A: The UV absorption spectrum of the Assay preparation obtained in the Assay exhibits maxima and minima at the same wavelengths as that of the Standard preparation, concomitantly measured.

B: Transfer a quantity of Injection, equivalent to about 50 mg of flunixin, to a 50-mL centrifuge tube. Add 10 mL of Acetate buffer, prepared as directed in the Assay, and extract with 25 mL of ethyl acetate. Use the upper phase as the test solution. Separately apply 10 µL of the test solution and 10 µL of a Standard solution of USP Flunixin Meglumine RS in methanol containing 3 mg per mL to a thin-layer chromatographic plate (see Chromatography 621) coated with a 0.25-mm layer of chromatographic silica gel mixture. Allow the spots to dry, and develop the chromatograms in a solvent system consisting of a mixture of toluene, ethyl acetate, glacial acetic acid, and water (75:25:10:1) until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the developing chamber, mark the solvent front, and allow the spots to air-dry. Examine the plate under short-wavelength UV light: the RF value of the principal spot obtained from the test solution corresponds to that obtained from the Standard solution.

Acetate buffer— Dissolve 4.1 g of anhydrous sodium acetate in 500 mL of water. Add 2.9 mL of glacial acetic acid, dilute with water to 1000 mL, and mix.

Standard preparation— Dissolve an accurately weighed quantity of USP Flunixin Meglumine RS in 0.1 N sodium hydroxide, and dilute quantitatively, and stepwise if necessary, with 0.1 N sodium hydroxide to obtain a solution having a known concentration of about 1.65 mg per mL. Transfer 4.0 mL of this solution to a 100-mL volumetric flask, dilute with 0.1 N hydrochloric acid to volume, and mix.

Assay preparation— Transfer an accurately measured volume of Injection, equivalent to about 100 mg of flunixin, to a 50-mL centrifuge tube. Add 20 mL of Acetate buffer, and extract with three 25-mL portions of ethyl acetate. Combine the extracts, filter, and evaporate to dryness on a steam bath under a stream of nitrogen. Dissolve the residue in 0.1 N sodium hydroxide, and transfer to a 100-mL volumetric flask with the aid of 0.1 N sodium hydroxide. Dilute with 0.1 N sodium hydroxide to volume, and mix. Transfer 4.0 mL of this solution to a second 100-mL volumetric flask, dilute with 0.1 N hydrochloric acid to volume, and mix.

Procedure— Concomitantly determine the absorbances of the Assay preparation and the Standard preparation at the wavelength of maximum absorbance at about 327 nm. Calculate the quantity, in mg, of flunixin (C14H11F3N2O2) in each mL of Injection taken by the formula: in which 296.25 and 491.46 are the molecular weights of flunixin and flunixin meglumine, respectively; C is the concentration, in mg per mL, of USP Flunixin Meglumine RS in the Standard preparation; V is the volume, in mL, of Injection taken to prepare the Assay preparation; and AU and AS are the absorbances of the Assay preparation and the Standard preparation, respectively.