Add the following:

Medium: 0.01M phosphate buffer, pH 7, with 0.5% of sodium dodecyl sulfate (prepared by dissolving 50 g of sodium dodecyl sulfate in approximately 9 L of water, adding 100 mL of 1 M monobasic sodium phosphate monohydrate, adjusting with sodium hydroxide to a pH of 7, and diluting with water to 10 L): 900 mL.

Apparatus 2: 50 rpm.

Time: 45 minutes.

Mobile phase— Prepare a degassed solution of acetonitrile, methanol, and water (53:35:12).

Standard stock solution— Prepare a 9 mg per mL solution of USP Ivermectin RS in Medium.

Standard solution— Using the accompanying table, dilute the Standard stock solution with Medium to volume, and mix.

Test solution— Pass a portion of the solution under test through a suitable filter, and use the filtrate.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 245-nm detector and a 4.6-mm × 10-cm column that contains 5-µm packing L1. The flow rate is about 1.2 mL per minute. The column temperature is maintained at 30. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.81 for H2B1b and 1.0 for H2B1a; the resolution, R, between the H2B1a and H2B1b peaks is not less than 1.5; the capacity factor, k¢, for the H2B1a peak is not less than 4; the column efficiency determined from both the H2B1a and H2B1b peaks is not less than 1500 theoretical plates; the tailing factor for the H2B1a peak is not more than 2; and the relative standard deviation for replicate injections for the H2B1a peak is not more than 2.0%.

Procedure— Separately inject equal volumes (about 100 µL) of the Test solution and the Standard solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the combined quantities, in percent, of H2B1a plus H2B1b dissolved based on the peak responses obtained from the Test solution and the Standard solution by the formula: in which AU is the total peak area of H2B1a plus H2B1b obtained from the Test solution; WS is the weight, in mg, of the USP Ivermectin RS taken to prepare the Standard stock solution; P is the purity of the USP Ivermectin RS (percent [w/w] H2B1a plus percent [w/w] H2B1b), expressed as a decimal; DU is the Test solution dilution factor; AS is the total peak area of H2B1a plus H2B1b obtained from the Standard solution; DS is the Standard solution dilution factor; and L is the label claim of ivermectin, in mg per Tablet.

Tolerances— Not less than 80% (Q) of the labeled amount of C48H74O14 (H2B1a ) plus C47H72O14 (H2B1b ) is dissolved in 45 minutes.USP32

procedure for content uniformity—

Chromatographic system (see Chromatography 621)— Proceed as directed in the Assay.

Procedure— Separately inject equal volumes (about 10 µL) of Standard solution A (for the 6 mg per Tablet dose) or Standard solution B (for the 3 mg per Tablet dose), the Sensitivity solution (0.2%), and the Test solution into the chromatograph, record the chromatograms, and measure the responses of the ivermectin peaks. Calculate the quantity as a percentage of the label claim of ivermectin per Tablet taken by the formula: in which AU is the peak area of H2B1a plus the peak area of H2B1b obtained from the Test solution; WS is the weight, in mg, of the USP Ivermectin RS taken to prepare Standard solution A or Standard solution B; P is the purity of USP Ivermectin RS (percent [w/w] H2B1a plus percent [w/w] H2B1b), expressed as a decimal; DU is the Test solution dilution factor; AS is the peak area of H2B1a plus the peak area of H2B1b obtained from Standard solution A or Standard solution B; DS is the Standard solution A or Standard solution B dilution factor; and L is the label claim of ivermectin, in mg per Tablet.

Mobile phase— Proceed as directed in the Assay.

BHA Working Standard solution— Dissolve an accurately weighed quantity of USP 3-tert-Butyl-4-hydroxyanisole RS in methanol, and dilute quantitatively, and stepwise if necessary, to obtain a solution having a known concentration of about 0.96 µg per mL.

