In recent plant hormone research, there is an increased demand for a highly sensitive
and comprehensive analytical approach to elucidate the hormonal signaling networks,
functions, and dynamics. We have demonstrated the high sensitivity of a comprehensive
and quantitative analytical method developed with nanoflow liquid chromatography-electrospray
ionization-ion trap mass spectrometry (LC-ESI-IT-MS/MS) under multiple-reaction monitoring
(MRM) in plant hormone profiling. Unlabeled and deuterium-labeled isotopomers of four
classes of plant hormones and their derivatives, auxins, cytokinins (CK), abscisic
acid (ABA), and gibberellins (GA), were analyzed by this method. The optimized nanoflow-LC-ESI-IT-MS/MS
method showed ca. 5-10-fold greater sensitivity than capillary-LC-ESI-IT-MS/MS, and
the detection limits (S/N=3) of several plant hormones were in the sub-fmol range.
The results showed excellent linearity (R(2) values of 0.9937-1.0000) and reproducibility
of elution times (relative standard deviations, RSDs, <1.1%) and peak areas (RSDs,
<10.7%) for all target compounds. Further, sample purification using Oasis HLB and
Oasis MCX cartridges significantly decreased the ion-suppressing effects of biological
matrix as compared to the purification using only Oasis HLB cartridge. The optimized
nanoflow-LC-ESI-IT-MS/MS method was successfully used to analyze endogenous plant
hormones in Arabidopsis and tobacco samples. The samples used in this analysis were
extracted from only 17 tobacco dry seeds (1mg DW), indicating that the efficiency
of analysis of endogenous plant hormones strongly depends on the detection sensitivity
of the method. Our analytical approach will be useful for in-depth studies on complex
plant hormonal metabolism.