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

B: Ultraviolet Absorption 197U—

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

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

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

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

Chromatographic system (see Chromatography 621)—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 G3 on 80- to 100-mesh support S1AB. The injection port and detector temperatures are maintained at 170 and 250, respectively, and the initial column temperature is 200 for a conditioning period of not less than 16 hours (not connected to the detector) and is then reduced to 165. Helium is used as the carrier gas at a flow rate of about 30 mL per minute, and hydrogen and air are introduced into the detector at rates of 25 mL and 500 mL per 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 1 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. The relative retention times for clioquinol and pyrene are about 0.6 and 1.0, respectively. Calculate the quantity, in mg, of C9H5ClINO in the Clioquinol 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.