A: The IRabsorption spectrum of a potassium bromide dispersion of it exhibits maxima only at the same wavelengths as that of a similar preparation of USP Cephalexin RS.

B: The UVabsorption spectrum of a solution (1in 50,000)exhibits maxima and minima at the same wavelengths as that of a similar solution of USP Cephalexin RS,concomitantly measured,and the absorptivity,calculated on the anhydrous basis,at the wavelength of maximum absorbance at about 262nm is not less than 95.0%and not more than 104.0%of that of USP Cephalexin RS,the potency of the Reference Standard being taken into account.

C: Prepare a solution of it in water,with the aid of 0.1Nhydrochloric acid,containing 25mg per mL(test solution).Separately apply 5µLof the test solution and 5µLof a Standard solution containing about 25mg of USP Cephalexin RSper mL,prepared by dissolving it in water with the aid of 0.1Nhydrochloric acid,to a suitable thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel mixture.Allow the spots to dry,place the plate in a saturated chamber containing a solvent system consisting of a mixture of ethyl acetate,water,acetonitrile,and glacial acetic acid (42:18:14:14)and lined with filter paper.Develop the chromatogram until the solvent front has moved about three-fourths of the length of the plate.Remove the plate from the developing chamber,mark the solvent front,allow the plate to air-dry,and examine under short-wavelength UVlight:the RFvalue of the principal spot obtained from the test solution corresponds to that obtained from the Standard solution.

Test solution: 5mg per mL,in pH4.4neutralized phthalate buffer (see Buffer Solutionsin the section Reagents,Indicators,and Solutions).

Solution A— Dissolve 1g of sodium 1-pentanesulfonate in a mixture of 1000mLof water and 15mLof triethylamine.Adjust with phosphoric acid to a pHof 2.5±0.1.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Solution B— Dissolve 1g of sodium 1-pentanesulfonate in a mixture of 300mLof water and 15mLof triethylamine.Adjust with phosphoric acid to a pHof 2.5±0.1,add 350mLof acetonitrile and 350mLof methanol,and mix.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Mobile phase— Use variable mixtures of Solution Aand Solution Bas directed for Chromatographic System.

Solvent— Dissolve 18g of monobasic potassium phosphate in 1000mLof water.

Standard solutions— Dissolve accurately weighed quantities of USP Cephalexin RSquantitatively in Solventto obtain solutions having known concentrations of about 0.08and 0.16mg of cephalexin (C16H17N3O4S)per mL,respectively,taking into account the stated potency of the USP Cephalexin RS.

Test solution— Transfer about 25mg of Cephalexin,accurately weighed,to a 5-mLvolumetric flask,dissolve in and dilute with Solventto 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 packing L1of low acidity.The flow rate is about 1mLper minute.The chromatograph is programmed as follows.

Procedure— Separately inject equal volumes (about 20µL)of the Standard solutionsand the Test solutioninto the chromatograph,record the chromatograms,and measure the responses for the cephalexin peaks in the chromatograms obtained from the Standard solutions and for all of the peaks,other than that from cephalexin,in the chromatogram obtained from the Test solution.Plot the responses of the cephalexin peaks in the chromatograms obtained from the Standard solutionsversus their concentrations,calculated on the anhydrous basis,in mg per mL,and draw a straight line through the two points and zero.From the line so obtained and the peak responses obtained from the Test solution,determine the concentration,I,in mg per mL,of each cephalexin-related substance obtained from the Test solutionother than the cephalexin peak.Calculate the percentage of each cephalexin-related substance by the formula: in which Wis the quantity,calculated on the anhydrous basis,in mg,of Cephalexin taken to prepare the Test solution:not more than 1.0%of any individual cephalexin-related substance is found;and the sum of all cephalexin-related substances found is not greater than 5.0%.

Mobile phase— Prepare 1015mLof a suitable mixture of water,acetonitrile,methanol,and triethylamine (850:100:50:15).Dissolve 1.0g of sodium 1-pentanesulfonate in this mixture,adjust with phosphoric acid to a pHof 3.0±0.1,and degas.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Internal standard solution— Transfer 300mg of 1-hydroxybenzotriazole to a 1000-mLvolumetric flask,dissolve in 10mLof methanol,dilute with Mobile phaseto volume,and mix.

Standard preparation— Dissolve an accurately weighed quantity of USP Cephalexin RSquantitatively in water to obtain a stock solution having a known concentration of about 1mg per mL.Transfer 10.0mLof this stock solution to a 50-mL,glass-stoppered flask,add 15.0mLof Internal standard solution,and mix.

Assay preparation— Transfer about 100mg of Cephalexin,accurately weighed,to a 100-mLvolumetric flask,dissolve in and dilute with water to volume,and mix.Transfer 10.0mLof this solution to a 50-mL,glass-stoppered flask,add 15.0mLof Internal standard solution,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 packing L1of low acidity.The flow rate is about 1.5mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed under Procedure:the resolution,R,between the internal standard and the analyte peaks is not less than 5,and the relative standard deviation for replicate injections is not more 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 0.35for 1-hydroxybenzotriazole and 1.0for cephalexin.Calculate the quantity,in µg,of C16H17N3O4Sper mg of the Cephalexin taken by the formula: in which Cis the concentration,in mg per mL,ofUSP Cephalexin RSin the stock solution used to prepare the Standard preparation,Pis the stated cephalexin content,in µg per mg,of USP Cephalexin RS,Mis the quantity,in mg,of Cephalexin taken to prepare the Assay preparation,and RUand RSare the ratios of the responses of the cephalexin peak to the 1-hydroxybenzotriazole peak obtained from the Assay preparationand the Standard preparation,respectively.