50A322/26,600b,

50A282/22,325b,

Citrate buffer— Dissolve 19.69g of sodium citrate dihydrate,0.1mLof pentachlorophenol,and 5mLof 2,2¢-thiodiethanol in 900mLof 0.2Nhydrochloric acid,adjust with hydrochloric acid to a pHof 2.2,dilute with water to 1000mL,and mix.

Ninhydrin reagent— Dissolve 18g of ninhydrin and 0.7g of hydrindantin in 675mLof dimethyl sulfoxide,add 225mLof 4Mlithium acetate solution previously adjusted with glacial acetic acid to a pHof 5.2,and mix.

Standard preparation— Dissolve an accurately weighed quantity of USPL-Lysine Hydrochloride RSinCitrate buffer to obtain a solution having a known concentration of about 91µg per mL(5×10-4M).

Test preparation— Transfer 1.0mLof Concentrate to a 10-mLvolumetric flask,dilute with water to volume,and mix.Transfer 1.0mLof this solution to an ampul,add 1.5mLof 6Nhydrochloric acid,and seal the ampul under nitrogen.Heat the ampul at 110for 22hours.Transfer the contents of the ampul to a round-bottom,50-mLflask,and dry by vacuum rotary evaporation.Dissolve the residue three times,using 5-mLportions of water,evaporating to dryness after each dissolution.Dissolve the residue in 10mLofCitrate buffer.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 1.75-mm ×50-cm column that contains a packing of 8-µm 8%cross-linked sulfonated divinylbenzene polystyrene cation-exchange resin.The column effluent is mixed continuously with flowingNinhydrin reagent,and the flowing mixture is heated at 130for 1.5minutes in a reaction coil.The absorbance of the reaction mixture is measured continuously by a 570-nm detector.Chromatograph theStandard preparation,and record the peak responses as directed forProcedure:the column efficiency determined from the analyte peak is not less than 1800theoretical plates,and the relative standard deviation for replicate injections is not more than 4.0%.

Procedure— Separately inject equal volumes (about 20µL)of theStandard preparation and theTest preparation into the chromatograph,record the chromatograms,and measure the responses for the major peaks.The retention time is about 57minutes for L-lysine.Calculate the molar concentration of lysine in the Concentrate taken by the formula: in which Cis the concentration,in µg per mL,of USPL-Lysine Hydrochloride RSin theStandard preparation;182.65is the molecular weight of anhydrous lysine hydrochloride;and rUand rSare the peak responses obtained from theTest preparation and theStandard preparation,respectively.Calculate the percentage of benzylpenicilloyl substitution taken by the formula: in which Bis the molar concentration of benzylpenicilloyl moiety in the Concentrate,as determined in theAssay;and Lis the molar concentration of lysine in the Concentrate:not less than 50%and not more than 70%is found.

Saline phosphate buffer— Dissolve 9g of sodium chloride and 1.38g of monobasic sodium phosphate in 900mLof water,adjust with 5Nsodium hydroxide or phosphoric acid to a pHof 7.6,dilute with water to 1000mL,and mix.

Mercuric chloride solution— Dissolve 35mg of mercuric chloride in 500mLof water,and mix.

Assay preparation— Transfer 1.0mLof Concentrate to a 500-mLvolumetric flask,dilute withSaline phosphate buffer to volume,and mix.

Procedure— Transfer 3.0mLofAssay preparation to a spectrophotometric cell.Using a suitable spectrophotometer and usingSaline phosphate buffer as the blank,determine the initial absorbance at the wavelength of maximum absorbance at about 282nm.Add 0.02mLofMercuric chloride solution to theAssay preparation in the spectrophotometric cell,mix,and determine the absorbance at the same wavelength after 1and 3minutes.Repeat the addition of 0.02-mLportions ofMercuric chloride solution until a maximum absorbance reading is obtained.Calculate the molar concentration of benzylpenicilloyl moiety in the Concentrate taken by the formula: in which Amis the highest absorbance observed;Aiis the initial absorbance,nis the number of 0.02-mLportions ofMercuric chloride solution added to theAssay preparation to obtain the maximum absorbance;22,325is the molar absorptivity of the penamaldate formed by the reaction of benzylpenicilloyl with mercuric chloride at pH7.6;and bis the length of the cell,in cm:between 0.0125Mand 0.020Mis found.