ChP HPLC Analysis of Clobetasol Propionate and Ketoconazole in Compound Ketoconazole Cream with Discovery^® HS C18 Column

Discovery^® HS C18 250 x 4.6 mm, 5 µm (568523-U)

[A] Water; [B] methanol
(A/B :26/74)

Isoctatic

1.0 mL/min

2150 psi (147 bar)

25 °C

UV, 239nm

10 µL

Accurately weigh ~ 4g compound ketoconazole cream into a 50-mL volumetric flask, add an appropriate amount of ethanol, place it in a water bath at 80 ℃ to dissolve ketoconazole and clobetasol propionate, cool down to room temperature. Add ethanol to tick mark of the volumetric flask. Shake well, cool in an ice bath for 2 h, filter, stand for HPLC analysis.

Compound ketoconazole cream is mainly used for superficial fungal infections of the skin, such as tinea manus, tinea pedis (beriberi), tinea corporis, tinea cruris, etc. Clobetasol propionate and ketoconazole are two of main active ingredients of Compound ketoconazole cream.

In this application, compound ketoconazole cream was dissolved in ethanol, extracted, filtered by PVDF filter, and finally separated by Discovery^® HS HPLC column.
When run a mixed standard containing 20 µg/mL of clobetasol propionate and 500 µg/mL of ketoconazole in ethanol, the theoretical plates calculated by the peak of ketoconazole is 3857 (> 2000). The resolution of the peaks between the two peaks is 3.82 (>2.0). This meets the requirements of the Chinese Pharmacopeia 2020.
For the tailing factor of the peak of ketoconazole, when the concentration decreases from 500 µg/mL to 10 µg/mL, it dramatically decreases from 2.17 to 1.08. This indicates the HPLC column is overloaded by a high concentration of ketoconazole following the Ch. P2020 monograph method.
The Limit of Detection (LOD) for clobetasol propionate is 0.16 µg/mL, and the limit of quantification (LOQ) is 0.47 µg/mL. The range from 0.2 to 50 µg/mL is linear, with an R² of 0.9998. The limit of detection (LOD) for ketoconazole is 0.98 µg/mL, and the limit of quantification (LOQ) is 2.98 µg/mL. The range from 1 to 800 µg/mL is linear, with an R² of 0.9998.

APP_529

Discovery is a registered trademark of Merck KGaA, Darmstadt, Germany