A: Dilute a volume of Injection,equivalent to about 50mg of bupivacaine hydrochloride,with 0.01Nhydrochloric acid to 25mL,and proceed as directed under Identification—Organic Nitrogenous Bases á181ñ,beginning with “Transfer the liquid to a separator.”The Injection meets the requirements of the test.

B: The retention time of the bupivacaine peak in the chromatogram of the Assay preparationcorresponds to that of the bupivacaine peak in the chromatogram of the Standard preparation,as obtained in the Assay.

pH6.8Phosphate buffer— Dissolve 1.94g of monobasic potassium phosphate and 2.48g of dibasic potassium phosphate in 1000mLof water.Adjust,if necessary,with 1Npotassium hydroxide or 1Mphosphoric acid to a pHof 6.8.

Mobile phase— Prepare a fresh solution of acetonitrile and pH6.8Phosphate buffer(65:35).Adjust,if necessary,with 1Mphosphoric acid to a pHof 7.7±0.2.Filter the solution through a membrane filter of 1-µm or finer porosity,and degas.

Internal standard solution— Prepare a solution of dibutyl phthalate in methanol containing about 1.3mg per mL.

Standard preparation— Dissolve about 50mg of USP Bupivacaine Hydrochloride RS,accurately weighed,in 10.0mLof water,using sonication if necessary,in a 100-mLvolumetric flask.Add 10mLof Internal standard solution,dilute with methanol to volume,and mix.

Assay preparation— Transfer an accurately measured volume of Injection,equivalent to about 50mg of bupivacaine hydrochloride,to a 100-mLvolumetric flask,add 10.0mLof Internal standard solution,dilute with methanol to volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 263-nm detector and a 4-mm ×30-cm column that contains packing L1.The flow rate is about 2mLper minute.Chromatograph three replicate injections of the Standard preparation,and record the peak responses as directed for Procedure:the relative standard deviation of the ratios of the bupivacaine hydrochloride peak to the dibutyl phthalate peak is not more than 1.0%,and the resolution R,factor,between bupivacaine hydrochloride and dibutyl phthalate is not less than 2.0.

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.The relative retention times are about 1.2for dibutyl phthalate and 1.0for bupivacaine hydrochloride.Calculate the quantity,in mg,of C18H28N2O·HCl in the volume of Injection taken by the formula: in which Wis the weight,in mg,of USP Bupivacaine Hydrochloride RS,calculated on the anhydrous basis,in the Standard preparation,and RUand RSare the ratios of the peak responses of bupivacaine hydrochloride to those of the internal standard obtained from the Assay preparationand the Standard preparation,respectively.