Leucovorin Calcium

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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C₂₀H₂₁CaN₇O₇ 511.50

l-Glutamic acid, N-[4-[[(2-amino-5-formyl-1,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-, calcium salt (1:1);

Calcium [4-({[(RS)-2-amino-5-formyl-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl]methyl}amino)benzoyl]-l-glutamate CAS RN®: 1492-18-8; UNII:

RPR1R4C0P4.

1 DEFINITION

Leucovorin Calcium contains NLT 95.0% and NMT 105.0% of leucovorin calcium (C H CaN O ), calculated on the anhydrous basis.

2 IDENTIFICATION

Change to read:

A. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197 (CN 1-May-2020) [Note—Methods described in 〈197K〉 or 〈197A〉 may be used.] (USP 1-Aug-2019) Do not dry specimens.

Acceptance criteria: Meets the requirements

Add the following:

B. Identification Tests—General 〈191〉, Chemical Identification Tests, Calcium: Meets the requirements (USP 1-Aug-2019)

Add the following:

C. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay. (USP

1-Aug-2019)

3 ASSAY

Change to read:

Procedure

(USP 1-Aug-2019) Use low-actinic glassware for solutions containing leucovorin calcium, and otherwise protect the solutions from unnecessary exposure to light. Complete the Assay without prolonged interruption.

Solution A: 1.0 M tetrabutylammonium hydroxide in methanol

Solution B: 276 mg/mL of monobasic sodium phosphate monohydrate in water

Mobile phase: Mix 15 mL of Solution A with 835 mL of water. Add 125 mL of acetonitrile, adjust with Solution B to a pH of 7.5 ± 0.1, dilute with water to 1000 mL, and filter. Adjust the concentration of acetonitrile, if necessary.

Diluent: Mix 15 mL of Solution A with 900 mL of water, and adjust with Solution B to a pH of 7.5 ± 0.1. Dilute with water to 1000 mL.

System suitability stock solution: 0.175 mg/mL of USP Folic Acid RS in Diluent

Standard solution: 0.175 mg/mL of USP Leucovorin Calcium RS in Diluent

Sample solution: 0.2 mg/mL of Leucovorin Calcium in Diluent

System suitability solution: System suitability stock solution and Standard solution (1:4)

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 254 nm

Column: 4-mm × 30-cm; 5-μm (USP 1-Aug-2019) packing L1

Flow rate: 1.5 mL/min (USP 1-Aug-2019)

Injection volume: 15 μL

Run time: 2.5 times the retention time of the leucovorin peak (USP 1-Aug-2019)

System suitability

Sample: System suitability solution

[Note—The relative retention times for leucovorin and folic acid are 1.0 and about 1.6, respectively.]

Suitability requirements

Resolution: NLT 3.6 between leucovorin

(USP 1-Aug-2019) and folic acid

Relative standard deviation: NMT 2.0% for the leucovorin peak (USP 1-Aug-2019)

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of leucovorin calcium (C₂₀H₂₁CaN₇O₇) in the portion of Leucovorin Calcium taken:

Result = (r_U/r_S)x(C_S/C_U) × 100

r_U = peak response from the Sample solution

r_S = peak response from the Standard solution

C_S = concentration of USP Leucovorin Calcium RS in the Standard solution (mg/mL)

C_U = concentration of Leucovorin Calcium in the Sample solution (mg/mL)

Acceptance criteria: 95.0%–105.0% on the anhydrous basis

4 IMPURITIES

Add the following:

Organic Impurities

Use low-actinic glassware for solutions containing leucovorin calcium, and otherwise protect the solutions from unnecessary exposure to light.

Buffer: Dissolve 2.6 mL of tetrabutylammonium hydroxide solution (40% in water) and 2.8 g of disodium hydrogen phosphate in 1 L of water.

Adjust with phosphoric acid to a pH of 7.8.

Mobile phase: Methanol and Buffer (150:850)

System suitability solution: 1.2 mg/mL of USP Leucovorin Calcium RS and 12 μg/mL each of 7,8-dihydrofolic acid and USP Folic Acid RS in water. Sonicate as necessary to dissolve.

Sensitivity solution: 0.4 μg/mL of USP Leucovorin Calcium RS in water

Standard solution: 0.12 mg/mL of USP Leucovorin Calcium RS in water. Sonicate as necessary to dissolve.

