A: Prepare a Standard solution as directed for Standard preparationin the Assay,except to use 1.0mLof pyridine instead of the Internal standard solution,and chromatograph as directed in the Assay:the chromatogram of the Assay preparationobtained in the Assayexhibits a peak for clioquinol,the retention time of which corresponds with that exhibited by the Standard solution.

B: Ultraviolet Absorption á197Uñ—

C: Heat 100mg with 5mLof sulfuric acid:copious violet vapors of iodine are evolved.

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

Standard preparation— Dissolve an accurately weighed quantity of USP Clioquinol RSin a mixture of pyridine and n-hexane (4:1)to obtain a Standard solution having a known concentration of about 3mg per mL.Transfer 1.0mLof the Standard solution to a screw-capped glass vial fitted with a septum,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,and then cool to ambient temperature.

Assay preparation— Transfer about 75mg of Clioquinol,previously dried and accurately weighed,to a 25-mLvolumetric flask,dissolve in a mixture of pyridine and n-hexane (4:1),dilute with the same solvent to volume,and mix.Transfer 1.0mLof this solution to a screw-capped glass vial fitted with a septum,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.

Chromatographic system (see Chromatography á621ñ)—The gas chromatograph is equipped with a flame-ionization detector,and contains a 1.83-m ×2-mm glass column packed with 3%liquid phase G3on 80-to 100-mesh support S1AB.The injection port and detector temperatures are maintained at 170and 250,respectively,and the initial column temperature is 200for a conditioning period of not less than 16hours (not connected to the detector)and is then reduced to 165.Helium is used as the carrier gas at a flow rate of about 30mLper minute,and hydrogen and air are introduced into the detector at rates of 25mLand 500mLper minute,respectively.Chromatograph the Standard preparation,and record the peak responses as directed under Procedure:the resolution,R,between the clioquinol and the internal standard peaks is not less than 3.

Procedure— Separately inject equal volumes (about 1µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.The relative retention times for clioquinol and pyrene are about 0.6and 1.0,respectively.Calculate the quantity,in mg,of C9H5ClINOin the Clioquinol 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.