Clioquinol and Hydrocortisone Ointment

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 DEFINITION 

Clioquinol and Hydrocortisone Ointment contains NLT 90.0% and NMT 110.0% of the labeled amounts of clioquinol (C₉H₅ClINO) and hydrocortisone (C₂₁H₃₀O₅) in a suitable ointment base. 

2 IDENTIFICATION 

A. The retention time of the clioquinol peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay for Clioquinol. 

B. The retention time of the hydrocortisone peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay for Hydrocortisone. 

3 ASSAY 

Clioquinol 

Internal standard solution: 2 mg/mL of pyrene in pyridine 

Standard stock solution: 3 mg/mL of USP Clioquinol RS in a mixture of pyridine and n-hexane (4:1) 

Standard solution: Transfer 1.0 mL of the Standard stock 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 min, and cool to room temperature. 

Sample solution: Transfer nominally 150 mg of clioquinol from Ointment to a 125-mL separator. Add 75 mL of n-hexane, insert the stopper in the separator, and mix until the specimen is completely dispersed. Extract with 25 mL of dimethylformamide, collecting the extract in a 50- mL volumetric flask. Repeat the extraction with two 10-mL portions of dimethylformamide, collecting the extracts in the 50-mL volumetric flask, and dilute with dimethylformamide to volume. Transfer 1.0 mL of this solution to a suitable size screw-capped vial, and evaporate the solution with the aid of nitrogen at 60° to dryness. Dissolve the residue in 1.0 mL of a mixture of pyridine and hexane (4:1), and pipet 1.0 mL of N,O-bis(trimethylsilyl)acetamide and 1.0 mL of Internal standard solution into the glass vial, fitted with a polytef-lined septum, and securely close. Heat the vial on a water bath at 50° for 15 min, and cool to room temperature. 

Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: GC 

Detector: Flame ionization 

Column: 2-mm × 1.8-m; packed with 3% liquid phase G3 on 80- to 100-mesh support SlAB 

Temperatures 

Column: 165° 

Injection port: 170° 

Detector: 250° 

Carrier gas: Dry helium 

Flow rate: 30 mL/min 

Injection volume: 1 µL 

System suitability 

Sample: Standard solution 

[Note—The relative retention times for clioquinol and pyrene are 0.6 and 1.0, respectively.] 

Suitability requirements 

Resolution: NLT 3.0 between the analyte and internal standard peaks 

Relative standard deviation: NMT 2.0% 

Analysis 

Samples: Standard solution and Sample solution 

Record the chromatograms to obtain NLT 40% of maximum recorder response, and measure the peak response of each component. Calculate the percentage of the labeled amount of clioquinol (C₉H₅ClINO) taken: 

Result = (R_U/R_S) × (C_S/C_U) × 100 

r_U = peak response ratio of clioquinol to the internal standard from the Sample solution

r_S = peak response ratio of clioquinol to the internal standard from the Standard solution

C_S = concentration of USP Clioquinol RS in the Standard solution (mg/mL)

C_U = nominal concentrationof clioquinol in the Sample solution (mg/mL)

Acceptance criteria: 90.0%–110.0% 

Hydrocortisone 

Mobile phase: Acetonitrile, methanol, and water (1:1:2.75) 

Standard stock solution: 1 mg/mL of USP Hydrocortisone RS in alcohol 

Standard solution: Standard stock solution and alcohol (1:9) 

Sample solution: Transfer nominally 10 mg of hydrocortisone from Ointment to a 50-mL centrifuge tube. Add 30 mL of alcohol, and heat on a steam bath just to boiling. Shake for 15 min, and centrifuge. 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. System suitability stock solution: 0.5 mg/mL of methylparaben in alcohol 

System suitability solution: Transfer 2 mL of System suitability stock solution and 20 mL of Standard stock solution into a 200-mL volumetric flask, and dilute with alcohol to volume. 

Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: LC 

Detector: UV 254 nm 

Columns 

Guard: Packing L2 

Analytical: 3.9-mm × 30-cm; packing L1 

Flow rate: 1 mL/min 

Injection volume: 10 µL 

System suitability 

Sample: System suitability solution 

[Note—The relative retention times for methylparaben and hydrocortisone are 0.6 and 1.0, respectively.] Suitability requirements 

Resolution: NLT 2.0 between the hydrocortisone and methylparaben peaks 

Relative standard deviation: NMT 2.0% 

Analysis 

Samples: Standard solution and Sample solution 

Calculate the percentage of the labeled amount of hydrocortisone (C₂₁H₃₀O₅) taken: 

Result = (r_U/r_S) × (C_S/C_U) × 100 

r_U = peak response from the Sample solution

r_S = peak response from the Standard solution

C_S = concentrationof USP Hydrocortisone RS in the Standard solution (mg/mL)

C_U = nominal concentration of hydrocortisone in the Sample solution (mg/mL)

F = conversion factor, 0.001 mg/µg 

Acceptance criteria: 90.0%–110.0% 

4 PERFORMANCE TESTS 

Minimum Fill 〈755〉: Meets the requirements 

5 ADDITIONAL REQUIREMENTS 

Packaging and Storage: Preserve in collapsible tubes or in tight, light-resistant containers. 

USP Reference Standards 〈11〉 

t USP Clioquinol RS 

USP Hydrocortisone RS