A: Infrared Absorption 197K.

B: Ultraviolet Absorption 197U.

Internal standard solution— Dissolve a sufficient quantity of o-nitroaniline in a small quantity of dichloromethane, and dilute with n-hexane to obtain a solution having a concentration of about 500 µg per mL.

Mobile phase— Prepare a degassed mixture of n-hexane, dichloromethane, and glacial acetic acid (82:12:6). Make adjustments if necessary (see System Suitability under Chromatography 621).

System suitability preparation— Prepare a solution containing 0.1 mg of USP Anthralin RS and 0.2 mg of danthron per mL in dichloromethane. Transfer 5 mL of this solution to a 25-mL volumetric flask. Add 5.0 mL of n-hexane, dilute with Mobile phase to volume, and mix.

Solvent blank solution— Mix Mobile phase, n-hexane, and dichloromethane (3:1:1).

Standard preparation— Dissolve an accurately weighed quantity of USP Anthralin RS in dichloromethane to obtain a solution having a known concentration of about 250 µg per mL. Pipet 5 mL of this solution and 5 mL of Internal standard solution into a 25-mL volumetric flask, dilute with Mobile phase to volume, and mix.

Assay preparation— Weigh accurately about 250 mg of Anthralin into a tared 100-mL flask, dilute with dichloromethane to volume after dissolution of the solid, and mix. Pipet 10 mL of the solution into a 100-mL volumetric flask, dilute with dichloromethane to volume, and mix. Pipet 5 mL of this solution and 5 mL of Internal standard solution into a 25-mL volumetric flask, dilute with Mobile phase to volume, and mix.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 354-nm detector and a 4.6-mm × 25-cm column that contains packing L3. The flow rate is about 2 mL per minute. Chromatograph replicate injections of the Standard preparation, and record the peak responses as directed for Procedure: relative retention times are 1.0 for anthralin, about 1.2 for danthron (if present), about 1.7 for dianthrone (if present), and about 2.3 for o-nitroaniline; and the relative standard deviation of the ratio of the peak responses is not more than 2.0%. Chromatograph the System suitability preparation: the resolution, R, is not less than 1.3; and the tailing factor, T, is not more than 1.5. Chromatograph the Solvent blank solution: no effect on the baseline is discernible at the retention time of anthralin.

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 areas for the major peaks. Calculate the quantity, in mg, of C14H10O3 in the portion of Anthralin taken by the formula: in which C is the concentration, in µg per mL, of USP Anthralin RS in the Standard preparation; and RU and RS are the response ratios of the anthralin peak to the o-nitroaniline peak obtained from the Assay preparation and the Standard preparation, respectively.