Leucovorin Calcium for Injection

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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1 DEFINITION

Leucovorin Calcium for Injection contains an amount of leucovorin calcium equivalent to NLT 90.0% and NMT 110.0% of the labeled amount of leucovorin (C₂₀H₂₃N₇O₇ ).

2 IDENTIFICATION

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A. Spectroscopic Identification Tests 〈197〉, Ultraviolet-Visible Spectroscopy: 197U (CN 1-May-2020)

Standard solution: 0.0215 mg/mL of USP Leucovorin Calcium RS in water

Sample solution: Nominally 0.02 mg/mL of leucovorin prepared by constituting Leucovorin Calcium for Injection in water

Acceptance criteria: Meets the requirements

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

3 ASSAY

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Procedure

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

Solution A: Dissolve 2.6 mL of tetrabutylammonium hydroxide solution (40% in water) and 2.8 g of disodium hydrogen phosphate in 1000 mL of water. Adjust with phosphoric acid to a pH of 7.8.

Mobile phase: Methanol and Solution A (150:850)

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

Sample solution: Nominally 0.1 mg/mL of leucovorin from NLT 4 vials 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

(ERR 1-May-2020)

System suitability

Sample: Standard solution

Suitability requirements

Tailing factor: NMT 2.0

Relative standard deviation: NMT 2.0%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of leucovorin (C₂₀H₂₃N₇O₇ ) in the portion of Leucovorin Calcium for Injection taken:

Result = (r_U/r_S)x(C_S/C_U) × (M_r1/M_r2) × 100

r_U = peak response of leucovorin 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 = nominal concentration of leucovorin in the Sample solution (mg/mL)

M_r1 = molecular weight of leucovorin, 473.45

M_r2 = molecular weight of leucovorin calcium, 511.50

Acceptance criteria: 90.0%–110.0%

4 PERFORMANCE TESTS

Uniformity of Dosage Units 〈905〉: Meets the requirements

5 IMPURITIES

Organic Impurities

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

Solution A, Mobile phase, Standard solution, and Chromatographic system: Proceed as directed in the Assay.

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

Sample solution: Nominally 1 mg/mL of leucovorin from NLT 4 vials in water

System suitability

Samples: Standard solution, System suitability solution, and Sensitivity 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 for Injection taken:

Result = (r_U/r_S)x(C_S/C_U) × (M_r1/M_r2) × (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 = nominal concentration of leucovorin in the Sample solution (mg/mL)

M_r1 = molecular weight of leucovorin, 473.45

M_r2 = molecular weight of leucovorin calcium, 511.50

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 Relative retention time may vary from 2.6 to 3.1 and may be compared with the result obtained from the System suitability solution.

6 SPECIFIC TESTS

pH 〈791〉: 6.5–8.5

Particulate Matter in Injections 〈788〉: Meets the requirements for small-volume injections

Sterility Tests 〈71〉: Meets the requirements

Bacterial Endotoxins Test 〈85〉: Meets the requirements

Other Requirements: It meets the requirements in Injections and Implanted Drug Products 〈1〉.

7 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in single-dose containers and protect from light. Store at controlled room temperature.

USP Reference Standards 〈11〉

USP Folic Acid RS

USP Leucovorin Calcium RS