A: Thin-Layer Chromatographic Identification Test 201—

Mobile phase— Prepare a filtered and degassed mixture of acetonitrile, methanol, and water (530:350:70). Make adjustments if necessary (see System Suitability under Chromatography 621). Increasing the proportion of water increases the resolution.

Standard preparation— Prepare a solution of USP Ivermectin RS in methanol having a known concentration of about 0.3 mg per mL.

Assay preparation— Transfer an accurately measured portion of Injection, equivalent to about 30 mg of ivermectin, to a 100-mL volumetric flask, dissolve in and dilute with methanol to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L1. The flow rate is about 1.2 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure [note—The relative retention times are about 0.8 for component B1b and 1.0 for component B1a.]: the resolution, R, between component B1b and component B1a is not less than 2.0; the column efficiency determined from the component B1a peak is not less than 2000 theoretical plates; and the relative standard deviation for replicate injections determined for the component B1a peak is not more than 1.0%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the area responses for the major peaks. Calculate the quantity, in mg, of ivermectin component B1a (C48H74O14) plus ivermectin component B1b (C47H72O14) in the portion of Injection taken by the formula: in which C is the concentration, in mg per mL, of USP Ivermectin RS in the Standard preparation; P is the designated percentage of the sum of component B1a plus component B1b in USP Ivermectin RS; and rU and rS are the sums of the peak area responses for ivermectin component B1a plus ivermectin component B1b obtained from the Assay preparation and the Standard preparation, respectively.

Mobile phase— Prepare a filtered and degassed mixture of chloroform, methanol, and water (900:100:1). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Prepare a solution of USP Clorsulon RS in methanol having a known concentration of about 2.4 mg per mL. Transfer 5.0 mL of this solution to a 50-mL volumetric flask, dilute with chloroform to volume, and mix.

Assay preparation— Transfer an accurately measured portion of Injection, equivalent to about 240 mg of clorsulon, to a 100-mL volumetric flask, dilute with methanol to volume, and mix. Transfer 5.0 mL of this solution to a 50-mL volumetric flask, dilute with chloroform to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L3. The flow rate is about 0.8 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure [note—The relative retention times are about 0.6 for ivermectin (the major components of ivermectin co-elute) and 1.0 for clorsulon.]: the column efficiency determined from the clorsulon peak is not less than 4000 theoretical plates; the tailing factor for the clorsulon peak is not more than 2.0; and the relative standard deviation for replicate injections determined for the clorsulon peak is not more than 1.0%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the area responses for the major peaks. Calculate the quantity, in mg, of clorsulon (C8H8Cl3N3O4S2) in the portion of Injection taken by the formula: in which C is the concentration, in mg per mL, of USP Clorsulon RS in the Standard preparation; and rU and rS are the clorsulon peak area responses obtained from the Assay preparation and the Standard preparation, respectively.