Methotrexate 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

Methotrexate for Injection is a sterile, freeze-dried preparation of methotrexate sodium with or without suitable added substances, buffers, and/or diluents. It contains NLT 95.0% and NMT 115.0% of the labeled amount of methotrexate (C₂₀H₂₂N₈O₅).

[Caution—Great care should be taken to prevent inhaling particles of methotrexate sodium and exposure to skin.]

2 IDENTIFICATION

Change to read:

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

Sample: 2.5 mg/mL of methotrexate in water from Methotrexate for Injection. Adjust with 0.1 N hydrochloric acid to a pH of 4.0. Place the slurry in a 50-mL centrifuge tube, and centrifuge. Decant the supernatant, add 25 mL of acetone, shake, and pass through a solvent-resistant membrane filter of 0.45-μm pore size. Air-dry the filtered precipitate.

Acceptance criteria: Meets the requirements

Add the following:

B. 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

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Procedure

Buffer: 0.2 M dibasic sodium phosphate and 0.1 M citric acid (63:37); adjusted, if necessary, with 0.1 M citric acid or 0.2 M dibasic sodium phosphate to a pH of 6.0

Mobile phase: Acetonitrile and Buffer (8:92) (USP 1-Aug-2019)

Standard solution: 0.1 mg/mL of USP Methotrexate RS in Mobile phase

Sample solution: Nominally equivalent to 0.1 mg/mL of methotrexate from 1 container of Methotrexate for Injection in Mobile phase

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 302 nm

Column: 4.6-mm × 25-cm; 5-μm (USP 1-Aug-2019) packing L1

Autosampler temperature: 5 ± 3° (USP 1-Aug-2019)

Flow rate: 1.2 mL/min

Injection volume: 10 μL

System suitability

Sample: Standard solution (USP 1-Aug-2019)

Suitability requirements

Tailing factor: NMT 2.0 (USP 1-Aug-2019)

Relative standard deviation: NMT 1.0% (USP 1-Aug-2019)

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of methotrexate (C₂₀H₂₂N₈O₅) in the portion of Methotrexate for Injection taken:

Result = (r_U/r_S) × (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 Methotrexate RS in the Standard solution (mg/mL)

C_U = nominal concentration of methotrexate in the Sample solution (mg/mL)

Acceptance criteria: 95.0%–115.0%

4 PERFORMANCE TESTS

Uniformity of Dosage Units 〈905〉: Meets the requirements

5 IMPURITIES

Add the following:

Organic Impurities

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

System suitability stock solution: 0.05 mg/mL each of USP Methotrexate Related Compound B RS and USP Methotrexate Related

Compound C RS prepared as follows. Transfer USP Methotrexate Related Compound B RS and USP Methotrexate Related Compound C RS into a suitable volumetric flask, add ammonium hydroxide equivalent to 2.5% of the final volume, and sonicate for 5 min. Add Mobile phase equivalent to 20% of the final volume and continue to sonicate until completely dissolved. Dilute with Mobile phase to volume.

System suitability solution: 2.5 μg/mL each of USP Methotrexate Related Compound B RS and USP Methotrexate Related Compound C RS in

Mobile phase from System suitability stock solution

Sensitivity solution: 0.25 μg/mL of USP Methotrexate RS in Mobile phase from Standard solution

Sample solution: Nominally equivalent to 0.5 mg/mL of methotrexate from 1 container of Methotrexate for Injection in Mobile phase

System suitability

Samples: System suitability solution and Sensitivity solution

Suitability requirements

Resolution: NLT 1.2 between the methotrexate related compound B and methotrexate related compound C peaks, System suitability

solution

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

Analysis

Sample: Sample solution

Calculate the percentage of each individual impurity in the portion of Methotrexate for Injection taken:

Result = r_U/{Σ[r_U × (1/F)] + r_M} × (1/F) × 100

r_U = peak area of each individual impurity from the Sample solution

F = relative response factor for each individual impurity (see Table 1)

r_M = peak area of methotrexate from the Sample solution

Table 1

Acceptance criteria: See Table 2. The reporting threshold is 0.05%.

Table 2

 (USP 1-Aug-2019)

6 SPECIFIC TESTS

pH 〈791〉

Sample: Constitute as directed in the labeling, except that water is used as the diluent.

Acceptance criteria: 7.0–9.0

Constituted Solution: At the time of use, it meets the requirements in Injections and Implanted Drug Products 〈1〉, Product Quality Tests Common to Parenteral Dosage Forms, Specific Tests, Completeness and Clarity of Solutions.

Change to read:

Bacterial Endotoxins Test 〈85〉: Meets the requirements (USP 1-Aug-2019)

Sterility Tests 〈71〉: Meets the requirements

Other Requirements: Meets the requirements in Labeling 〈7〉, Labels and Labeling for Injectable Products

7 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve as described in Packaging and Storage Requirements 〈659〉, Injection Packaging, protected from light.

Change to read:

USP Reference Standards 〈11〉

USP Methotrexate RS

USP Methotrexate Related Compound B RS

(S)-2-{4-[(2,4-Diaminopteridin-6-yl)methylamino]benzamido}pentanedioic acid.

C₁₉H₂₀N₈O₅ 440.41

USP Methotrexate Related Compound C RS

(S)-2-(4-{[(2-Amino-4-oxo-1,4-dihydropteridin-6-yl)methyl](methyl)amino}benzamido)pentanedioic acid.

C₂₀H₂₁N₇O₆ 455.42 (USP 1-Aug-2019)