A: Ultraviolet Absorption á197Uñ—

B: Transfer a volume of Injection equivalent to about 50mg of xylazine to a separator,add 1mLof sodium carbonate solution (1in 20),and extract with four 10-mLportions of methylene chloride,combining the methylene chloride extracts in a beaker and evaporating to dryness.Add 10mLof methanol to the beaker,and swirl to dissolve the residue.The test solution thus obtained responds to Identificationtest Bunder Xylazine Hydrochloride.

Mobile phase— Dissolve 6.0g of sodium 1-heptanesulfonate in 3000mLof water,adjust to a pHof 3.0by dropwise addition of phosphoric acid.Add 1000mLof acetonitrile,mix,and pass through a filter having a 0.5-µm or finer porosity.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed portion of USP Xylazine Hydrochloride RSquantitatively in Mobile phaseto obtain a solution having a known concentration of about 1.2mg per mL.Transfer 5.0mLof this solution to a 50-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.Pass a portion of this solution through a filter having a 0.5-µm or finer porosity,discarding the first 3mLof the filtrate.Use the clear filtrate as the Standard preparation.This solution contains about 0.12mg of USP Xylazine Hydrochloride RSper mL.

Assay preparation— Transfer an accurately measured volume of Injection,equivalent to about 200mg of xylazine,to a 100-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.Transfer 5.0mLof this solution to a second 100-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.Pass a portion of this solution through a filter having a 0.5-µm or finer porosity,discarding the first 3mLof the filtrate.Use the clear filtrate as the Assay preparation.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector,a 2-mm ×2-cm guard column that contains packing L2,and a 4.6-mm ×25-cm analytical column that contains packing L1and is maintained at a constant temperature of about 40.The flow rate is about 1mLper minute.Chromatograph the Assay preparation,and record the peak responses as directed for Procedure:the resolution,R,between the main xylazine peak and the closest eluting other peak,if any,is not less than 2.5;and the tailing factor for the xylazine peak is not more than 2.0.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative standard deviation for replicate injections is not more than 2.0%.[NOTE—After daily use,rinse the column with 100mLof water and with 100mLof methanol,and store the column containing methanol.]

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of xylazine (C12H16N2S)in each mLof the Injection taken by the formula: in which 220.34and 256.80are the molecular weights of xylazine and xylazine hydrochloride,respectively;Cis the concentration,in mg per mL,of USP Xylazine Hydrochloride RSin the Standard preparation;Vis the volume,in mL,of Injection taken to prepare the Assay preparation;and rUand rSare the areas of the xylazine peak responses in the chromatograms obtained from the Assay preparationand the Standard preparation,respectively.