Leucovorin Calcium Tablets

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 Tablets contain NLT 90.0% and NMT 110.0% of the labeled amount of leucovorin (C₂₀H₂₃N₇O₇ ).

2 IDENTIFICATION

A.

Sample: Equivalent to 200 mg of leucovorin calcium from finely powdered Tablets

Analysis: Transfer the Sample to a conical flask. Add 10 mL of water, shake vigorously, sonicate for 10 min, and filter. Transfer the filtrate to a stoppered centrifuge tube, add 125 mg of ammonium oxalate, shake vigorously, and centrifuge until a clear supernatant is obtained.

Transfer the supernatant to another stoppered centrifuge tube, add 1 mL of methanol and 3 drops of hydrochloric acid, and shake vigorously. If the preparation is cloudy, add methanol until a clear solution is obtained, and filter if necessary to remove any undissolved material. Cool the preparation at 0° until a precipitate forms, and centrifuge for 1–2 min. [Note—The cooling and centrifuging steps may be repeated if necessary to increase the amount of precipitate collected.] Decant the supernatant, add 2 mL of methanol to the tube, shake vigorously to dissolve the precipitate, and transfer the contents to a beaker. Evaporate under a current of air to dryness, and dry the residue at 50° for 30 min.

Acceptance criteria: The IR absorption spectrum of a potassium bromide dispersion of the residue exhibits maxima only at the same wavelengths as that of a similar preparation of USP Leucovorin Calcium RS.

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

Procedure

Diluent: Methanol and water (20:80)

Mobile phase: 5 mM tetrabutylammonium phosphate in Diluent. Adjust with 50% (w/v) sodium hydroxide to a pH of 7.5.

Standard solution: 0.5 mg/mL of USP Leucovorin Calcium RS and 10 μg/mL of USP 10-Formylfolic Acid RS in water

Sample solution: Transfer finely powdered Tablets (NLT 20), equivalent to 50 mg of leucovorin, to a 100-mL volumetric flask. Add 50 mL of water, sonicate for 30 min, dilute with water to volume, mix, and filter.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 254 nm

Column: 4.6-mm × 15-cm; packing L1

Flow rate: 2.0 mL/min

Injection volume: 20 μL

System suitability

Sample: Standard solution

[Note—The relative retention times for leucovorin and 10-formylfolic acid are about 1.0 and 2.3, respectively.]

Suitability requirements

Resolution: NLT 1.5 between leucovorin and 10-formylfolic acid

Relative standard deviation: NMT 2.0% for leucovorin

Analysis

Samples: Standard solution and Sample solution

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

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

r_U = peak area of the Sample solution

r_S = peak area of 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

Dissolution 〈711〉

Medium: Water; 900 mL

Apparatus 2: 50 rpm

Time: 30 min

Detector: UV, at a maximum of about 284 nm

Standard solution: USP Leucovorin Calcium RS in Medium

Sample solution: Use

ltered portion of solution under test, and dilute with water if necessary to a concentration similar to that of the

Standard solution.

Calculate the percentage of the labeled amount of leucovorin (C₂₀H₂₃N₇O₇ ) dissolved:

Result = (A_U/A_S) × C_S × V × D × (M_r1/M_r2) × (1/L) × 100

A_U = absorbance of the Sample solution

A_S = absorbance of the Standard solution

C_S = concentration of the Standard solution (mg/mL)

V = volume of Medium, 900 mL

D = dilution factor

M_r1 = molecular weight of leucovorin, 473.45

M_r2 = molecular weight of leucovorin calcium, 511.50

L = label claim (mg/Tablet)

Tolerances: NLT 75% (Q) of the labeled amount of leucovorin (C₂₀H₂₃N₇O₇ ) is dissolved.

Change to read:

Uniformity of Dosage Units 〈905〉: Meet the requirements (CN 1-Aug-2023)

Analysis for content uniformity

Standard solution: 10 μg/mL of USP Leucovorin Calcium RS

Sample solution: 10 μg/mL of leucovorin calcium, use individual intact Tablets.

Blank: Water

Instrumental conditions

(See Ultraviolet-Visible Spectroscopy 〈857〉.)

Mode: UV

Cell: 1 cm

Analytical wavelength: UV, at maxima about 284 nm

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of leucovorin (C₂₀H₂₃N₇O₇ ) in each Tablet taken:

Result = (A_U/A_S) × (C_S/C_U) × (M_r1/M_r2 ) × 100

A_U = absorbance of the Sample solution

A_S = absorbance of the Standard solution

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

C_U = nominal concentration of leucovorin in the Sample solution (μg/mL)

M_r1 = molecular weight of leucovorin, 473.45

M_r2 = molecular weight of leucovorin calcium, 511.50

5 IMPURITIES

Organic Impurities

Diluent, Mobile phase, Standard solution, Sample solution, Chromatographic system, and System suitability: Proceed as directed in the Assay.

Analysis

Sample: Sample solution

Calculate the percentage of each impurity in the portion of Tablets taken:

Result = (r_U/r_T) × 100

r_U = response of each impurity peak

r_T= sum of the responses of all the peaks

Acceptance criteria

Individual impurities: NMT 2.5%

Total impurities: NMT 4.0 %

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in well-closed containers, protected from light, at controlled room temperature.

USP Reference Standards 〈11〉

USP 10-Formylfolic Acid RS

USP Leucovorin Calcium RS