Warfarin Sodium 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)

DOWNLOAD PDF HERE

1 DEFINITION

Warfarin Sodium for Injection is a sterile, freeze-dried mixture of Warfarin Sodium and suitable added substances. It contains NLT 95.0% and NMT 105.0% of the labeled amount of warfarin sodium (C₁₉H₁₅NaO₄). It may contain a suitable buffer.

2 IDENTIFICATION

A. SPECTROSCOPIC IDENTIFICATION TESTS (197), Infrared Spectroscopy: 197K

Sample: Dissolve 100 mg in 25 mL of water, and adjust with hydrochloric acid to a pH of less than 3, using short-range pH indicator paper. Stir the mixture, and allow the precipitate to coagulate. Filter the mixture. Wash the precipitate with four 5-mL portions of water, and dry under vacuum over phosphorus pentoxide for 4 h. Use the warfarin obtained.

Standard: Use USP Warfarin 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

Buffer: Transfer 1.36 g of monobasic potassium phosphate to a 200-ml volumetric flask, and dissolve in 50 mL of water. Add 39.1 mL of 0.2 N sodium hydroxide, and dilute with water to volume. Adjust with sodium hydroxide or phosphoric acid to a pH of 7.4 ± 0.1.

Mobile phase: Methanol, glacial acetic acid, and water (64:1:36)

Standard stock solution: 0.94 mg/mL of USP Warfarin RS prepared as follows. Transfer USP Warfarin RS to a suitable volumetric flask, and dissolve in 0.1 N sodium hydroxide equivalent to 39% of the final volume. Add 0.2 M monobasic potassium phosphate, equivalent to 25% of the final volume, and dilute with water to volume.

Standard solution: Transfer 5 mL of Standard stock solution into a 50-mL volumetric flask, and dilute with Buffer to volume.

Sample solution: Nominally 0.1 mg/mL of warfarin sodium in Buffer prepared as follows. Dissolve the contents of NLT 10 containers of Warfarin Sodium for Injection in Buffer.

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 280 nm

Column: 4.6 - mm x 25 - 4 cm; packing L7

Flow rate: 1.4 mL/min

Injection volume: 20 µL

System suitability

Sample: Standard solution

Suitability requirements

Relative standard deviation: NMT 1.0%

Analysis

Samples: Standard solution and Sample solution.

Calculate the percentage of the labeled amount of warfarin sodium (C₁₉H₁₅NaO₄) in the portion of Warfarin Sodium for Injection taken:

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

r_U = peak response of warfarin from the Sample solution

r_S = peak response of warfarin from the Standard solution

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

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

M_r1 = molecular weight of warfarin sodium, 330.31

M_r2 = molecular weight of warfarin, 308.33 

Acceptance criteria: 95.0%-105.0%

4 PERFORMANCE TESTS

UNIFORMITY OF DOSAGE UNITS (905): Meets the requirements

5 IMPURITIES

ORGANIC IMPURITIES

Diluent: Methanol and water (25:75)

Mobile phase: Methanol, tetrahydrofuran, glacial acetic acid, and water (53:1:1:47)

Standard stock solution 1: 0.126 mg/mL of USP Warfarin RS prepared as follows. Transfer USP Warfarin RS to a suitable volumetric flask, and add 0.1 N sodium hydroxide and methanol equivalent to 2% and 25% of the final volume, respectively. Sonicate to dissolve. Dilute with water to volume.

Standard stock solution 2: 2.4 µg/mL of USP Warfarin RS in Diluent from Standard stock solution 1

Standard solution: 1.2 µg/mL of USP Warfarin RS in Diluent from Standard stock solution 1

System suitability stock solution 1: 0.24 mg/mL of USP Warfarin Related Compound A RS in methanol

System suitability stock solution 2: 2.4 µg/mL of USP Warfarin Related Compound A RS in Diluent from System suitability stock solution 1

System suitability solution: 1.2 µg/mL of each of USP Warfarin RS and USP Warfarin Related Compound A RS in Diluent from Standard stock solution 2 and System suitability stock solution 2

Sample solution: Nominally equivalent to 0.5 mg/mL of warfarin sodium in Diluent prepared as follows. Transfer the contents of NLT 10 vials of Warfarin Sodium for Injection into a suitable volumetric flask, dissolve, and dilute with Diluent to volume.

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 280 nm

Column: 4.6-mm x 25-cm; packing L11

Column temperature: 35°

Flow rate: 1.5 mL/min

Injection volume: 100 µL

Run time: NLT 4 times the retention time of warfarin

System suitability

Samples: Standard solution and System suitability solution

[NOTE-The relative retention times for warfarin and warfarin related compound A are about 1.0 and 1.4, respectively.]

Suitability requirements

Resolution: NLT 2 between warfarin and warfarin related compound A peaks, System suitability solution

Relative standard deviation: NMT 5.0%, Standard solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of Alice's ketone in the portion of Warfarin Sodium for Injection taken:

                         Result = (r_U/r_S) × (C_S/C_U) x (M_r1/M_r2) × (1/F) x 100

r_U = peak response of warfarin related compound A from the Sample solution

r_S = peak response of warfarin from the Standard solution

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

C_U = concentration of warfarin sodium in the Sample solution (mg/mL)

M_r1 = molecular weight of warfarin sodium, 330.31

M_r2 = molecular weight of warfarin, 308.33

F = relative response factor for Alice's ketone, 0.9

[NOTE-Alice's ketone is a sodium salt of warfarin related compound A, 3-(o-hydroxyphenyl)-5-phenyl-2-cyclohexen-1-one sodium salt.]

Acceptance criteria: NMT 0.5%

6 SPECIFIC TESTS

WATER DETERMINATION, Method 1 (921): NMT 4.5%

pH (791)

Sample solution: 10 mg/mL.

Acceptance criteria: 7.2-8.3

CONSTITUTED SOLUTION: Meets the requirements in Injections and Implanted Drug Products (1). Specific Tests. Completeness and clarity of solutions

BACTERIAL ENDOTOXINS TEST (85): NMT 24.0 of USP Endotoxin Units/mg of warfarin sodium

STERILITY TESTS (71): Meets the requirements

7 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve in light-resistant containers as described in Packaging and Storage Requirements (659), Injection Packaging. Packaging for constitution. Store at controlled room temperature.

LABELING: Meets the requirements in Injections and Implanted Drug Products (1), Specific Tests. Completeness and clarity of solutions

Change to read:

USP REFERENCE STANDARDS (11)

USP Warfarin RS

USP Warfarin Related Compound A RS

3-(^▲2_{▲(ERR 1-Feb-2022)} -Hydroxyphenyl)-5-phenyl-2-cyclohexen-1-one.

C₁₈H₁₆O₂          ^▲264.32_{▲(ERR 1-Feb-2022)}