Internal standard solution , Standard preparation, and Chromatographic system—Proceed as directed in the Assay under Clioquinol.

Assay preparation— Transfer an accurately weighed portion of Ointment, equivalent to about 150 mg of clioquinol, to a 125-mL separator. Add 75 mL of n-hexane, and mix. Add 15 mL of dimethylformamide, and mix for 1 minute. Allow the layers to separate, and transfer the lower layer to a 50-mL volumetric flask. Repeat the extraction with separate 15-mL and 10-mL portions of dimethylformamide, and transfer the lower layers to the 50-mL volumetric flask. Dilute with dimethylformamide to volume, and mix. Transfer 1.0 mL of this solution to a screw-capped glass vial fitted with a septum, and evaporate at about 60 under a stream of nitrogen to dryness. Add 1.0 mL of a mixture of pyridine and n-hexane (4:1) to the residue, add 1.0 mL of bis(trimethylsilyl)acetamide and 1.0 mL of Internal standard solution, attach the cap, and mix. Heat in a water bath at 50 for 15 minutes, then cool to ambient temperature.

Procedure— Proceed as directed for Procedure under Clioquinol. Calculate the quantity, in mg, of C9H5ClINO in the portion of Ointment taken by the formula: in which C is the concentration, in mg per mL, of USP Clioquinol RS in the Standard solution used to prepare the Standard preparation; and RU and RS are the ratios of the peak responses of the clioquinol peak to the internal standard peak obtained from the Assay preparation and the Standard preparation, respectively.