A: Thin-Layer Chromatographic Identification Test á201ñ—

B: The retention time and UVspectrum of the major peak in the chromatogram of the Assay preparationcorrespond to those in the chromatogram of the Standard preparation,as obtained in the Assay.

Medium: pH5.0buffer,prepared by mixing 10.2g of citric acid monohydrate and 18.16g of dibasic sodium phosphate dihydrate with 1liter of water;900mL.

Apparatus 2: 75rpm.

Time: 30minutes.

Buffer solution— Prepare a monobasic potassium phosphate solution (1.5in 1000),and adjust with phosphoric acid to a pHof 2.3.

Mobile phase— Prepare a filtered and degassed mixture of acetonitrile,Buffer solution,and methanol (49:40:11).

Standard solution— Transfer about 22mg of USP Repaglinide RS,accurately weighed,to a 100-mLvolumetric flask,dissolve in and dilute with methanol to volume,and mix.Transfer 5.0mLof this solution to a 100-mLvolumetric flask,and dilute with methanol to volume.Transfer 5.0mLof the resulting solution to a 100-mLvolumetric flask,add 25mLof methanol,dilute with Mediumto volume,and mix.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a fluorometric detector,set at an excitation wavelength of 244nm and an emission wavelength of 348nm,and a 4.0-mm ×12.5-cm column that contains 10-µm packing L1.The column temperature is maintained at 40.The flow rate is about 1.0mLper minute.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the capacity factor,k¢,is about 1.8;the tailing factor is between 0.5and 2.0;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20µL)of the Standard solutionand a filtered portion of the solution under test into the chromatograph,record the chromatograms,and measure the areas for the major peaks.Calculate the quantity of C27H36N2O4dissolved by comparing the measured peak responses of the Standard solutionand the solution under test.

Tolerances— Not less than 70%(Q)of the labeled amount of C27H36N2O4is dissolved in 30minutes.

pH4.0Phosphate buffer,pH2.5Phosphate buffer,Diluent,and Mobile phase— Proceed as directed in the Assay.

Standard solution 1,Standard solution 2,and System suitability solution— Proceed as directed in the Assayfor Standard preparation 1,Standard preparation 2,and System suitability preparation,respectively.

Standard solution 3— Transfer 2.5mLof Standard solution 2to a 1-liter volumetric flask,dilute with Diluentto volume,and mix.

Test solution— Use the Assay preparation.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 210-nm diode array detector and a 4.0-mm ×6-cm column that contains 5-µm packing L1.The column temperature is maintained at 40.The flow rate is about 1.0mLper minute.Chromatograph the System suitability solution,and record the peak responses as directed for Procedure:the capacity factors,k¢,for repaglinide and repaglinide related compound Aare about 4.9and 1.2,respectively;the resolution,R,between the two peaks is not less than 7.0;and the tailing factor is between 0.8and 2.0.Chromatograph Standard solution 3,and record the peak responses as directed for Procedure:the relative standard deviation for replicate injections is not more than 10%.

Procedure— Separately inject equal volumes (about 20µL)of Standard solution 2and the Test solutioninto the chromatograph,record the chromatograms,and measure all of the peak responses.Calculate the percentage of each impurity in the portion of Tablets taken by the formula: in which riis the peak response for each impurity in the Test solution;and rSis the repaglinide peak response obtained from Standard solution 2:not more than 0.5%of total impurities is found.

pH4.0Phosphate buffer— Prepare a monobasic ammonium phosphate solution (2in 1000),and adjust with phosphoric acid to a pHof 4.0.

pH2.5Phosphate buffer— Prepare a monobasic ammonium phosphate solution (2in 1000),and adjust with phosphoric acid to a pHof 2.5.

Diluent— Prepare a mixture of methanol and pH4.0Phosphate buffer(7:3).

Mobile phase— Prepare a filtered and degassed mixture of methanol and pH2.5Phosphate buffer(7:3).

Standard preparation 1— Dissolve an accurately weighed quantity of USP Repaglinide RSin methanol to obtain a solution having a known concentration of about 800µg per mL.

Standard preparation 2— Transfer 5.0mLof Standard preparation 1to a 50-mLvolumetric flask,dilute with Diluentto volume,and mix.

System suitability preparation— Dissolve an accurately weighed quantity of USP Repaglinide Related Compound A RSin methanol to obtain a solution having a known concentration of about 80µg per mL.Transfer 1.0mLof this solution to a 50-mLvolumetric flask,add 5.0mLof Standard preparation 1,dilute with Diluentto volume,and mix.

Assay preparation— Transfer 8whole Tablets to a suitable volumetric flask,and dissolve in and dilute with Diluentto volume to obtain a solution having a concentration of about 80µg per mL.Stir for 20minutes with the aid of a magnetic stirrer,and filter a portion of the solution.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 245-nm diode array detector and a 4.0-mm ×6-cm column that contains 5-µm packing L1.The column temperature is maintained at 40.The flow rate is about 1.0mLper minute.Chromatograph the System suitability preparation,and record the peak responses as directed for Procedure:the capacity factors,k¢,for repaglinide and repaglinide related compound Aare about 4.9and 1.2,respectively;the resolution,R,between the two peaks is not less than 7.0;and the tailing factor is between 0.8and 2.0.Chromatograph Standard preparation 2,and record the peak responses as directed for Procedure:the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20µL)of Standard preparation 2and the Assay preparationinto the chromatograph,record the chromatograms,and measure the areas for the major peaks.Calculate the quantity,in mg,of repaglinide (C27H36N2O4)in each of the Tablets taken by the formula: in which Vis the volume,in mL,of Diluentused in the Assay preparation;Cis the concentration,in mg per mL,of USP Repaglinide RSin Standard preparation 2;and rUand rSare the peak responses obtained from the Assay preparationand Standard preparation 2,respectively.