Dispersive Matrix Solid-Phase Extraction Method Coupled with High Performance Liquid Chromatography-Tandem Mass Spectrometry for Ultrasensitive Quantification of Endogenous Brassinosteroids in Minute Plants and Its Application for Geographical Distribution Study.

An ultrasensitive analysis method for quantification of endogenous brassinosteroids in fresh minute plants was developed based on dispersive matrix solid-phase extraction coupled with high performance liquid chromatography-tandem mass spectrometry. During the dispersive matrix solid-phase extraction, plant samples were first ground with solid sorbent (dispersant) in one microcentrifuge tube and then centrifuged after adding extraction solvent and cleanup materials (another type of sorbent). Three protocols based on dispersive matrix solid-phase extraction were compared and discussed for plant samples with different matrix complexity. The choice of any protocol was a compromise of increasing purification efficiency and decreasing sample loss. Under optimized conditions, the limits of detection were 1.38-6.75 pg mL for five brassinosteroids in the oilseed rape samples. The intraday and interday precisions were in the range of 0.8%-9.8% and 4.6%-17.3%, respectively. The proposed method was successfully applied to detection of endogenous brassinosteroids in milligram oilseed rape (2.0 mg) and submilligram Arabidopsis thaliana seedlings (0.5 mg). Finally, the geographical distribution of five endogenous brassinosteroids of Brassica napus L. oilseed rape in different provinces of origin in the Yangtze River basin was described.