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

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

Procedure— Proceed as directed for Procedure under Clioquinol.Calculate the quantity,in mg,of C9H5ClINOin the portion of Ointment taken by the formula: in which Cis the concentration,in mg per mL,of USP Clioquinol RSin the Standard solution used to prepare the Standard preparation,and RUand RSare the ratios of the peak responses of the clioquinol peak to the internal standard peak obtained from the Assay preparationand the Standard preparation,respectively.