Test solution— Use the Assay preparation.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 280-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L1. The column temperature is maintained at 30. The flow rate is about 1.2 mL per minute. Chromatograph the BHA Working Standard solution and the Test solution, and record the peak responses as directed for Procedure: the relative retention times at 280 nm are about 0.24 for BHA, 0.77 for 8a-oxo-H2B1a, and 1.0 for H2B1a.

Procedure— Separately inject equal volumes (about 10 µL) of the BHA Working Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the percentage of 8a-oxo-H2B1a as a percentage of the label claim of ivermectin in the portion of Tablets taken by the formula: in which AD is the peak area of 8a-oxo-H2B1a obtained from the Test solution; WS is the weight of USP 3-tert-Butyl-4-hydroxyanisole RS, in mg, taken to prepare the BHA Working Standard solution; P is the purity of USP 3-tert-Butyl-4-hydroxyanisole RS, expressed as a decimal; DU is the Test solution dilution factor; CF is the correction factor (equal to 0.98) used to convert mg of 8a-oxo-H2B1a to mg of ivermectin; AS is the peak area of BHA obtained from the BHA Working Standard solution; DS is the BHA Working Standard solution dilution factor; N is the number of Tablets taken to prepare the Test solution; L is the label claim of ivermectin, in mg per Tablet; and F is the relative response factor (equal to 1.0): not more than 2.0% of 8a-oxo-H2B1a is found. The correction factor, CF, (equal to 0.98) is calculated by the following formula:

Mobile phase— Prepare a mixture of acetonitrile, methanol, and water (53:35:12). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Ivermectin RS in methanol to obtain a solution containing 0.25 mg per mL.

Stock sensitivity solution (1%)— Quantitatively prepare a 1 in 100 dilution of the Standard solution in methanol.

Sensitivity solution (0.2%)— Quantitatively prepare a 1 in 5 dilution of the Stock sensitivity solution (1%) in methanol.

Assay preparation— Transfer the appropriate number of Tablets into a 250-mL volumetric flask according to the accompanying table: Add approximately 25 mL of water, and sonicate for 10 minutes. Add methanol to fill the flask three-quarters full, sonicate for 5 minutes or until the Tablets are completely disintegrated, and shake until mixed well. Allow the solution to cool to room temperature. Dilute with methanol to volume, add a magnetic stirrer, and mix until no lumps are present in the solution. Pass a portion of this solution through a 1.0- to 1.2-µm chemically resistant filter prior to injection.

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 1.2 mL per minute. The column temperature is maintained at 30. Chromatograph the Sensitivity solution (0.2%) and the Standard preparation at 245-nm detection, and record the peak responses as directed for Procedure: the signal-to-noise ratio for the ivermectin peak obtained from the Sensitivity solution (0.2%) is not less than 10; obtained from the Standard preparation, the relative retention times are about 0.82 and 1.0 for components H2B1b and H2B1a, respectively; the capacity factor, k ¢, for the component H2B1b is not less than 3; the column efficiency for component H2B1a is not less than 1500 theoretical plates; the tailing factor for component H2B1a is not more than 2; and the relative standard deviation for the area response for total ivermectin (H2B1a plus H2B1b) for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak areas for component H2B1a plus component H2B1b. Calculate the percentage of component H2B1a (C48H74O14) plus component H2B1b (C47H72O14) as a percentage of the label claim of ivermectin per Tablet taken by the formula: in which AU is the total peak response of H2B1a plus H2B1b obtained from the Assay preparation; WS is the weight of the USP Ivermectin RS, in mg, taken to prepare the Standard preparation; P is the purity of the USP Ivermectin RS (percent [w/w] H2B1a plus percent [w/w] H2B1b), expressed as a decimal; DU is the sample dilution factor; AS is the total peak area of H2B1a plus H2B1b obtained from the Standard preparation; DS is the Standard dilution factor; N is the number of Tablets taken to prepare the Assay preparation; and L is the label claim of ivermectin, in mg per Tablet.