Determination of Endogenous Brassinosteroids in Plant Tissues Using Solid-phase Extraction with Double Layered Cartridge Followed by High-performance Liquid Chromatography-Tandem Mass Spectrometry

Introduction Brassinosteroids (BRs) are a group of important phytohormones that play vital roles in plant growth, development and a series of physiological phenomena. In order to understand biosynthesis, degradation and metabolic pathways of BRs, a reliable analytical method of BRs with effective sample pre-treatment process is favourable. Objective The development of a quick and effective method for the quantification of endogenous BRs in plant tissue with the aid of double layered solid-phase extraction (SPE) cartridges (graphite carbon black and primary secondary amine silica sorbent: GCB/PSA). Method The method involved an initial extraction of BRs with acetonitrile, a dehydration process with anhydrous MgSO4 and NaCl, a SPE purification process with a double layered cartridge, and a further clean-up step utilising liquid-liquid extraction (LLE). The purification process was mainly realised on the GCB/PSA cartridge. GCB could eliminate hydrophobic compounds, especially those containing a system, and PSA was introduced to remove the polar interferences. Endogenous BRs were quantified by HPLC-ESI-MS/MS. Results Good linearities were obtained in the range of 0.4-500ng/mL (0.0124-15ng), with the correlation coefficients above 0.9957. The relative recoveries of BRs of this method were in the range of 71.1-113.1%, with intra- and interday relative standard deviations (RSDs) less than 16.3%. With the proposed method, the requirement of plant tissue amount was minimised to 1g fresh weight, which is the smallest amount reported so far, to our knowledge. Copyright (c) 2013 John Wiley & Sons, Ltd.