Analysis of selected withanolides in plant extract by capillary electrochromatography and microemulsion electrokinetic chromatography

Microemulsion electrokinetic chromatography (MEEKC) coupled with a diode-array detector was developed for the simultaneous analysis of natural steroidal compounds, withanolides including withaferin A, withacnistin and iochromolide. Optimal resolution was obtained with a microemulsion consisting of 70 mm octane, 800 mm 1-butanol, 100 mm sodium dodecyl sulfate (SIDS), and 10 mm phosphate-borate buffer (pH 7) using a fused-silica capillary at 25 W and 40degreesC. Since this technique is not compatible with mass spectrometry detection, a capillary electrochromatographic method was developed to separate the investigated withanolides. The effects of mobile phase composition and pH were systematically investigated. Complete separation was obtained with a capillary electrochromatography (CEC) Hypersil C18 bonded silica column (packed length, 25 cm x 100 mum ID and 375 mum OD), packed with 3 mum particles. The mobile phase consisted of formic acid-ammonia, pH 8/acetonitrile (40/60 v/v); the voltage was set at 25 kV and the temperature at 20degreesC. Under these conditions, resolution of these closely related compounds, including the critical pair withacnistin and iochromolide, was achieved in less than 5 min. The separations by MEEKC and CEC were compared with that obtained by reversed-phase liquid chromatography and showed similar retention order, indicating the analogy of the retention mechanism of these techniques. To further improve specificity and sensitivity, the developed CEC method was interfaced with electrospray ionization mass spectrometry using a Teflon connection between the CEC column and a void fused-silica capillary. Finally, the described methods were applied to the qualitative analysis of withanolides in lochroma gesnerioides plant extract.