Packaging and storage- Preserve in single-dose or in multipledose containers, preferably of Type I glass. Injection labeled to contain 0.5% or less of bupivacaine hydrochloride may be packaged in 50-mL multiple-dose containers. 

USP Reference standards- USP Bupivacaine Hydrochloride RS. USP Endotoxin RS. 

Identification: 

A: Dilute a volume of Injection, equivalent to about 50 mg of bupivacaine hydrochloride, with 0.01 N hydrochloric acid to 25 mL, and proceed as directed under Identification-Organic Nitrogenous Bases, 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 preparation corresponds to that of the bupivacaine peak in the chromatogram of the Standard preparation obtained as directed in the Assay. 

Bacterial endotoxins: It contains not more than 2.5 USP Endotoxin Units por mg of bupivacaíne hydrochloride. 

pH: between 4.0 and 6.5. 

Other requirements:  It meets the requirements under Injections (Particulate matter, Sterility) 

Assay:  

pH 6.8 phosphate buffer- Dissolve 1.94 g of monobasic potassium phosphate and 2.48 g of dibasic potassium phosphate in 1000 mL of water. Adjust, if necessary, with 1 N potassium hydroxide or 1 N phosphoric acid to a pH of 6.8. 

Mobile phase-Prepare a fresh solution of acetonitrile and pH 6.8 phosphate buffer (65:35). Adjust, if necessary, with 1 N phosphoric acid to a pH of 7.7 ± 0.2. Filter the solution through a membrane filter of 1 mm or finer porosity, and degas. 

Internal standard solution-Prepare a solution of dibutylphthalate in methanol containing about 1.3 mg por mL. 

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

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

Chromatographic system- The liquid chromatograph is equipped with a 263-nm detector and a 4-mm X 30-cm column that contains packing LI. The flow rate is about 2 mL por minute. Chromatograph three replicate injections of the Standard preparation, and record the peak responses as directed under 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 factor between bupivacaine hydrochloride and dibutyl phthalate is not less than 2.0. 

Procedure- Separately inject equal volumes (about 20 mL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. The relative retention times are about 1.2 for dibutyl phthalate and 1.0 for bupivacaine hydrochloride. Calculate the quantity, in mg, of C₁₈H₂₈N₂O.HCl in the volume of Injection taken by the formula: 

W(R_U/R_S), 

in which W is the weight, in mg, of USP Bupivacaine Hydrochloride RS, calculated on the anhydrous basis, in the Standard preparation, and R_U and R_S are the ratios of the peak responses of bupívacaine hydrochloride to those of the internal standard obtained from the Assay preparation and the Standard preparation, respectively.