, Standard solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of creatine (C4H9N3O2) in the portion of Creatine taken:
 Result = (rU/rS) × (CS/CU) × 100

rU = peak response of creatine from the Sample solution

rS = peak response of creatine from the Standard solution

CS = concentration of USP Creatine RS in the Standard solution (mg/mL)

CU = concentration of Creatine in the Sample solution (mg/mL)

Acceptance criteria: 98.0%–102.0% on the dried basis

IMPURITIES
• Residue on Ignition 〈281〉: NMT 0.1%
• Chloride and Sulfate 〈221〉, Sulfate
Standard solution: 0.10 mL of 0.020 M sulfuric acid
Sample: 200 mg of Creatine
Acceptance criteria: NMT 0.1%

PROCESS-RELATED IMPURITIES
[Note—On the basis of information regarding the manufacturing process, perform either Procedure 1 or Procedure 2.]

• Procedure 1
Mobile phase, Diluent, System suitability solution, Chromatographic system, and System suitability: Proceed as directed in the Assay.
Standard solution: 2.5 µg/mL of USP Creatine RS and 2.5 µg/mL of USP Creatinine RS in Diluent
Sample solution: 2.5 mg/mL of Creatine in Diluent
Analysis

L
Samples: Standard solution and Sample solution
Calculate the percentage of creatinine (C4H7N3O) in the portion of Creatine taken:
IA
Result = (rU/rS) × (CS/CU) × 100

rU = peak response of creatinine from the Sample solution

rS = peak response of creatinine from the Standard solution

IC

CS = concentration of USP Creatinine RS in the Standard solution (mg/mL)

CU = concentration of Creatine in the Sample solution (mg/mL)

FF

Separately calculate the percentage of dicyandiamide and dihydrotriazine in the portion of Creatine taken:

Result = (rU/rS) × (CS/CU) × (1/F) × 100

rU = peak response of dicyandiamide or dihydrotriazine from the Sample solution

O

rS = peak response of creatine from the Standard solution

CS = concentration of USP Creatine RS in the Standard solution (mg/mL)

CU = concentration of Creatine in the Sample solution (mg/mL)

F = relative response factor (see Table 1)

Calculate the percentage of any unspecified impurity in the portion of Creatine taken:

Result = (rU/rS) × (CS/CU) × 100

rU = peak response of any unspecified impurity from the Sample solution

rS = peak response of creatine from the Standard solution

CS = concentration of USP Creatine RS in the Standard solution (mg/mL)

 CU = concentration of Creatine in the Sample solution (mg/mL)

Acceptance criteria: See Table 1.

Table 1

Relative Relative Acceptance

Retention Response Criteria,
Name Time Factor NMT (%)

Dicyandiamide 0.8 68.5 0.1

Creatine 1.0 1.0 —

Dihydrotriazine 1.3 44.5 0.0005

Creatinine 2.0 — 0.1

Any unspecified impurity — 1.0 0.1

Total unspecified impurities — — 1.5

Total impurities — — 2.0

[Note—On the basis of information regarding the manufacturing process, perform either Procedure 1 or Procedure 2.]
• Procedure 2

L
Buffer solution: 10 mM of monobasic sodium phosphate in water
Mobile phase: Acetonitrile and Buffer solution (83:17)
IA
Diluent: Acetonitrile and water (50:50)
System suitability solution: 0.05 mg/mL of USP Creatine RS, 0.02 mg/mL of USP Creatinine RS, and 2 mg/mL of urea
Standard solution: 2 µg/mL of USP Creatine RS and 2 µg/mL of USP Creatinine RS in Diluent
Sample solution: 2 mg/mL of Creatine in Diluent
IC

Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
Mode: LC
Detector: UV 208 nm
Column: 4.6-mm × 15-cm; 5-µm packing L3
FF

Column temperature: 30°

Flow rate: 1.5 mL/min
Injection volume: 10 µL
System suitability
Sample: System suitability solution
O

[Note—The relative retention times for urea, creatinine, and creatine are 0.15, 0.30, and 1.0, respectively.]
Suitability requirements
Resolution: NLT 3.0 between urea and creatinine
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of creatinine (C4H7N3O) in the portion of Creatine taken:

Result = (rU/rS) × (CS/CU) × 100

rU = peak response of creatinine from the Sample solution

rS = peak response of creatinine from the Standard solution

CS = concentration of USP Creatinine RS in the Standard solution (mg/mL)

CU = concentration of Creatine in the Sample solution (mg/mL)

 Separately calculate the percentage of dicyandiamide, urea, and sarcosine in the portion of Creatine taken:

Result = (rU/rS) × (CS/CU) × (1/F) × 100

rU = peak response of dicyandiamide, urea, or sarcosine from the Sample solution

rS = peak response of creatine from the Standard solution

CS = concentration of USP Creatine RS in the Standard solution (mg/mL)

CU = concentration of Creatine in the Sample solution (mg/mL)

F = relative response factor (see Table 2)

Calculate the percentage of any unspecified impurity in the portion of Creatine taken:

Result = (rU/rS) × (CS/CU) × 100

rU = peak response of any unspecified impurity from the Sample solution

rS = peak response of creatine from the Standard solution

CS = concentration of USP Creatine RS in the Standard solution (mg/mL)

CU = concentration of Creatine in the Sample solution (mg/mL)

Acceptance criteria: See Table 2.

Table 2

Relative

L Relative Acceptance
IA
Retention Response Criteria,
Name Time Factor NMT (%)

Dicyandiamide 0.11 3.0 0.1

IC

Urea 0.15 0.016 0.1

Creatinine 0.28 — 0.1

Sarcosine 0.73 0.016 0.1

FF

Creatine 1.0 — —

Any unspecified impurity — 1.0 0.1

O

Total impurities — — 2.0

SPECIFIC TESTS
• Loss on Drying 〈731〉
Analysis: Dry at 105° for 2 h.
Acceptance criteria: 10.5%–12.5%

ADDITIONAL REQUIREMENTS
• Packaging and Storage: Preserve in tight, light-resistant containers.

• Labeling: If Procedure 2 for Process-Related Impurities is used, then the labeling states that the article complies with Process-Related
Impurities, Procedure 2.
• USP Reference Standards 〈11〉
USP Creatine RS
USP Creatinine RS
▲ (USP 1-May-2020)
1 Use ACS reagent grade.

Auxiliary Information - Please check for your question in the FAQs before contacting USP.

Topic/Question Contact Expert Committee

Fatkhulla K Tadjimukhamedov NBDS2020 Non-botanical Dietary

CREATINE
Associate Scientific Liaison Supplements

Chromatographic Database Information: Chromatographic Database

Most Recently Appeared In:

Pharmacopeial Forum: Volume No. PF 44(3)

Current DocID: GUID-FB300FEA-1F49-49E9-AEF0-EDB7C7165AC9_2_en-US

DOI: https://doi.org/10.31003/USPNF_M20457_02_01
DOI ref: rml6h

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