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Printed by: USP NF Official Date: Official as of 01-Nov-2022 Document Type: DIETARY SUPPLEMENTS @2024 USPC
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1
Table 1
Calcium L-5-Methyltetrahydrofolate
Time Solution A Solution B
(min) (%) (%)
0 100 0
14 45 55
17 0 100
24 0 100
24.01 100 0
33 100 0
[NOTE—After analysis the column should be flushed and
C20H23CaN7O6 · xH2O stored in a mixture of methanol and water (85:15).]
System suitability solution: Transfer 25 mg of USP Folic
C20H23CaN7O6 (anhydrous) 497.52 Acid RS and 25 mg of USP 4-Aminobenzoylglutamic Acid
N-[4-[[(2-Amino-1,4,5,6,7,8-hexahydro-5-methyl-4-oxo- RS to a 100-mL volumetric flask. Add about 15 mg each of
(6S)-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid, sodium hydrogen carbonate and sodium carbonate to the
calcium salt (1:1); flask, add sufficient water, sonicate to dissolve, and dilute
N-{4-[[((6S)-2-Amino-1,4,5,6,7,8-hexahydro-5-methyl-4- with water to volume. Transfer 1.0 mL of this solution to a
oxo-6-pteridinyl)methyl]amino]-benzoyl}-L-glutamic acid, second 100-mL volumetric flask containing 50 mg of ▲USP
al
calcium salt (1:1) CAS RN®: 151533-22-1. Calcium d,l-5-Methyltetrahydrofolate RS▲ (ERR 1-Nov-2022),
DEFINITION dissolve, and dilute with water to volume.
Calcium L-5-Methyltetrahydrofolate contains NLT 95.0% and [NOTE—The following Standard and Sample solutions
NMT 102.0% of calcium 5-methyltetrahydrofolate must be injected immediately after preparation and
(C20H23CaN7O6), the sum of the L- and D-diastereoisomers, injected only once.]
Standard solution: 0.5 mg/mL of ▲USP Calcium d,l-
calculated on the anhydrous and solvent-free basis, of which
NMT 1.0% corresponds to calcium D-
5-methyltetrahydrofolate.
ci 5-Methyltetrahydrofolate RS▲ (ERR 1-Nov-2022) in water
Sample solution: 0.5 mg/mL of Calcium L-
5-Methyltetrahydrofolate in water
IDENTIFICATION Chromatographic system
(See Chromatography á621ñ, System Suitability.)
ffi
Change to read: Mode: LC
• A. SPECTROSCOPIC IDENTIFICATION TESTS á197ñ, Infrared Detector: UV 280 nm
Spectroscopy: 197K Column: 4.6-mm × 25-cm; 5-µm packing L1
[NOTE—If the spectra obtained show differences, Column temperature: 32°
dissolve the substance to be examined and the ▲USP Flow rate: 1.1 mL/min
Injection volume: 10 µL
O
Calcium d,l-
5-Methyltetrahydrofolate RS▲ (ERR 1-Nov-2022) separately System suitability
in the minimum quantity of water, and add dropwise Samples: System suitability solution and Standard solution
sufficient acetone to produce a precipitate. Allow to [NOTE—For the System suitability solution the relative
stand for 15 min, centrifuge to collect the precipitate, retention times of the component peaks are listed in
wash the precipitate twice with a minimum quantity Table 2. The L- and D-isomers of
of acetone, and dry. Record new spectra using the 5-methyltetrahydrofolate co-elute as a single peak.
residues.] The 4a-hydroxy-5-methyltetrahydrofolic acid,
• B. IDENTIFICATION TESTS—GENERAL, Calcium á191ñ: A 5-methyltetrahydropteroic acid, and
5-mg/mL solution meets the requirements. dimethyltetrahydrofolic acid are included as minor
• C. HPLC: The retention time of the major peak of the components in ▲USP Calcium d,l-
Sample solution corresponds to that of the Standard 5-Methyltetrahydrofolate RS▲ (ERR 1-Nov-2022).]
