Profiling of phytohormones and their major metabolites in rice using binary solid-phase extraction and liquid chromatography-triple quadrupole mass spectrometry

• Analysis of 43 phytohormones and their major metabolites using LC–MS/MS.
• Using a binary solid-phase extraction effectively reduced matrix effects.
• Base-labile compounds were stabilized by diluting extract prior to purification.
• Twenty tested compounds were successfully quantified in a rice sample.

A high-throughput method was developed using liquid chromatography-triple quadrupole mass spectrometry (LC–MS/MS) for the profiling and quantification of 43 phytohormones and their major metabolites, including auxins, abscisic acid, jasmonic acid, salicylic acid, cytokinins and gibberellins in a single sample extract. Considerable matrix effects (MEs) were observed (with most ME values in the range of 29%–84%, but maximum MEs of more than 115%, even up to 206%, existed) in sample extracts for most of the compounds studied. The application of the proposed binary solid-phase extraction using polymer anion and polymer cation exchange resins, was performed to purify 25 acidic and 18 alkaline phytohormones and their major metabolites prior to the LC–MS/MS analysis, which markedly reduced the MEs to acceptable levels, with ME values in the range of ±15%. Moreover, all of the isomers of cytokinins and their metabolites were fully separated on a sub-2 μm particle C18 reverse-phase column with the optimized mobile phase consisting of methanol and 5 mM ammonium formate. The method showed good linearity for all 43 analytes with regression coefficients (R2) > 0.991. Limits of detection ranged from 0.19 to 7.57 fmol for auxin, gibberellins, abscisic acid and their metabolites, 29.7 fmol for jasmonic acid, 18.1 fmol for salicylic acid, and from 0.03 to 0.31 fmol for cytokinins and their metabolites. The mean recoveries for all of the analytes were from 70.7 to 118.5%, and the inter-day precisions (n = 6) were less than 18.7%, with intra-day precisions (n = 6) within 25.4%. Finally, 20 compounds were successfully quantified in rice sample profiles using the proposed method, which will greatly facilitate the understanding of hormone-related regulatory networks that influence rice growth and development. To our knowledge, there are limited reports that measure this level of phytohormone species in rice samples using a single analysis.