A: Thin-Layer Chromatographic Identification Test 201—

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

Medium: pH 5.0 buffer, prepared by mixing 10.2 g of citric acid monohydrate and 18.16 g of dibasic sodium phosphate dihydrate with 1 L of water; 900 mL.

Apparatus 2: 75 rpm.

Time: 30 minutes.

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

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

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

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a fluorometric detector, set at an excitation wavelength of 244 nm and an emission wavelength of 348 nm, 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 1.0 mL per 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 0.5 and 2.0; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard solution and 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 C27H36N2O4 dissolved by comparing the measured peak responses of the Standard solution and the solution under test.

Tolerances— Not less than 70% (Q) of the labeled amount of C27H36N2O4 is dissolved in 30 minutes.

pH 4.0 Phosphate buffer, pH 2.5 Phosphate 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 Assay for Standard preparation 1, Standard preparation 2, and System suitability preparation, respectively.

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

Test solution— Use the Assay preparation.

Chromatographic system (see Chromatography 621)— 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 1.0 mL per 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 A are about 4.9 and 1.2, respectively; the resolution, R, between the two peaks is not less than 7.0; and the tailing factor is between 0.8 and 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 20 µL) of Standard solution 2 and the Test solution into 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 ri is the peak response for each impurity in the Test solution; and rS is the repaglinide peak response obtained from Standard solution 2: not more than 0.5% of total impurities is found.

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

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

Diluent— Prepare a mixture of methanol and pH 4.0 Phosphate buffer (7:3).

Mobile phase— Prepare a filtered and degassed mixture of methanol and pH 2.5 Phosphate buffer (7:3).

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

Standard preparation 2— Transfer 5.0 mL of Standard preparation 1 to a 50-mL volumetric flask, dilute with Diluent to volume, and mix.

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

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

Chromatographic system (see Chromatography 621)— 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 1.0 mL per 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 A are about 4.9 and 1.2, respectively; the resolution, R, between the two peaks is not less than 7.0; and the tailing factor is between 0.8 and 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 20 µL) of Standard preparation 2 and the Assay preparation into 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 V is the volume, in mL, of Diluent used in the Assay preparation; C is the concentration, in mg per mL, of USP Repaglinide RS in Standard preparation 2; and rU and rS are the peak responses obtained from the Assay preparation and Standard preparation 2, respectively.