Ultrasensitive quantification of endogenous brassinosteroids in milligram fresh plant with a quaternary ammonium derivatization reagent by pipette-tip solid-phase extraction coupled with ultra-high-performance liquid chromatography tandem mass spectrometr

•A simple and ultra-sensitive PT-SPE-UPLC-MS/MS method was established for BRs.•A quaternary ammonium derivatization reagent was first developed.•A pipette-tip solid-phase extraction strategy was proposed.•MDA of 27–94 amol was achieved for BRs.•Endogenous BRs were quantified in 0.5 mg FW of rice leaf segments.

Determination of endogenous brassinosteroids (BRs) in limited sample amount is vital to elucidating their tissue- and even local tissue-specific signaling pathway and physiological effects on plant growth and development. In this work, an ultra-sensitive quantification method was established for endogenous BRs in milligram fresh plant by using pipette-tip solid-phase extraction coupled with ultra-performance liquid chromatography tandem mass spectrometry (PT-SPE-UPLC-MS/MS), in which a quaternary ammonium phenyl boronic acid, 4-borono-N,N,N-trimethylbenzenaminium iodide (BTBA) was first developed for chemical derivatization of BRs. Due to the cationic quaternary ammonium group of BTBA, the ionization efficiencies of the BRs chelates with BTBA (BTBA-BRs) were enhanced by 1190–448785 times, which is the highest response enhancement factor among all derivatization reagents reported for BRs. In addition, PT-SPE packed with C18 sorbent was first used for purifying BRs from plant extracts, so the required sample amount was minimized, and recoveries higher than 91% were achieved. Under the optimized conditions, the minimal detectable amounts (MDA) of five target BRs were in the range of 27–94 amol, and the correlation coefficients (R2) were >0.9985 over four orders of magnitude. The relative recoveries of 75.8–104.9% were obtained with the intra- and inter-day relative standard deviations (RSDs) less than 18.7% and 19.6%, respectively. Finally, three BRs were successfully quantified in only 5 mg fresh rice plant samples, and 24-epiBL can even be detected in only 0.5 mg FW rice leaf segments. It is the first time that the BRs content in sub-milligram fresh plant sample has been quantified.