solution, as obtained in the Assay. It complies with the Suitability requirements
acceptance criteria of the test for Enantiomeric Purity. Resolution: System suitability solution
NLT 6 between 4-aminobenzoylglutamic acid and
ASSAY 4a-hydroxy-5-methyltetrahydrofolic acid
NLT 8 between folic acid and 5-methyltetrahydrofolic
Change to read: acid
• PROCEDURE NLT 15 between 5-methyltetrahydrofolic acid and
Buffer: 7.8 g/L of sodium dihydrogen phosphate dihydrate dimethyltetrahydrofolic acid
in water Relative standard deviation: Prepare three separate
Solution A: Adjust the Buffer with 32% (w/v) sodium Standard solutions, and inject each immediately and only
hydroxide solution to a pH of 6.5. one time. NMT 2.0%; peak response factor from three
Solution B: Methanol and Buffer (35:65). Adjust with 32% injections
(w/v) sodium hydroxide solution to a pH of 8.0. Analysis
Mobile phase: Gradient elution. See Table 1. Samples: Standard solution and Sample solution
Calculate the percentage of calcium
5-methyltetrahydrofolate (C20H23CaN7O6), the sum of the
L- and D-diastereoisomers, in the portion of Calcium L-
5-Methyltetrahydrofolate taken:
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Printed by: USP NF Official Date: Official as of 01-Nov-2022 Document Type: DIETARY SUPPLEMENTS @2024 USPC
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Result = (rU/rS) × (CS/CU) × 100 rU = peak response of the corresponding impurity
from the Sample solution
rU = peak response from the Sample solution rS = peak response of the principal peak from the
rS = peak response from the Standard solution Standard solution
CS = concentration of ▲USP Calcium d,l- CS = concentration of ▲USP Calcium d,l-
5-Methyltetrahydrofolate RS▲ (ERR 1-Nov-2022) in the 5-Methyltetrahydrofolate RS▲ (ERR 1-Nov-2022) in the
Standard solution (mg/mL) Standard solution (mg/mL)
CU = concentration of Calcium L-5-Methyltetra CU = concentration of Calcium L-5-Methyltetra
hydrofolate in the Sample solution (mg/mL) hydrofolate in the Sample solution (mg/mL)
F = relative response factor for the corresponding
Acceptance criteria: 95.0%–102.0% on the anhydrous and impurity peak (see Table 2)
solvent-free basis Mr1 = molecular weight of L-5-methyltetrahydrofolic
acid, 459.46
IMPURITIES Mr2 = molecular weight of calcium L-5-methyltetra
• CHLORIDE hydrofolate, 497.52
Sample: 300 mg
Blank: Mix 1 mL of nitric acid with 75 mL of water. Acceptance criteria
Titrimetric system [NOTE—Disregard any impurity peak less than 0.05%.]
(See Titrimetry á541ñ.) Individual impurities: See Table 2.
