A: The retention time of the major peak obtained from the Assay preparation corresponds to that obtained from the Standard preparation, both relative to the internal standard, obtained as directed in the Assay.

B: Infrared Absorption 197K—Prepare the test specimen as follows. Triturate a quantity of finely powdered Tablets, equivalent to about 200 mg of warfarin sodium, with 50 mL of water, centrifuge, and filter the supernatant. Extract with 50 mL of ether, transfer the aqueous layer to a second separator, and discard the ether. Adjust with hydrochloric acid to a pH of less than 3, using short-range pH indicator paper, and extract with 50 mL of chloroform. Transfer the chloroform layer to another separator, extract with 50 mL of sodium hydroxide solution (1 in 250), and discard the chloroform. Transfer the aqueous layer to a beaker, and adjust with hydrochloric acid to a pH of less than 3 (using the pH indicator paper) to precipitate the warfarin. Stir the mixture and allow the precipitate to coagulate. Filter, and wash the precipitate with four 5-mL portions of water. If the precipitate is not white or practically white, dissolve it in a minimum volume of sodium hydroxide solution (1 in 250), dilute with water to 50 mL, and repeat the foregoing procedure, beginning with “Extract with 50 mL of ether.” Dry the warfarin so obtained in vacuum over phosphorus pentoxide for 4 hours.

Medium: water; 900 mL.

Apparatus 2: 50 rpm.

Time: 30 minutes.

Mobile phase and Chromatographic system—Proceed as directed in the Assay.

Internal standard solution— Prepare a solution of propylparaben in water containing, in each mL, an amount of propylparaben equivalent to 0.0025 times the labeled amount, in mg, of warfarin sodium in each Tablet. [NOTE—A small amount of methanol may be used, if necessary, to dissolve the propylparaben.]

Standard stock solution— Dissolve an accurately weighed quantity of USP Warfarin RS in water to obtain a solution having a known concentration of about 0.0011L mg per mL, L being the labeled amount, in mg, of warfarin sodium in the Tablets. [NOTE—Use a small amount of 0.1 N sodium hydroxide to aid in dissolution.]

Standard solution— To 3.0 mL of Standard stock solution, add 1.0 mL of Internal standard solution, and mix.

Test solution— To a filtered 3.0-mL aliquot of the solution under test, add 1.0 mL of Internal standard solution, and mix.

Procedure— Separately inject equal volumes (about 40 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of warfarin sodium dissolved by the formula: in which C is the concentration, in mg per mL, of USP Warfarin RS in the Standard stock solution, 330.32 and 308.34 are the molecular weights of warfarin sodium and warfarin, respectively; and RU and RS are the ratios of the peak responses of warfarin to those of propylparaben obtained from the Test solution and the Standard solution, respectively.

Tolerances— Not less than 80% (Q) of the labeled amount of C19H15NaO4 is dissolved in 30 minutes.

(Official January 1, 2007)

pH 7.4 Buffer and Chromatographic system—Proceed as directed in the Assay under Warfarin Sodium.

Solvent mixture— Prepare a mixture of pH 7.4 Buffer and acetonitrile (85:15).

Mobile phase— Prepare a filtered and degassed mixture of methanol, water, and glacial acetic acid (68:32:1). Make adjustments if necessary (see System Suitability under Chromatography 621).

Internal standard solution— Prepare a solution of propylparaben in acetonitrile having a concentration of 1 mg per mL.

Standard preparation— Transfer about 62.5 mg of USP Warfarin RS, accurately weighed, to a 200-mL volumetric flask, and dissolve in 78 mL of 0.1 N sodium hydroxide. Add 50 mL of 0.2 M monobasic potassium phosphate, dilute with water to volume, and mix. Transfer 15.0 mL of this solution to a 50-mL volumetric flask. Add 5.0 mL of Internal standard solution, and dilute with Solvent mixture to volume.

Assay preparation— Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 5 mg of warfarin sodium, to a 50-mL volumetric flask, and add 5 mL of Internal standard solution and about 30 mL of Solvent mixture. Sonicate for 10 minutes, and then shake by mechanical means for 60 minutes. Dilute with Solvent mixture to volume, and filter.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C19H15NaO4 in the portion of Tablets taken by the formula: in which 330.32 and 308.34 are the molecular weights of warfarin sodium and warfarin, respectively; C is the concentration, in mg per mL, of USP Warfarin RS in the Standard preparation; and RU and RS are the ratios of the peak responses of warfarin to those of propylparaben obtained from the Assay preparation and the Standard preparation, respectively.