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

Abstract

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 (SDS), and 10 mM phosphate-borate buffer (pH 7) using a fused-silica capillary at 25 kV and 40 degrees C. 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 cmx100 microm ID and 375 microm OD), packed with 3 microm 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 20 degrees C. 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 Iochroma gesnerioides plant extract

Recent advances in microemulsion electrokinetic chromatography

“…SDS, an anionic surfactant, is usually used in MEEKC at concentrations of 3.3% w/w (118 mM). Increasing the SDS concentration affects solute migration times depending on analyte charge, and also by reducing the EOF level [9,18,23,28,34]. If the solute ion-pairs with the droplet, increasing surfactant concentration increases the droplet charge density and increases analyte migration and peak resolution [18,31,32].…”

Recent applications of microemulsion electrokinetic chromatography

“…No real samples were analyzed. Another chemical group of plant constituents with biological activities, the withanolides, was analyzed by MEEKC as well as by capillary electrokinetic chromatography coupled to mass spectrometry (CEC-MS) [52]. The MEEKC separation was by far the best regarding separation efficiency, however, MEEKC is not suited for on-line coupling to MS due to the high surfactant concentration.…”

Recent advances in the development and application of microemulsion EKC

“…SDS, which is an anionic surfactant, is usually employed in MEEKC at a concentration of 3.3% (typically 118 mM). Increasing the SDS concentration effects solute migration times depending on the analytes charge and also by reducing the EOF [10,28,[30][31][32]. Depending on its interaction with the surfactant, increasing its concentration can either decrease [33], or increase analyte migration time [10] which affects peak resolution [33].…”