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

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

(Official January 1, 2007)

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

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

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

Assay preparation— Transfer an accurately weighed quantity of Cream, equivalent to about 150 mg of clioquinol, to a 60-mL separator. Place the separator on its side in a vacuum oven at about 45 for 4 hours. Remove the separator, cool to room temperature, and add 15.0 mL of 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-mL volumetric flask, rinse the separator with two 15-mL portions 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 1 mL of 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 1.0 mL of a mixture of pyridine and hexane (4:1), add 1 mL each of N,O-bis(trimethylsilyl)acetamide and Internal standard solution to the screw-capped glass vial, fitted with a polytef-lined septum, and mix. Heat on a water bath at 50 for 15 minutes, and cool to room temperature.

Chromatographic system (see Chromatography 621)—The gas chromatograph is equipped with a flame-ionization detector and a 2-mm × 1.8-m column packed with 3% liquid phase G3 on 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 30 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative retention times are about 0.6 for clioquinol and 1.0 for 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 1 µL) of the Standard preparation and the Assay preparation into 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 C9H5ClINO in the portion of Cream taken by the formula: in which C is the concentration, in mg per mL, of USP Clioquinol RS in the Standard preparation; and RU and RS are the peak response ratios obtained from the Assay preparation and 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 Suitability under Chromatography 621).

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

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

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

Chromatographic system— (see Chromatography 621). 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 1 mL per minute. Chromatograph the Resolution solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.6 for methylparaben and 1.0 for 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 10 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C21H30O5 in the portion of Cream taken by the formula: in which C is the concentration, in µg per mL, of USP Hydrocortisone RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.