A: The retention time of the major peak for cilastatin in the chromatogram of the Test solution,as obtained in the test for Chromatographic purity,corresponds to that in the chromatogram of a similar preparation of USP Cilastatin Ammonium Salt RS.

B: Ignite a small portion of it on a platinum wire in a nonluminous flame:an intense yellow color is imparted to the flame.

Test solution: 10mg per mL,in a mixture of methanol and hydrochloric acid (120:1).

Internal standard solution— Transfer 0.5mLof n-propyl alcohol to a 1000-mLvolumetric flask,dilute with water to volume,and mix.

Standard solution— Transfer 2.0mLof acetone,0.50mLof methanol,and 0.50mLof mesityl oxide to a 1000-mLvolumetric flask,dilute with water to volume,and mix.Transfer 2.0mLof this solution and 2.0mLof Internal standard solutionto a 10-mLvolumetric flask,dilute with water to volume,and mix.This solution contains 316µg of acetone,79µg of methanol,and 86µg of mesityl oxide per mL.

Test solution— Transfer about 200mg of Cilastatin Sodium,accurately weighed,to a 10-mLvolumetric flask,add 2.0mLof Internal standard solutionand about 5mLof water,and dissolve by shaking.Dilute with water to volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The gas chromatograph is equipped with a flame-ionization detector and a 0.53-mm ×30-m capillary column,the internal wall of which is coated with a 1.0-µm film of liquid phase G16.The column temperature is maintained at 50for 2.5minutes,then increased at a rate of 8per minute to 70,and maintained at 70for 0.5minute;the injection port temperature is maintained at 160;the detector temperature is maintained at 250;and helium is used as the carrier gas at a flow rate of about 9mLper minute.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the relative retention times are about 0.26for acetone,0.35for methanol,0.67for n-propyl alcohol,and 1.0for mesityl oxide;and the relative standard deviation for replicate injections,determined from peak area ratios of each analyte to n-propyl alcohol,is not more than 5.0%.

Procedure— Separately inject equal volumes (about 1µL)of the Standard solutionand the Test solutioninto the chromatograph,using the solvent (water)flush technique;record the chromatograms;and measure the areas for the acetone,methanol,n-propyl alcohol,and mesityl oxide peaks.Calculate the percentages of acetone,methanol,and mesityl oxide in the portion of Cilastatin Sodium taken by the formula: in which Cis the concentration,in µg per mL,of the appropriate analyte in the Standard solution;Wis the quantity,in mg,of Cilastatin Sodium taken to prepare the Test solution;and RUand RSare the peak area ratios of the corresponding analyte to n-propyl alcohol obtained from the Test solutionand the Standard solution,respectively.Not more than 1.0%of acetone is found;not more than 0.5%of methanol is found;and not more than 0.4%of mesityl oxide is found.

Solvent— Use water.

Solution A— Prepare a mixture of dilute phosphoric acid (1in 1000)and acetonitrile (700:300),pass through a filter having a 0.5-µm or finer porosity,and degas.

Solution B— Use dilute phosphoric acid (1in 1000).Pass through a filter having a 0.5-µm or finer porosity,and degas.

Mobile phase— Use variable mixtures of Solution Aand Solution Bas directed for Chromatographic system.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Test solution— Prepare a solution of Cilastatin Sodium in Solventhaving a concentration of about 1.6mg per mL.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 210-nm detector and a 4.5-mm ×25-cm column containing packing L1.The column is maintained at a constant temperature of about 50.The flow rate is about 2mLper minute.The chromatograph is programmed as follows. Chromatograph the Test solution,and measure the peak responses as directed for Procedure:the capacity factor,k¢,is not less than 10;the column efficiency determined from the cilastatin peak is not less than 3000theoretical plates;and the tailing factor is not more than 4.5.

Procedure— Separately inject equal volumes (about 20µL)of the Test solutionand the Solventinto the chromatograph,record the chromatograms,and measure the areas of the peaks.Calculate the chromatographic purity,in percentage,of the portion of Cilastatin Sodium taken by the formula: in which rCis the area of the cilastatin peak obtained from the Test solution;rTis the sum of the areas of all the peaks obtained from the Test solution;rBis the sum of the areas of all the peaks obtained from the Solvent;and rAis the response of the peak,if any,of nonretained substances,such as acetone,at the solvent front obtained from the Test solution:not less than 98.5%is found.Calculate the percentage of each impurity in the portion of Cilastatin Sodium taken by the formula: in which riis the peak area for each impurity in the chromatogram obtained from the Test solutionand the other terms are as defined above:not more than 0.5%of any individual impurity is found.