Calcium l-5-Methyltetrahydrofolate Capsules
DEFINITION
Calcium l-5-Methyltetrahydrofolate Capsules contain NLT 90.0% and NMT 110.0% of the labeled amount of calcium l-5-methyltetrahydrofolate (C20H23CaN7O6).
IDENTIFICATION
• A. HPLC:
The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Strength, and to the l-isomer of the Standard solution in the test for Enantiomeric Purity.
STRENGTH
• Procedure
Buffer:
7.8 g/L of sodium dihydrogen phosphate dihydrate in water
Solution A:
Adjust the Buffer with 32% (w/v) sodium hydroxide solution to a pH of 6.5.
Solution B:
Methanol and Buffer (35:65). Adjust with 32% (w/v) sodium hydroxide solution to a pH of 8.0.
Mobile phase:
Gradient elution. See Table 1.
Table 1
| Time (min) |
Solution A (%) |
Solution B (%) |
|---|---|---|
| 0 | 100 | 0 |
| 14 | 45 | 55 |
| 17 | 0 | 100 |
| 24 | 0 | 100 |
| 24.01 | 100 | 0 |
| 33 | 100 | 0 |
System suitability solution:
Transfer 25 mg of USP Folic Acid RS to a 100-mL volumetric flask. Add about 15 mg each of sodium hydrogen carbonate and sodium carbonate to the flask, add sufficient water, sonicate to dissolve, and dilute with water to volume. Transfer 1.0 mL of this solution to a second 100-mL volumetric flask containing 50 mg of USP Calcium d,l-5-Methyltetrahydrofolate RS, dissolve, and dilute with water to volume.
[Note—The following Standard solution and Sample solution must be injected immediately after preparation and injected only once. ]
Standard solution:
0.1 mg/mL of USP Calcium d,l-5-Methyltetrahydrofolate RS in Solution A
Sample solution:
Remove, as completely as possible, the contents of NLT 30 Capsules, and weigh accurately. Prepare solution, nominally equivalent to 0.1 mg/mL of calcium l-5-methyltetrahydrofolate, from the combined contents of Capsules, in Solution A, and filter.
Chromatographic system
Mode:
LC
Detector:
UV 280 nm
Column:
4.6-mm × 25-cm; 5-µm packing L1
Column temperature:
32
Flow rate:
1.1 mL/min
Injection volume:
10 µL
System suitability
Samples:
System suitability solution and Standard solution
[Note—For the System suitability solution the relative retention times of folic acid and l- and d-isomers of 5-methyltetrahydrofolate, which co-elute as a single peak, are 0.85 and 1.0, respectively. ]
Suitability requirements
Resolution:
NLT 8 between folic acid and 5-methyltetrahydrofolic acid, System suitability solution
Relative standard deviation:
NMT 2.0%, Standard solution
Analysis
Samples:
Standard solution and Sample solution
Calculate the percentage of the labeled amount of calcium l-5-methyltetrahydrofolate (C20H23CaN7O6) in the portion of Capsules taken:
Result = (rU/rS) × (CS/CU) × 100
| rU | = | = peak response from the Sample solution |
| rS | = | = peak response from the Standard solution |
| CS | = | = concentration of USP Calcium d,l-5-Methyltetrahydrofolate RS in the Standard solution (mg/mL) |
| CU | = | = nominal concentration of calcium l-5-methyltetrahydrofolate in the Sample solution (mg/mL) |
Acceptance criteria:
90.0%–110.0%
IMPURITIES
• Enantiomeric Purity
Buffer:
4.54 g/L of sodium dihydrogen phosphate dihydrate in water
Mobile phase:
Acetonitrile and Buffer (3:97). Adjust with 32% (w/v) sodium hydroxide to a pH of 6.8.
Standard solution:
0.4 mg/mL of USP Calcium d,l-5-Methyltetrahydrofolate RS in water
Sample solution:
Filtered portion, equivalent to 0.4 mg/mL of calcium l-5-methyltetrahydrofolate, from the contents of NLT 30 Capsules, in water
System suitability solution:
Transfer 0.2 mL of Standard solution to a 10-mL volumetric flask, and dilute with Sample solution to volume.
Chromatographic system
Mode:
LC
Detector:
UV 280 nm
Column:
4.0-mm × 15-cm; 5-µm packing L791
Column temperature:
40
Flow rate:
1.0 mL/min
Injection volume:
10 µL
System suitability
Sample:
System suitability solution
[Note—The relative retention times of l-5-methyltetrahydrofolate and d-5-methyltetrahydrofolate are about 1 and 1.5, respectively. ]
Suitability requirements
Resolution:
NLT 1.5 between l-5-methyltetrahydrofolate and d-5-methyltetrahydrofolate
Analysis
Sample:
Sample solution
Calculate the percentage of d-5-methyltetrahydrofolate in the portion of calcium l-5-methyltetrahydrofolate taken:
Result = [rD/(rD + rL)] × 100
| rD | = | = peak response of d-5-methyltetrahydrofolate from the Sample solution |
| rL | = | = peak response of l-5-methyltetrahydrofolate from the Sample solution |
Acceptance criteria:
NMT 1.0%
PERFORMANCE TESTS
• Disintegration and Dissolution 
2040
:
Meet the requirements for Disintegration
2040:
Meet the requirements for Disintegration
• Weight Variation 2091:
Meet the requirements
2091:
Meet the requirements
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Store in a tight, light-resistant container, in a cool and dry place.
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1
A chiral-recognition protein, human serum albumin (HSA), chemically bonded to silica particle, about 5 µm in diameter. For example: Chromtech Chiral HSA, available at www.chromtech.com.
Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Natalia Davydova
Scientific Liaison (301) 816-8328 |
(DS2010) Monographs - Dietary Supplements and Herbal Medicines |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP38–NF33 Page 5935
Pharmacopeial Forum: Volume No. 39(3)