A: Ultraviolet Absorption á197Uñ—

Solution: 25µg per mL.

Medium: water.

B: It responds to the tests for Sodium á191ñ.

Test solution: a known amount of specimen,equivalent to about 50mg of cephalothin,per mL,in water.

Mobile phase ,Resolution solution,and Chromatographic system—Proceed as directed in the Assay.

Standard solution— Use the Standard preparation,prepared as directed in the Assay,transfer 1.0mLto a 100-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.

Test solution— Use the Assay preparationprepared as directed in the Assay.

Procedure— Proceed as directed for the Procedurein the Assay,except to inject equal volumes (about 20µL)of the Standard solutionand the Test solutionand to continue the chromatography of the Test solutionfor at least 4times the retention time of the main cephalothin peak.The area of any peak in the chromatogram obtained from the Test solution,except the main peak,is not greater than the area of the main peak in the chromatogram obtained from the Standard solution(1.0%),and the sum of the areas of any such peaks is not greater than 3times the area of the main peak in the chromatogram obtained from the Standard solution(3.0%).[NOTE—Any peak in the chromatogram obtained from the Test solution with an area less than one-tenth that of the main peak in the chromatogram obtained from the Standard solution is disregarded.]

Mobile phase— Dissolve 17g of sodium acetate in 790mLof water,add 0.6mLof glacial acetic acid,and if necessary adjust with 0.1Nsodium hydroxide or glacial acetic acid to a pHof 5.9±0.1.Add 150mLof acetonitrile and 70mLof alcohol,and mix.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Cephalothin Sodium RSquantitatively in Mobile phaseto obtain a solution having a known concentration of about 1mg per mL.

Resolution solution— Heat a 5-mLportion of the Standard preparationin a water bath at 90for 10minutes.Cool the solution,and immediately inject a portion of it into the chromatograph as directed under Chromatographic system.

Assay preparation— Transfer about 25mg of Cephalothin Sodium,accurately weighed,to a 25-mLvolumetric flask,add about 15mLof Mobile phase,swirl to dissolve,dilute with Mobile phaseto volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm ×25-cm column that contains 5µm packing L1and is maintained at a constant temperature of about 40.The flow rate is about 1mLper minute.Chromatograph the Resolution solution,and record the peak responses as directed under Procedure:the resolution between the two principal peaks is not less than 9.0.Chromatograph the Standard preparation,and record the peak responses as directed under Procedure:the tailing factor is not more than 1.8,and the relative standard deviation for replicate injections is not more than 1.0%.

Procedure— [NOTE—Use peak areas where peak responses are indicated.]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 µg,of cephalothin (C16H16N2O6S2)in each mg of Cephalothin Sodium taken by the formula: in which Cis the concentration,in mg per mL,of USP Cephalothin Sodium RSin the Standard preparation,Pis the assigned potency,in µg of cephalothin per mg,of USP Cephalothin Sodium RS,Wis the quantity,in mg,of Cephalothin Sodium taken to prepare the Assay preparation,and rUand rSare the cephalothin peak responses obtained from the Assay preparationand the Standard preparation,respectively.