A: Thin-Layer Chromatographic Identification Test 201—

Test solution —Disperse a quantity of Ivermectin Paste containing about 5 mg of ivermectin, accurately weighed, in 10 mL of methanol, sonicating if necessary until completely dispersed.

Application volume: 2 µL.

Developing solvent system: a mixture of methylene chloride, methanol, and ammonium hydroxide (90:9:1).

Procedure —Develop the chromatogram in an unsaturated chamber. Remove the plate, allow to air-dry, and examine under short- and long-wavelength UV light.

B: The retention times of the two principal component peaks of ivermectin in the chromatogram of the Assay preparation correspond to those of the two principal component peaks of ivermectin in the chromatogram of the Standard preparation, as obtained in the Assay.

Mobile phase and Chromatographic system— Proceed as directed in the Assay.

Standard solution— Dissolve an accurately weighed quantity of USP Ivermectin RS in methanol, and dilute quantitatively, and stepwise if necessary, with methanol to obtain a solution having a concentration of 0.004 mg per mL.

Test solution— Disperse a quantity of Ivermectin Paste, accurately weighed, in methanol, sonicating if necessary, to obtain a solution containing 0.4 mg of ivermectin per mL, based on the label claim.

Procedure— Inject equal volumes (about 20 µL) of the Test solution and the Standard solution into the chromatograph, record the chromatogram, and measure all of the peak responses. Calculate the percentage of each impurity in the portion of Paste taken by the formula: in which CS is the concentration, in mg per mL, of ivermectin in the Standard solution; CT is the concentration, in mg per mL, of ivermectin in the Test solution; ri is the peak response for each impurity obtained from the Test solution; and rS is the ivermectin peak response obtained from the Standard solution. Not more than 2.7% of the peak with a relative retention time of about 1.3 to 1.5 (relative to that of the principal peak) is found; not more than 1.0% of any other impurity is found; and not more than 6.0% of total impurities is found. Disregard any peak below 0.05%.

Mobile phase— Prepare a filtered and degassed mixture of acetonitrile, methanol, and water (106:55:39). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Ivermectin RS in methanol, and dilute quantitatively, and stepwise if necessary, with methanol to obtain a solution having a known concentration of about 0.4 mg per mL.

Assay preparation— Disperse a quantity of Ivermectin Paste, accurately weighed, in methanol, sonicating, if necessary, to obtain a solution containing 0.4 mg of ivermectin per mL.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 245-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L1. The flow rate is about 1.5 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the resolution, R, between the first peak (component H2B1b) and the second peak (component H2B1a) is not not less than 3.0; and the relative standard deviation for replicate injections is not more than 2.0%, determined from the component H2B1a peak.

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 responses for the component H2B1a plus component H2B1b. Calculate the percentage of the label claim for ivermectin (H2B1a + H2B1b) in the portion of Paste taken by the formula: in which CS is the concentration, in mg per mL, of USP Ivermectin RS in the Standard preparation; CU is the concentration, in mg per mL, of ivermectin in the Assay preparation; rU is the total response of the H2B1a plus H2B1b peaks obtained from the Assay preparation; and rS is the total peak response of the H2B1a plus H2B1b peaks obtained from the Standard preparation. Calculate the ratio of the contents, in percentage, of H2B1a / (H2B1a + H2B1b) in the portion of Paste taken by the formula: in which r1a is the peak response of H2B1a obtained from the Assay preparation, and rU is as described above.