Mode: Direct titration
Titrant: 0.005 M silver nitrate VS Table 2
Endpoint detection: Potentiometric
Relative Relative Acceptance
Analysis: Dissolve the Sample in 75 mL of water (heat to
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Retention Response Criteria,
maximum of 40°), add 1 mL of nitric acid, and titrate Name Time Factor NMT (%)
with the Titrant. Perform a Blank determination, and make
4-Aminobenzoylgluta-
any necessary correction. mic acida 0.29 0.91 0.5
Calculate the percentage of chloride (Cl) in the Sample
taken: 4a-Hydroxy-5-methyl
VS
Result = [(VS − VB) × M × F/W] × 100
= volume of Titrant consumed by the Sample (mL)
ci -
tetrahydrofolic acidb
(6R)-Mefox c, d
0.37
0.49
1.09
1.05
1.0
1.0 (sum of 6R
VB = volume of Titrant consumed by the Blank (mL) (6S)-Mefoxc, d 0.50 1.05 and 6S)
ffi
M = actual molarity of the Titrant (mmol/mL) e
F = equivalency factor, 35.45 mg/mmol Tetrahydrofolic acid 0.65 1.00k 0.5
W = Sample weight (mg) 7,8-Dihydrofolic acidf 0.83 0.95 0.5
Acceptance criteria: NMT 0.5% Folic acidg 0.85 0.83 0.5
• ELEMENTAL IMPURITIES—PROCEDURES á233ñ 5,10-Methylenetetrahydro-
O
Acceptance criteria folic acidh 0.88 1.00k 0.5
Boron: NMT 50 µg/g
5-Methyltetrahydrop
Platinum: NMT 10 µg/g -
Arsenic: NMT 1.5 µg/g teroic acidi 1.10 0.67 0.5
Cadmium: NMT 0.5 µg/g
Dimethyltetrahydrofol-
Lead: NMT 1.0 µg/g ic acidj 1.25 1.00k 0.15
Mercury: NMT 1.5 µg/g
• RESIDUAL SOLVENTS á467ñ Total impurities — — 2.5
Acceptance criteria a N-(4-Aminobenzoyl)-L-glutamic acid.
Ethanol: NMT 0.5% b N-[4-({[(6S)-2-Amino-4a-hydroxy-5-methyl-4-oxo-1,4,4a,5,6,7,8,8a-
2-Propanol: NMT 0.5% octahydropteridin-6-yl]methyl}amino)benzoyl]-L-glutamic acid.
[NOTE—For acceptance criteria for any other residual c 2-Amino-8-methyl-4,9-dioxo-7-methyl-p-aminobenzoyl-glutamate-6,7,8,9-
solvents, see Residual Solvents á467ñ.] tetrahydro-4H-pyrazino-(1,2-a)-s-triazine.
d Report the impurity Mefox as the sum of 6S- and 6R-Mefox.
e N-[4-({[(S)-2-Amino-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl]methyl}
Change to read:
amino)benzoyl]-L-glutamic acid.
• RELATED COMPOUNDS f N-(4-{[(2-Amino-4-oxo-1,4,7,8-tetrahydropteridin-6-yl)methyl]amino}
Solution A, Solution B, Mobile phase, System suitability benzoyl)-L-glutamic acid.
g N-(4-{[(2-Amino-4-oxo-1,4-dihydropteridin-6-yl)methyl]amino}benzoyl)-L-
solution, Standard solution, Sample solution, glutamic acid.
Chromatographic system, System suitability, and h N-(4-(3-Amino-1-oxo-5,6,6a,7-tetrahydroimidazo[1,5-f]pteridin-8(1H,4H,
Suitability requirements: Proceed as directed in the 9H)-yl)benzyl)-L-glutamic acid.
Assay. i (S)-4-{[(2-Amino-5-methyl-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl)methyl]
Analysis amino}benzoic acid.
j N-[4-({[(S)-5-Methyl-2-(methylamino)-4-oxo-1,4,5,6,7,8-hexahydropteridin-
Samples: Standard solution and Sample solution
[NOTE—The impurities are listed in Table 2.] 6-yl]methyl}amino)benzoyl]-L-glutamic acid.
k Estimated factor.
Calculate the percentage of each impurity, as free acid, in
the portion of Calcium L-5-Methyltetrahydrofolate taken:
Result = (rU/rS) × (CS/CU) × F × (Mr1/Mr2) × 100
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Printed by: USP NF Official Date: Official as of 01-Nov-2022 Document Type: DIETARY SUPPLEMENTS @2024 USPC
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Change to read: Titrimetric system
(See Titrimetry á541ñ.)
