Identification—
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
—
Solution:
5 µg per mL.
Medium:
3 N hydrochloric acid.
Absorptivities at 267 nm, calculated on the dried basis, do not differ by more than 3.0%.
C:
Heat 100 mg with 5 mL of sulfuric acid: copious violet vapors of iodine are evolved.
Free iodine and iodide—
Shake 1.0 g with 20 mL of water for 30 seconds, allow to stand for 5 minutes, and filter. To 10 mL of the filtrate add 1 mL of 2 N sulfuric acid, then add 2 mL of chloroform, and shake: no violet color appears in the chloroform (
free iodine). To the mixture add 5 mL of 2 N sulfuric acid and 1 mL of
potassium dichromate TS, and shake for 15 seconds: the color in the chloroform layer is no deeper than that produced in a control test made in the following manner: Dilute 2.0 mL of potassium iodide solution (1 in 6000) with water to 10 mL, add 6 mL of 2 N sulfuric acid, 1 mL of
potassium dichromate TS, and 2 mL of chloroform, and shake for 15 seconds (
0.05% of iodide).
Assay—
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 C
9H
5ClINO in the Clioquinol taken by the formula:
25C(RU / RS),
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.
;)
