Mobile phase andChromatographic system— Prepare as directed in the Assay.

Standard piperacillin solution— Transfer about 40mg of USP Piperacillin RS,accurately weighed,to a 100-mLvolumetric flask,add a few drops of methanol to dissolve it,dilute with Mobile phaseto volume,and mix.Transfer 5.0mLof this solution to a 50-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.[NOTE—Use these solutions within 1hour.]

Standard ampicillin solution— Prepare a solution of USP Ampicillin RSin Mobile phasehaving a known concentration of about 0.08mg per mL.

Test solution— Use the Assay preparation.

Procedure— Separately inject equal volumes (about 10µL)of the Test solution,the Standard ampicillin solution,and the Standard piperacillin solution,and proceed as directed in the Assay.Calculate the percentage of ampicillin in the portion of Piperacillin taken by the formula: in which Cis the concentration,in mg per mL,of USP Ampicillin RSin the Standard ampicillin solution;Pis the designated potency,in µg of ampicillin per mg,of USP Ampicillin RS;Wis the weight,in mg,of Piperacillin taken to prepare the Test solution;and rUand rSaare the peak responses of ampicillin obtained from the Test solutionand the Standard ampicillin solution,respectively:not more than 0.2%of ampicillin is found.

Mobile phase— Prepare a mixture of methanol,water,0.2Mmonobasic sodium phosphate,and 0.4Mtetrabutylammonium hydroxide (615:282:100:3).Adjust with phosphoric acid to a pHof 5.50±0.02,and degas.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard stock solution— Transfer about 20mg of USP Piperacillin RS,accurately weighed,to a 50-mLvolumetric flask,add a few drops of methanol to dissolve it,dilute with Mobile phaseto volume,and mix.[NOTE—Use this solution within 1hour.]

Standard solution— Transfer 5.0mLof the Standard stock solutionto a 50-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.[NOTE—Use this solution within 1hour.]

Test solution— Transfer about 40mg of Piperacillin,accurately weighed,to a 100-mLvolumetric flask,add a few drops of methanol to dissolve it,dilute with Mobile phaseto volume,and mix.[NOTE—Use this solution within 1hour.]

Chromatographic system (seeChromatography á621ñ)— The liquid chromatograph is equipped with a 220-nm detector and a 4.6-mm ×25-cm column that contains packing L1.The flow rate is about 1mLper minute.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the relative retention times are about 1.0for piperacillin and 2.55for 6-[2-[6-[2-(4-ethyl-2,3-dioxo-1-piperazinecarboxamido)-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxamido]-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid (piperacillin related compound D);and the relative standard deviation for replicate injections is not more than 2%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the peak responses for piperacillin and piperacillin related compound D.Calculate the percentage of piperacillin related compound Din the portion of Piperacillin taken by the formula: in which Cis the concentration,in mg per mL,of USP Piperacillin RSin the Standard solution;1.47is the relative response factor for piperacillin related compound D;Pis the designated potency,in µg of piperacillin per mg,of USP Piperacillin RS;Wis the weight,in mg,of Piperacillin taken to prepare the Test solution;riis the impurity peak response obtained from the Test solution;and rSis the peak response of piperacillin obtained from the Standard solution:not more than 2.0%of piperacillin related compound Dis found.The sum of all impurities found in Test 1and Test 2is not more than 3.8%.

100(CP/W)(rU/rS),