• ENANTIOMERIC PURITY Mode: Direct titration
Buffer: 4.54 g/L of sodium dihydrogen phosphate dihydrate Titrant: 0.05 M edetate disodium VS
in water Endpoint detection: Visual
Mobile phase: Acetonitrile and Buffer (3:97). Adjust with Analysis: Dissolve the Sample in 150 mL of water, add 15 mL
32% (w/v) sodium hydroxide to a pH of 6.8. of 1 N sodium hydroxide and 300 mg of hydroxy naphthol
Standard solution: 0.5 mg/mL of ▲USP Calcium d,l- blue, and titrate with the Titrant until the solution is deep
5-Methyltetrahydrofolate RS▲ (ERR 1-Nov-2022) in water blue in color. Perform a Blank determination, and make any
Sample solution: 0.5 mg/mL of Calcium L- necessary correction.
5-Methyltetrahydrofolate in water Calculate the percentage of calcium (Ca) in the Sample
System suitability solution: Transfer 1.0 mL of Standard taken:
solution to a 50-mL volumetric flask, and dilute with Sample
solution to volume. Result = [(VS − VB) × M × F/W] × 100
Chromatographic system
(See Chromatography á621ñ, System Suitability.) VS = volume of Titrant consumed by the Sample (mL)
Mode: LC VB = volume of Titrant consumed by the Blank (mL)
Detector: UV 280 nm M = actual molarity of the Titrant (mmol/mL)
Column: 4.0-mm × 15-cm; 5-µm packing L79 F = equivalency factor, 40.08 mg/mmol
Column temperature: 40° W = Sample weight (mg)
Flow rate: 1.0 mL/min
Injection volume: 10 µL Acceptance criteria: 7.0%–8.5% on the anhydrous and
solvent-free basis
al
System suitability
Sample: System suitability solution • WATER DETERMINATION, Method Ic á921ñ
[NOTE—The relative retention times of L- Sample: Transfer 40 mg of Calcium L-
5-methyltetrahydrofolate and D- 5-Methyltetrahydrofolate to a 20-mL headspace vial, and
5-methyltetrahydrofolate are about 1 and 1.5, cap tightly. Heat the vial in a suitable Karl Fischer oven at
respectively.] 250°.
Suitability requirements
Resolution: NLT 1.5 between L-
5-methyltetrahydrofolate and D-
ci Analysis: The released and evaporated water is transferred
into the titration-cell in a stream of dry nitrogen at a flow
rate of about 40 mL/min as directed in Water Determination,
5-methyltetrahydrofolate Method Ic á921ñ.
Analysis Acceptance criteria: 6.0%–17.0%
ffi
Sample: Sample solution
ADDITIONAL REQUIREMENTS
Calculate the percentage of D-5-methyltetrahydrofolate in
• PACKAGING AND STORAGE: Store in a tight container, in a
the portion of Calcium L-5-Methyltetrahydrofolate taken:
cool and dry place.
Result = [(rD/(rD + rL) × 100]
Change to read:
O
rD = peak response of D-5-methyltetrahydrofolate
• USP REFERENCE STANDARDS á11ñ
from the Sample solution
USP 4-Aminobenzoylglutamic Acid RS
rL = peak response of L-5-methyltetrahydrofolate
N-(4-Aminobenzoyl)-L-glutamic acid.
from the Sample solution
C12H14N2O5 266.25
Acceptance criteria: NMT 1.0% of D-5-methyltetra
▲
USP Calcium d,l-
hydrofolate 5-Methyltetrahydrofolate RS▲ (ERR 1-Nov-2022)
N[4-[[(2-Amino-1,4,5,6,7,8-hexahydro-5-methyl-4-oxo-
SPECIFIC TESTS 6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid,
• CALCIUM calcium salt (1:1).
Sample: 250 mg C20H23CaN7O6 497.52
Blank: 150 mL of water, 15 mL of 1 N sodium hydroxide, USP Folic Acid RS
and 300 mg of hydroxy naphthol blue
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