Mobile phase— Prepare a filtered and degassed mixture of methanol,water,and glacial acetic acid (70:30:1).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Internal standard solution— Dissolve a quantity of sodium benzoate in water to obtain a solution containing 33mg per mL.

Diluent— Prepare a mixture of methanol and water (7:3).

Standard preparation— Transfer about 83mg of USP Flunixin Meglumine RS,accurately weighed,to a 50-mLcentrifuge tube.Add 5.0mLof Internal standard solution,20.0mLof water,and 10.0mLof methanol to the tube,and mix to dissolve.Transfer 10.0mLof this solution to a 25-mLvolumetric flask,dilute with Diluentto volume,and mix.

Assay preparation— Transfer an accurately weighed quantity of Paste,equivalent to about 50mg of flunixin,to a 50-mLcentrifuge tube.Add 5.0mLof Internal standard solutionand 20.0mLof water to the tube,and rotate for 20minutes.Add 10.0mLof methanol,and mix.Heat the tube in a water bath at 60for 5minutes,with occasional shaking.Continue rotating the tube until cool,and centrifuge.Transfer 10.0mLof the clear supernatant to a 25-mLvolumetric flask,dilute with Diluentto volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 4-mm ×30-cm column that contains packing L1.The flow rate is about 1mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative retention times are about 0.5for sodium benzoate and 1.0for flunixin meglumine;the resolution,R,between sodium benzoate and flunixin meglumine is not less than 1.9;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard preparation and the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of flunixin (C14H11F3N2O2)in the portion of Paste taken by the formula: in which 296.25and 491.46are the molecular weights of flunixin and flunixin meglumine,respectively;Cis the concentration,in mg per mL,of USP Flunixin Meglumine RSin the Standard preparation;and RUand RSare the ratios of the peak responses for flunixin and sodium benzoate obtained from the Assay preparationand the Standard preparation,respectively.