Solution A— Prepare a filtered and degassed solution in water containing 17 g of tetrabutylammonium hydrogen sulfate per L. Make adjustments if necessary (see System Suitability under Chromatography 621).

Solution B— Use filtered and degassed acetonitrile. Make adjustments if necessary (see System Suitability under Chromatography 621).

Mobile phase— Use variable mixtures of Solution A and Solution B as directed for Chromatographic system.

Standard solution— Dissolve an accurately weighed quantity of USP Astemizole RS in methanol, and dilute quantitatively, and stepwise if necessary, with methanol to obtain a solution having a known concentration of about 25 µg per mL.

Resolution solution— Dissolve an accurately weighed quantity of USP Astemizole RS and ketoconazole in methanol, and dilute quantitatively, and stepwise if necessary, with methanol to obtain a solution having a known concentration of about 25 µg and 250 µg per mL, respectively.

Test solution— Transfer about 100 mg of Astemizole, accurately weighed, to a 10-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 278-nm detector and a 4.6-mm × 10-cm column that contains base-deactivated 3-µm packing L1. The flow rate is about 1 mL per minute. Equilibrate the system with acetonitrile and then with 95% Solution A and 5% Solution B, and hold at that composition for 5 minutes prior to injection. After injection, linearly change the composition to 80% Solution A and 20% Solution B over a period of 15 minutes. Maintain this composition for an additional 3 minutes. Purge the column with 100% Solution B for 5 minutes, and then equilibrate the system to the initial composition for 5 minutes prior to the following injection. Chromatograph the Resolution solution, and record the peak responses as directed for Procedure: the resolution, R, between the astemizole and ketoconazole peaks is not less than 1.5.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the peak responses. Calculate the percentage of each impurity in the portion of Astemizole taken by the formula: in which ri is the peak response for each impurity, and rS is the peak response of the Standard solution: not more than 0.25% of any individual impurity is found, and not more than 0.5% of total impurities is found.

Mobile phase— Prepare a filtered and degassed mixture of methanol, 0.13 M ammonium acetate, acetonitrile, and diethylamine (470:300:230:1.0), and adjust with glacial acetic acid to a pH of 7.5. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Astemizole RS in Mobile phase, and dilute quantitatively, and stepwise if necessary, with Mobile phase to obtain a solution having a known concentration of about 1.0 mg per mL.

Assay preparation— Transfer about 50 mg of Astemizole, accurately weighed, to a 50-mL volumetric flask, dissolve in and dilute with Mobile phase to volume, and mix.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 220-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 2 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the column efficiency is not less than 4000 theoretical plates; the tailing factor is not more than 1.8; and the relative standard deviation for replicate injections is not more than 1.5%.

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 responses for the major peaks. Calculate the quantity, in mg, of C28H31FN4O in the portion of Astemizole taken by the formula: in which C is the concentration, in mg per mL, of USP Astemizole RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.