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.