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— Prepare a solution of pyrene in pyridine containing about 2mg per mL.

Standard solution— Transfer about 75mg of USP Clioquinol RSto a 25-mLvolumetric flask,add a mixture of pyridine and hexane (4:1)to volume,and mix to obtain a Standard solutionhaving a known concentration of about 3mg of USP Clioquinol RSper mL.

Standard preparation— Transfer 1.0mLof Standard solution,1.0mLof N,O-bis(trimethylsilyl)acetamide and 1.0mLof Internal standard solutionto a suitable screw-capped glass vial,fitted with a polytef-lined septum,and mix.Heat on a water bath at 50for 15minutes,and cool to room temperature.

Assay preparation— Transfer an accurately weighed quantity of Cream,equivalent to about 150mg of clioquinol,to a 60-mLseparator.Place the separator on its side in a vacuum oven at about 45for 4hours.Remove the separator,cool to room temperature,and add 15.0mLof a mixture of pyridine and hexane (4:1).Insert the stopper in the separator,and mix until the specimen is completely dispersed.Quantitatively transfer the contents of the separator to a 50-mLvolumetric flask,rinse the separator with two 15-mLportions of a mixture of pyridine and hexane (4:1),collecting the rinsings in the volumetric flask,dilute with the same solvent mixture to volume,and mix.Immediately transfer 1mLof this solution to a dry,screw-capped glass vial,and evaporate with the aid of gentle heat and a stream of nitrogen to dryness.Dissolve the residue in 1.0mLof a mixture of pyridine and hexane (4:1),add 1mLeach ofN,O-bis(trimethylsilyl)acetamide and Internal standard solutionto the screw-capped glass vial,fitted with a polytef-lined septum,and mix.Heat on a water bath at 50for 15minutes,and cool to room temperature.

Chromatographic system (see Chromatography á621ñ)—The gas chromatograph is equipped with a flame-ionization detector and a 2-mm ×1.8-m column packed with 3%liquid phase G3on 80-to 100-mesh support SlAB.The column,injection port,and detector block temperatures are maintained at 165,170,and 250,respectively.Dry helium is used as the carrier gas at a flow rate of about 30mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative retention times are about 0.6for clioquinol and 1.0for pyrene;the resolution,R,between the analyte and internal standard peaks is not less than 3.0;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Inject equal volumes (about 1µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms so as to obtain not less than 40%of maximum recorder response,and measure the peak response of each component.Calculate the quantity,in mg,of C9H5ClINOin the portion of Cream 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— Prepare a filtered and degassed solution of water,acetonitrile,and methanol (2.75:1:1).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Hydrocortisone RSin alcohol to obtain a solution having a known concentration of about 1mg per mL(Solution A).Pipet 1mLof this solution into a 10-mLvolumetric flask,dilute with alcohol to volume,and mix to obtain a solution having a known concentration of about 100µg of USP Hydrocortisone RSper mL.

Resolution solution— Dissolve an accurately weighed quantity of methylparaben in alcohol to obtain a solution having a known concentration of about 0.5mg of methylparaben per mL.Pipet 2mLof this solution and 20mLof Solution Ainto a 200-mLvolumetric flask,dilute with alcohol to volume,and mix.

Assay preparation— Transfer an accurately weighed quantity of Cream,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.

Chromatographic system— (see Chromatography á621ñ).The liquid chromatograph is equipped with a 254-nm detector,a 3.9-mm ×30-cm column that contains packing L1,and a guard column that contains packing L2.The flow rate is about 1mLper minute.Chromatograph the Resolution solution,and record the peak responses as directed for Procedure:the relative retention times are about 0.6for methylparaben and 1.0for hydrocortisone;the resolution,R,between the hydrocortisone and methylparaben peaks is not less than 2.0;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of C21H30O5in the portion of Cream 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.