Magnetic solid phase extraction coupled with in situ derivatization for the highly sensitive determination of acidic phytohormones in rice leaves by UPLC-MS/MS

Abstract

A simple, rapid and sensitive method based on magnetic solid phase extraction (MSPE) coupled with in situ derivatization (ISD) was developed for the determination of endogenous acidic phytohormones in rice leaves by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. With this method, acidic phytohormones were extracted onto the surface of a TiO₂/magnetic hollow mesoporous silica sphere (MHMSS) through hydrophilic interaction, and then in situ derivatization was performed by the subsequent addition of 3-bromoactonyltrimethylammonium bromide (BTA). Thus, the process integrated extraction, purification, and derivatization into one step. Additionally, the permanent positively charged moiety from BTA significantly improved the ionization efficiencies of the acidic phytohormones. Several parameters affecting the efficiencies of the extraction, derivatization, and desorption were evaluated. The signal intensities of acidic phytohormones increased by 2 to 481 fold after treatment with MSPE-ISD. Under the optimized conditions, several endogenous acidic phytohormones, including GA₄, GA₉, GA₂₀, JA, IAA, and ABA, were identified and quantified in rice leaves by the MSPE-ISD method. The limits of detection (LODs) were in the range of 1.03–91.21 pg mL⁻¹. The relative recoveries ranged from 71.6–112.8%, with the intra- and interday relative standard deviations (RSDs) being less than 14.9% and 16.2%, respectively. Taken together, the proposed method provides a novel approach of combining magnetic solid phase extraction and in situ derivatization for the highly sensitive determination of endogenous acidic phytohormones.