Leucovorin Calcium 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 Injection is a sterile solution of leucovorin calcium (C₂₀H₂₁CaN₇O₇ ) in Water for Injection. It contains NLT 90.0% and NMT 120.0% of the labeled amount of leucovorin (C₂₀H₂₃N₇O₇ ).

2 IDENTIFICATION

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A. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197K (CN 1-May-2020)

Analysis: Transfer a volume of Injection, equivalent to 6 mg of leucovorin calcium, to a glass-stoppered, 50-mL centrifuge tube. Add 40 mL of acetone, mix, centrifuge for a few min, and decant and discard the liquid phase. Repeat the washing process with an additional 40 mL of acetone. Dry the precipitate so obtained with a stream of dry nitrogen.

Acceptance criteria: Meets the requirements

3 ASSAY

3.1 Procedure

Use only freshly deionized water wherever water is specified throughout this Procedure. 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: 250 mg/mL of 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 an apparent 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.

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

Sample solution: Transfer a measured volume of Injection, equivalent to 9 mg of leucovorin, to a 50-mL volumetric flask, and dilute with

Diluent to volume. Pipet 25 mL of this solution into a 60-mL separator, add 25 mL of methylene chloride, shake the mixture, allow the layers to separate, and discard the methylene chloride extract. Repeat the extraction with two more 25-mL portions of methylene chloride, discarding the methylene chloride extracts. Filter the aqueous layer, discarding the first 5 mL of the filtrate, and collect the remaining filtrate in a glass-stoppered conical flask.

System suitability stock solution: 0.175 mg/mL of folic acid in Diluent

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

3.2 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 254 nm

Column: 4-mm × 30-cm; packing L1

Flow rate: 1–2 mL/min

Injection volume: 15 μL

3.3 System suitability

Sample: System suitability solution

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

3.4 Suitability requirements

Resolution: NLT 3.6 between leucovorin calcium and folic acid

Relative standard deviation: NMT 2.0%

3.5 Analysis

Samples: Standard solution and Sample solution

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

Result = (r_U/r_S)x(C_S/C_U) × (M_r1/M_r2) × 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 = 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%–120.0%

4 SPECIFIC TESTS

pH 〈791〉: 6.5–8.5

Bacterial Endotoxins Test 〈85〉: NMT 1.95 USP Endotoxin Units/mg of leucovorin calcium

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

5 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in single-dose, light-resistant containers, preferably of Type I glass.

USP Reference Standards 〈11〉

USP Leucovorin Calcium RS