A: Infrared Absorption á197Kñ.

B: The retention times of the three main isomeric peaks in the chromatogram of the Assay preparationcorrespond to those in the chromatogram of the Standard preparation,as obtained in the Assay.

Buffer solution,Solution A,Solution B,and Mobile phase— Prepare as directed in the Assay.

Standard solution— Prepare a solution of methyl benzenesulfonate in acetonitrile having a known concentration of about 0.2mg per mL.Quantitatively dilute a portion of this solution with Solution Ato obtain a solution having a known concentration of about 1µg per mL.

Test solution— Transfer about 100mg of Atracurium Besylate,accurately weighed,to a 10-mLvolumetric flask,dissolve in and dilute withSolution Ato volume,and mix.

Resolution solution— Transfer 1mLof the Test solutionand 5mLof a solution containing 0.2mg of methyl benzenesulfonate per mLto a 100-mLvolumetric flask,dilute with Solution Ato volume,and mix.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 217-nm detector and a 4.6-mm ×25-cm column that contains base-deactivated packing L1.The flow rate is about 1mLper minute.The chromatograph is programmed as follows. Chromatograph the Resolution solution,and record the peak responses as directed for Procedure:the resolution,R,between the trans-transisomer and methyl benzenesulfonate is not less than 12.0.Chromatograph duplicate injections of the Standard solution,and record the peak responses as directed for Procedure:the responses for duplicate injections do not differ from each other by more than 12%.

Procedure— Separately inject equal volumes (about 100µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the responses for the methyl benzenesulfonate peaks:the peak response obtained from the Test solutionis not greater than that obtained from the Standard solution.Not more than 0.01%of methyl benzenesulfonate is found.

Test Solution— Prepare as directed for Method Iunder Organic Volatile Impurities á467ñ.

Standard solution— Prepare a solution containing 100µg of toluene per mL.

Chromatographic System— Proceed as directed for Method Iunder Organic Volatile Impurities á467ñ,except to use the Standard solution.

Procedure— Separately inject equal volumes (about 1µL)of the Standard solutionand the Test Solutioninto the chromatograph,record the chromatograms,and measure the responses for the major peaks:the toluene peak from the Test Solutionis not greater than the toluene peak obtained from the Standard solution.Not more than 0.5%of toluene is found.

Buffer solution,Solution A,Solution B,and Mobile phase— Proceed as directed in the Assay.

Standard solution— Transfer 1.0mLof the Standard preparation,prepared as directed in the Assay,to a 100-mLvolumetric flask,dilute with Solution Ato volume,and mix.

Test solution— Use the Assay preparation.

Chromatographic system (see Chromatography á621ñ)— Prepare as directed in the Assay.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the responses of the cis-cisisomers from not fewer than two injections do not differ by more than 10%.

Procedure— Separately inject equal volumes (about 20µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure all of the peak responses,except the three main isomeric peaks.Calculate the percentage of each impurity in the portion of Atracurium Besylate taken by the formula: in which Cis the concentration,in mg per mL,of the cis-cisisomer in the Standard solution;Wis the weight,in mg,of Atracurium Besylate taken to prepare the Test solution;riis the peak response for each impurity obtained from the Test solution;and rSis the peak response for the cis-cisisomer obtained from the Standard solution:not more than 1.5%of any individual impurity is found,and not more than 3.5%of total impurities is found.

Buffer solution— Transfer about 10.2g of monobasic potassium phosphate to a 1000-mLvolumetric flask,and dissolve in about 950mLof water.While stirring,adjust with phosphoric acid to a pHof 3.1,dilute with water to volume,and mix.

Solution A— Prepare a mixture of Buffer solution,acetonitrile,and methanol (75:20:5).

Solution B— Prepare a mixture of Buffer solution,methanol,and acetonitrile (50:30:20).

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

Standard preparation— Dissolve an accurately weighed quantity of USP Atracurium Besylate RSin Solution Ato obtain a solution having a known concentration of about 1.0mg per mL.

Assay preparation— Transfer about 100mg of Atracurium Besylate,accurately weighed,to a 100-mLvolumetric flask,dissolve in and dilute with Solution Ato volume,and mix.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 280-nm detector and a 4.6-mm ×25-cm column that contains base-deactivated packing L1.The flow rate is about 1mLper minute.The chromatograph is programmed as follows. Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative retention times are about 0.8,0.9,and 1.0for the trans-transisomer,the cis-transisomer,and the cis-cisisomer,respectively;the resolution,R,between the trans-transisomer and the cis-transisomer and between the cis-transisomer and the cis-cisisomer is not less than 1.1;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 three isomeric peaks.Calculate the quantity,in mg,of C65H82N2O18S2in the portion of Atracurium Besylate taken by the formula: in which Cis the concentration,in mg per mL,of USP Atracurium Besylate RSin the Standard preparation;and rUand rsare the sums of the peak responses for the trans-transisomer,the trans-cisisomer,and the cis-cisisomer obtained from the Assay preparationand the Standard preparation,respectively.