Sample solution: 1.1 mg/mL of Leucovorin Calcium in water

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 280 nm

Column: 4.6-mm × 25-cm; 5-μm packing L1

Temperatures

Autosampler: 10°

Column: 50°

Flow rate: 1 mL/min

Injection volume: 10 μL

Run time: 4 times the retention time of leucovorin

System suitability

Samples: System suitability solution, Sensitivity solution, and Standard solution

Suitability requirements

Resolution: NLT 1.5 between leucovorin and 7,8-dihydrofolic acid, System suitability solution

Relative standard deviation: NMT 2.0%, Standard solution

Signal-to-noise ratio: NLT 10, Sensitivity solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of each impurity in the portion of Leucovorin Calcium taken:

Result = (r_U/r_S)x(C_S/C_U) × (1/F) × 100

r_U = peak response of each impurity from the Sample solution

r_S = peak response of leucovorin from the Standard solution

C_S = concentration of USP Leucovorin Calcium RS in the Standard solution (mg/mL)

C_U = concentration of Leucovorin Calcium in the Sample solution (mg/mL)

F = relative response factor (see Table 1)

Acceptance criteria: See Table 1. Disregard any impurity peaks less than 0.03%.

Table 1

^a 4-{[(2-Amino-5-formyl-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl)methyl]amino}benzoic acid.

^b N-(4-Aminobenzoyl)-l-glutamic acid.

^c (4-{N-[(2-Amino-4-oxo-1,4,7,8-tetrahydropteridin-6-yl)methyl]formamido}benzoyl)-l-glutamic acid.

^d (4-{N-[(2-Amino-5-formyl-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl)methyl]formamido}benzoyl)-l-glutamic acid.

^e N-(4-{[(2-Amino-4-oxo-1,4,7,8-tetrahydropteridin-6-yl)methyl]amino}benzoyl)-l-glutamic acid.

^f (4-{N-[(2-Amino-4-oxo-1,4-dihydropteridin-6-yl)methyl]formamido}benzoyl)-l-glutamic acid.

^g RRT may vary from 2.6 to 3.1 and may be compared with the result obtained from the System suitability solution.

^h Includes all impurities in Table 1 and 5-(γ-folinoyl)tetrahydrofolate from the test for Limit of 5-(γ-Folinoyl)tetrahydrofolate if the latter is possible from the manufacturing process.

Add the following:

Limit of 5-(γ-Folinoyl)tetrahydrofolate (if present)

The chemical name of 5-(γ-folinoyl)tetrahydrofolate is {4-[({(S)-2-amino-5-[(S)-4-(4-{[(2-amino-4-oxo-1,4-dihydropteridin-6-yl)methyl]amino}benzamido)-4-carboxybutanoyl]-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl}methyl)amino]benzoyl}-l-glutamic acid.

Solution A: Acetonitrile

Solution B: Methanol

Solution C: Dissolve 3.25 g of tetrabutylammonium hydroxide solution (40% in water) and 1.36 g of potassium dihydrogen phosphate in 1000 mL of water. Adjust with phosphoric acid to a pH of 7.0.

Mobile phase: See Table 2.

Table 2

Standard solution: 1.0 mg/mL of USP Leucovorin Calcium RS in water

Sample solution: 1.0 mg/mL of Leucovorin Calcium in water

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 287 nm

Column: 2.1-mm × 10-cm; 3.5-μm packing L1

Column temperature: 40°

Flow rate: 0.3 mL/min

Injection volume: 5 μL

System suitability

Sample: Standard solution

[Note—The relative retention times for leucovorin and 5-(γ-folinoyl)tetrahydrofolate are 1.0 and about 1.7, respectively.]

Suitability requirements

Tailing factor: NMT 2.0

Relative standard deviation: NMT 2.0%

Analysis

Sample: Sample solution

Calculate the percentage of 5-(γ-folinoyl)tetrahydrofolate in the portion of Leucovorin Calcium taken:

Result = (r_U/r_T) × 100

r_U = peak response of 5-(γ-folinoyl)tetrahydrofolate from the Sample solution

r_T = sum of all the peak responses from the Sample solution

Acceptance criteria: NMT 0.3%. It is included in the Total impurities in Table 1 if it is possible from the manufacturing process. (USP 1-Aug-

2019)

5 SPECIFIC TESTS

Water Determination 〈921〉, Method I: NMT 17.0%

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in well-closed, light-resistant containers.

Change to read:

USP Reference Standards 〈11〉

USP Folic Acid RS (USP 1-Aug-2019)

USP Leucovorin Calcium RS