DEVELOPMENT AND VALIDATION OF A NEW HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD FOR ENANTIOSEPARATION OF DORZOLAMIDE HYDROCHLORIDE ON A COATED CELLULOSE PHENYLCARBAMATE CHIRAL STATIONARY PHASE

Abstract

A simple, accurate, precise, and sensitive method using coated cellulose phenylcarbamate chiral stationary phase for separation of dorzolamide hydrochloride (4S,6S) and its enantiomer (4R,6R) in commercial drops has been established. The mobile phase consisted of a mixture of n-hexane and 2-propanol (50:50 v/v) containing 0.1% diethylamine and used a flow rate of 1 mL min⁻¹. The influence of temperature, flow rate, type, and concentration of organic components and diethylamine in the mobile phase on the retention and enantioselectivity was investigated. The method was validated for linearity, repeatability, accuracy, and limits of detection (LOD) and quantification (LOQ). The calibration range of quantitation for dorzolamide (4S,6S) and its enantiomer (4R,6R) was 0.5–10 µ g mL⁻¹ and 0.2–5 µg mL⁻¹, respectively. The LOD and LOQ were found to be 0.2 and 0.5 µ g mL⁻¹ for dorzolamide hydrochloride (4S,6S) and 0.1 and 0.2 µg mL⁻¹ for its enantiomer (4R,6R), respectively. Repeatability (n = 6) showed less than 0.5% relative standard deviation.

Keywords:

• cellulose phenylcarbamate chiral stationary phase
• dorzolamide hydrochloride
• enantiomers
• enantioseparation
• high performance liquid chromatography
• validation

ACKNOWLEDGMENTS

The support of this work by Research Council of Tarbiat Modares University and Iran National Science Foundation (INSF) is gratefully acknowledged. The authors would like to thank Jaber Ebne Hayyan Pharmaceutical Industry, Tehran, Iran, for the excellent collaboration.

Notes

Conditions: mobile phase, n-hexane-alcohol 50:50 v/v% + 0.1% DEA; flow rate, 1 mL min⁻¹; column temperature, 25°C.

Conditions: mobile phase, n-hexane/2-propanol 50:50 v/v% + X% DEA; flow rate, 1 mL min⁻¹; column temperature, 25°C.

Conditions: mobile phase, n-hexane/2-propanol 50:50 v/v% + 0.1% DEA; flow rate, 1 mL min⁻¹.

Conditions: mobile phase, n-hexane/2-propanol 50:50 v/v% + 0.1% DEA; flow rate, 1 mL min⁻¹; column temperature, 25°C.