0.005M Tetrabutylammonium hydroxide— Dilute 6.6mLof a 40%solution of tetrabutylammonium hydroxide with water to obtain 1800mLof solution.Adjust with 1Mphosphoric acid to a pHof 5.0±0.1,dilute with water to 2000mL,and mix.

Mobile phase— Prepare a filtered and degassed mixture of 0.005M Tetrabutylammonium hydroxideand acetonitrile (1650:350).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Quantitatively dissolve accurately weighed quantities of USP Ampicillin RSand USP Sulbactam RSin Mobile phaseto obtain a solution having known concentrations of about 0.6mg of ampicillin per mLand 0.3mg of sulbactam per mL.[NOTE—Inject this solution promptly.]

Resolution solution— Prepare a solution of USP Sulbactam RSin 0.01Nsodium hydroxide containing 0.3mg per mL,and allow to stand for 30minutes.Adjust with phosphoric acid to a pHof 5.0±0.1.Transfer 5mLof the solution to a 25-mLvolumetric flask,add 4.25mLof acetonitrile,dilute with 0.005M Tetrabutylammonium hydroxideto volume,and mix.Transfer 1mLof this solution to a second 25-mLvolumetric flask,add 15mg of USP Ampicillin RS,dilute with Mobile phaseto volume,and mix.[NOTE—Inject this solution promptly.]

Assay preparation 1— Mix the contents of a container of Ampicillin and Sulbactam for Injection.Quantitatively dissolve an accurately weighed portion of the powder in Mobile phaseto obtain a solution having a concentration of about 1mg of the powder per mL.[NOTE—Inject this solution promptly.]

Assay preparation 2(where it is represented as being in a single-dose container)— Constitute a container of Ampicillin and Sulbactam for Injection with a volume of water,accurately measured,corresponding to the volume of solvent specified in the labeling.Withdraw the total withdrawable contents from the container,using a suitable hypodermic needle and syringe,and dilute quantitatively,and stepwise if necessary,with Mobile phaseto obtain a solution containing about 0.6mg of ampicillin per mLand 0.3mg of sulbactam per mL.[NOTE—Inject this solution promptly.]

Assay preparation 3(where the label states the quantities of ampicillin and sulbactam in a given volume of constituted solution)— Constitute a container of Ampicillin and Sulbactam for Injection with a volume of water,accurately measured,corresponding to the volume of solvent specified in the labeling.Dilute an accurately measured volume of the constituted solution quantitatively,and stepwise if necessary,with Mobile phaseto obtain a solution containing about 0.6mg of ampicillin per mLand 0.3mg of sulbactam per mL.[NOTE—Inject this solution promptly.]

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 230-nm detector and a 4-mm ×30-cm column containing packing L1.The flow rate is about 2mLper minute.Chromatograph the Resolution solution,and record the responses as directed for Procedure:the relative retention times are about 0.7for ampicillin and 1.0for sulbactam alkaline degradation product;and the resolution,R,between ampicillin and sulbactam alkaline degradation product is not less than 4.0.Chromatograph the Standard preparation,and record the responses as directed for Procedure:the relative retention times are about 0.35for ampicillin and 1.0for sulbactam;the column efficiency determined from the sulbactam peak is not less than 3500theoretical plates;the tailing factor is not more than 1.5;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard preparationand the appropriate Assay preparationinto the chromatograph,record the chromatograms,and measure the areas for the major peaks.Calculate the quantities,in µg,of ampicillin (C16H19N3O4S)and of sulbactam (C8H11NO5S)in the portion of Ampicillin and Sulbactam for Injection taken by the same formula: in which CSis the concentration,in mg per mL,of the appropriate USP Reference Standard in the Standard preparation;Pis the assigned content,in µg per mg,of the appropriate USP Reference Standard;CUis the concentration,in mg per mL,of Ampicillin and Sulbactam for Injection in Assay preparation 1,based on the weight,in mg,of powder removed from the container and the extent of dilution;and rUand rSare the peak areas for the appropriate analyte obtained from Assay preparation 1and the Standard preparation,respectively.Calculate the quantities of ampicillin (C16H19N3O4S)and of sulbactam (C8H11NO5S)withdrawn from the container,or in the volume of constituted solution taken by the same formula: in which Lis the labeled quantity,in mg,of ampicillin or sulbactam,as appropriate,in the container or in the volume of constituted solution taken;Dis the concentration,in mg per mL,of ampicillin or sulbactam in Assay preparation 2or Assay preparation 3,on the basis of the labeled quantity,in mg,of ampicillin or sulbactam,as appropriate,in the container and the extent of dilution;rUand rSare the peak areas for the appropriate analyte obtained from Assay preparation 2or Assay preparation 3and the Standard preparation,respectively;and the other terms are as defined above.