LC-MS Method for Structural Characterization of Ecdysteroids in Sida rhombifolia L.

Abstract

The genus Sida is used extensively in the herbal medicine industry [1]. Roots and aerial parts of S. rhombifolia are used in the treatment of gonorrhoea, anti-soud, diuretic, aphrodisiac and rheumatism [1]. Ecdysteroids are insect moulting hormones [2], and are generally considered to have a significance and specific role in the plant-insect chemical interactions [2]. Ecdysteroids isolated from Sida rhombifolia L. were studied by electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn) in the positive ion mode using ion trap analyzers and high performance liquid chromatography coupled with photodiode array detector (HPLC-PDA). The HPLC experiments were performed by means of a reversed-phase C18 column and a binary mobile phase system consisting of water (containing 0.05% formic acid) and acetonitrile (containing 0.05% formic acid) under gradient elution conditions. According to the substitution at C-2, C-5, C-20 and C-24, ecdysteroids in S. rhombifolia were classified into 4 sub-groups. Structural identification of these four sub-groups of ecdysteroids was established by multi-stage ion trap mass spectrometry. The fragmentation patterns of ecdysteroids yielded ions of successive loss of 1–3 water molecules. Furthermore, ions corresponding to the complete loss of the side chain at C-17 will help to identify the sub-groups of ecdysteroids in addition to containing a hydroxy moiety at one of the above mentioned positions. Based on the HPLC retention behavior, the diagnostic UV spectra and the molecular structural information provided by electrospray multi-stage mass spectrometry spectra, a total of eleven naturally occurring ecdysteroids were identified, of these four are identified for the first time in S. rhombifolia. Acknowledgements: This research is funded in part by “Science Based Authentication of Dietary Supplements” funded by the Food and Drug Administration grant number 2 U01 FD 002071-07.