A: The chromatogram of the Assay preparationobtained as directed in the Assay for clioquinolexhibits a peak for clioquinol,the retention time of which corresponds to that exhibited by the Standard preparation.

B: The chromatogram of the Assay preparationobtained as directed in the Assay for hydrocortisoneexhibits a peak for hydrocortisone,the retention time of which corresponds to that exhibited by the Standard preparation.

Internal standard solution ,Standard solution,Standard preparation,and Chromatographic system—Prepare as directed in the Assay for clioquinolunder Clioquinol and Hydrocortisone Cream.

Assay preparation— Transfer an accurately weighed quantity of Ointment,equivalent to about 150mg of clioquinol,to a 125-mLseparator.Add 75mLof n-hexane,insert the stopper in the separator,and mix until the specimen is completely dispersed.Extract with 25mLof dimethylformamide,collecting the extract in a 50-mLvolumetric flask.Repeat the extraction with two 10-mLportions of dimethylformamide,collecting the extracts in the 50-mLvolumetric flask,dilute with dimethylformamide to volume,and mix.Transfer 1.0mLof this solution to a suitable size screw-capped vial,and evaporate the solution with the aid of nitrogen at about 60to dryness.Dissolve the residue in 1.0mLof a mixture of pyridine and hexane (4:1),and pipet 1.0mLof N,O-bis(trimethylsilyl)acetamide and 1.0mLof Internal standard solutioninto the glass vial,fitted with a polytef-lined septum,securely close,and mix.Heat the vial on a water bath at 50for 15minutes,and cool to room temperature.

Procedure— Proceed as directed for Procedurein the Assay for clioquinolunder Clioquinol and Hydrocortisone Cream.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 preparation;and RUand RSare the peak response ratios obtained from the Assay preparationand the Standard preparation,respectively.

Mobile phase,Standard preparation,Resolution solution,andChromatographic system— Prepare as directed in the Assay for hydrocortisoneunder Clioquinol and Hydrocortisone Cream.

Assay preparation— Transfer an accurately weighed quantity of Ointment,equivalent to about 10mg of hydrocortisone,to a 50-mLcentrifuge tube.Add 30mLof alcohol and heat on a steam bath just to boiling.Shake for 15minutes,and centrifuge.Quantitatively transfer the supernatant extract to a 100-mLvolumetric flask.Repeat the extraction with two 20-mLportions of alcohol,combining the extracts in the 100-mLvolumetric flask.Add alcohol to volume,mix,and filter.

Procedure— Proceed as directed for Procedurein the Assay for hydrocortisoneunder Clioquinol and Hydrocortisone Cream.Calculate the quantity,in mg,of C21H30O5in the portion of Ointment taken by the formula: in which Cis the concentration,in µg per mL,of USP Hydrocortisone RSin the Standard preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.