Rapid liquid chromatographic-mass spectrometric analysis of withanolides in crude plant extracts by use of a monolithic column

Abstract

A rapid analytical method has been developed for the mutual resolution of three steroidal compounds, withaferin A, iochromolide, and withacnistin. Liquid chromatography was performed on a Chromolith analytical column (4.6 mm i.d.×50 mm), made from a cylindrical silica rod, operated at a flow rate of 4 mL min−1 with a simple linear gradient prepared from 0.1% aqueous formic acid and 0.1% formic acid in acetonitrile. Under optimum conditions simultaneous separation of the compounds was achieved in less than 7 min, one eighth the time required for conventional LC separation. The overall analysis time was reduced without sacrificing chromatographic performance—essential for the resolution of positional isomers such as iochromolide and withacnistin. The column was coupled to a single-quadrupole mass spectrometer and the method was characterized by good performance in terms of repeatability, selectivity, linearity, and sensitivity. Detection limits in the single-ion-monitoring mode were 0.15 μg mL−1 or below. Finally, the developed method was successfully applied to the determination of withanolides in extracts fromlochroma gesnerioides obtained by three different processes—traditional Soxhlet extraction and two faster methods, microwave-assisted extraction and pressurized solvent extraction

LC-MS/MS method for the quantification of withaferin-A in plant extracts ofWithaniaspp.

“…Thus, our limit of detection (LOD) in the standards was found to be 0.6 ng/mL (ppb), and applying the method to plant extracts, the LOD was approximately 10 times higher than in the standards, being 6 ng/g. Examples of reported detection limits for withaferin-A include 500 ng/mL using single-ion monitoring [10], 190 ng/mL using photodiode array [5] and 120 ng/mL using HPTLC [12], thus confirming that the LOD decreases in MS/MS compared to other methods. We required an analysis time of 10 min, which is less than in other reported studies on withanolides, in which total analysis times are 24 min (retention time for withaferin-A 14 min), 35 min (retention time for withaferin-A 21.35 min) and 50 min (retention time for withaferin-A 19.2 min) [4,9,10], respectively.…”

“…Examples of reported detection limits for withaferin-A include 500 ng/mL using single-ion monitoring [10], 190 ng/mL using photodiode array [5] and 120 ng/mL using HPTLC [12], thus confirming that the LOD decreases in MS/MS compared to other methods. We required an analysis time of 10 min, which is less than in other reported studies on withanolides, in which total analysis times are 24 min (retention time for withaferin-A 14 min), 35 min (retention time for withaferin-A 21.35 min) and 50 min (retention time for withaferin-A 19.2 min) [4,9,10], respectively. Similar to our case, Bandyopadhyay et al [13] reported an analysis time of 10 min and a retention time of 5.45 min for withaferin A in W. somnifera root extracts.…”

“…Until now, no studies about the identification and quantification of withaferin-A in plant extracts using MS/MS detection have been described. Only Khajuria et al [9] and Kaufmann et al [10] have reported on withaferin-A MS but in the selected ion mode (SIM), which is less specific than MRM and high-resolution mass spectrometry identification. The MS/MS detection mode has the advantage of a higher sensitivity than other methods.…”

“…Recently, withaferin-A content of the aerial parts and roots of W. somnifera and W. coagulans has been analysed by HPTLC [8]. Quantitative liquid chromatography/mass spectrometry (LC/MS) analyses of withanolides have been reported by several authors, but generally using the less specific single-ion monitoring (SIM) mode or full scan methods [9,10] and not the MS/MS method, which is more sensitive and reliable.…”

Development of Rapid Analytical Methods in the Laboratory of Pharmaceutical Analytical Chemistry (LCAP)

“…The LCAP has already developed different methods for the rapid analysis of drugs with the above-mentioned strategies [2] [3]. Research is in progress to determine advantages and drawbacks in terms of efficiency, robustness, cost, and sensitivity.…”