Quantification of endogenous brassinosteroids in sub-gram plant tissues by in-line matrix solid-phase...

A matrix solid-phase dispersion (MSPD)-tandem mixed mode anion exchange (MAX)-mixed mode cation exchange (MCX) solid phase extraction-high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) method was developed for quantification of six endogenous brassinosteroids (BRs) (24-epibrassinolide, 24-epicastasterone, 6-deoxo-24-epicastasterone, dolichosterone, teasterone and typhasterol) in rice plant tissues. Non-polar interferences were removed effectively by C8 dispersant used in MSPD, while the following tandem MAX-MCX process facilitated the elimination of polar and ionizable compounds. The weak reversed-phase retention feature of MAX-MCX leaded to good compatibility of the elution solvents in the in-line coupled MSPD-MAX-MCX system. This system was optimized for extraction and purification of BRs in plant samples. The effects of the type of solid phase, the elution solvent, the extraction temperature and the clean-up material were studied. Before HPLC separation, BRs purified were derivatized by m-aminophenylboronic acid to enhance the sensitivity of MS/MS to BRs. Compared with traditional liquid-liquid extraction and solid phase extraction (LLE-SPE), the proposed MSPD-MAX-MCX method showed higher extraction efficiency, lower matrix effect, and advantages of easy manipulation and time-saving. The in-line MSPD-MAX-MCX coupled with HPLC-MS/MS method provided a linear response over two orders of magnitude of BRs concentration with correlation coefficients above 0.9982, limits of detection between 0.008 and 0.04ngmL(-1), relative standard deviations (RSDs) below 29.4%, and recoveries above 77.8%. The proposed method has been successfully applied to analysis of endogenous BRs in rice plant at booting stage and maturity stage.