On-line preconcentration by analyte focusing micelle collapse in capillary electrophoretic separation of steroids

Abstract

Analyte focusing by micelle collapse (AFMC) is an on-line sample preconcentration of neutral analyte for capillary electrophoresis (CE). The purpose of this research is to study the separation of six neutral steroids; prednisolone, prednisone, betamethasone, testosterone, androstenedione, and methyltestosterone by AFMC-micellar electrokinetic chromatography (MEKC) technique. The separation of neutral steroids using MEKC was also conducted as a comparison of enhancement factor to AFMC-MEKC. The use of a mixture of 20 mM sodium dodecyl sulphate (SDS) and methanol 10% (v/v) in 25 mM acetate buffer (pH 9.0) with a positive applied voltage of 25 kV and pressure injection of 40 mbar for 1 sec at 25°C was able to separate the steroids by MEKC. AFMC-MEKC was employed for enhancement in terms of sensitivity. The selected steroids were prepared in a solution containing 7 mM SDS and 250 mM acetate buffer with an optimized conductivity ratio of 0.34. The separation of the six steroids by AFMC-MEKC showed enhancement of sensitivity for each steroids with 2.4 to 3-fold enhancement with good repeatability (RSD 3.5-7.1% , n = 3) and reproducibility (RSD 2.6-12.7%, n = 3). The limits of detection for the six steroids ranged from 1.1-5.7 mg/L. The optimized method was successfully applied to analyze steroids in pharmaceutical tablets and urine samples. Combination of AFMC-MEKC with solid-phase extraction was used to determine six selected steroids in spiked urine sample while liquid-liquid extraction pretreatment was used for the determination of prednisolone and betamethasone in pharmaceutical tablets. The average recoveries of the selected steroids in spiked urine samples and pharmaceutical tablets were good ranging from 93 - 101% with RSD of 0.8 - 11.2% (n = 3). AFMC-MEKC separation on selected steroids showed triple fold enhancement in sensitivity compared to MEKC.