Simultaneous determination of multi-class phytohormones in sub-milligram plant samples by one-pot multi-functional derivatization assisted liquid chromatography-tandem mass spectrometry.

Phytohormones play crucial roles in every aspect of plant life, and their regulatory functions rely on complex crosstalk networks among the different classes of phytohormones in an antagonistic or synergistic manner. Therefore, the simultaneous determination of multiclass phytohormones is important for studies of phytohormone functions and networks. However, due to the heterogeneity of the sensitivity resulting from structural diversity and the low content in the plant samples, simultaneous determination of multiclass phytohormones is challenging, especially in very tiny plant tissues or organs. Here, we describe a novel method for the simultaneous determination of 31 phytohormones from different classes in a single run. This uses a one-pot multifunctional derivatization coupled to liquid chromatography tandem mass spectrometry (LC-MS/MS). N,N-Diethyl ethylenediamine (DEED) and 2-methyl-4-phenylaminomethyl-benzeneboronic acid (2-methy-4-PAMBA) were used for derivatization of carboxylated phytohormones and brassinosteroids (BRs), respectively, to simultaneously improve detection sensitivities of carboxylated phytohormones and BRs. The method was fully validated for the 31 targeted phytohormones and could quantify multiclass endogenous phytohormones in small amounts of fresh plant samples (0.02-5 mg, FW). Finally, we used this method to investigate the spatial-temporal distribution of multiple phytohormones in reproductive organs of a single flower of Arabidopsis thaliana for the first time. This method could be a powerful auxiliary tool for studies of phytohormone functions and regulatory networks.