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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 π 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 0.4-500 ng/mL (0.0124-15 ng), 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 1 g fresh weight, which is the smallest amount reported so far, to our knowledge.