Improved methodology for analysis of multiple phytohormones using sequential magnetic solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry

•A simple and efficient method was developed for simultaneous analysis of multiple phytohormones.•IAA, ABA, JA, GAs, BRs and CKs were purified by sequential magnetic solid-phase extraction.•16 endogenous phytohormones could be detected in 100 mg (fresh weight) flower of Brassica napus L.

Phytohormones are special small molecules that play important role in plant growth and development at trace levels. Quantification of multiple phytohormones will be great helpful for researches about cross-talks that plant hormones regulate the responses of plants against both biotic and abiotic stresses by means of synergistic or antagonistic interactions. In the current study, we developed a method for profiling of phytohormones in one small sample, including indole-3-acetic acid, abscisic acid, jasmonic acid, gibberellins, cytokinins and brassinosteroids. These phytohormones mentioned above were firstly purified and separated by sequential magnetic solid-phase extraction (MSPE) and then analyzed by ultra-high performance liquid chromatography-electrospray tandem mass spectrometry (UHPLC-MS/MS). Under the optimized extraction conditions, good linearity was obtained with correlation coefficients (r) ranging from 0.9961 to 0.9998. The limits of detection (LODs, S/N = 3) were ranged from 0.45 to 126.1 pg mL−1. The recoveries were between 85.0% and 116.2%. The relative standard deviations (RSDs) were ranged from 2.7% to 16.1%. With the proposed strategy, 23 phytohormones could be purified and analyzed from a single plant extract. Finally, 16 phytohormones could be detected in 100 mg (fresh weight) flower of Brassica napus L., including IAA, ABA, JA, 4 GAs, 3 BRs and 6 CKs with the concentration ranged from 0.09 to 305.23 ng g−1.

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