High-throughput miniaturised solid phase extraction - supercritical fluid chromatography tandem mass spectrometry workflow for acidic phytohormone profiling.

Acidic phytohormones, such as abscisates, auxins, jasmonates, and salicylic acid, are low-abundance signalling molecules in plants. A high-throughput workflow coupling reverse phase based miniaturised solid phase extraction (RP-μSPE) with ultra-high performance supercritical fluid chromatography tandem mass spectrometry method (SFC-MS/MS) was established for their profiling, introducing complementary selectivity in sample clean-up and chromatographic separation. The SFC-MS/MS method was developed through stationary phase selection, design of experiment screening, and optimisation of chromatographic conditions, ion-source parameters, make-up solvent, injection volume, and gradient conditions. The Viridis HSS C18 SB column was selected among five stationary phases of different chemistries, supercritical CO2 and methanol as a co-solvent, containing 0.1% ammonia and 3% water, were employed as the mobile phase. Direct injection of 5 μL μSPE eluate enabled satisfactory peak shapes without an evaporation step. Methanol was used as the make-up solvent at 0.25 mL/min. The workflow was validated, providing accuracies within 85-115%, precision of 0.2-14.9%, matrix effects of 87-109%, limits of detection 2.5-250 fmol, linear ranges spanning 3-4 orders of magnitude, R2 0.9946-0.9999, and carry-over not exceeding 20%, for 19 acidic phytohormones passing the validation criteria. Compared to the routine RP-LC workflow, the SFC approach reduced chromatographic run time from 19 to 11 min, offered complementary selectivity, and decreased matrix effects. However, in SFC, the detection sensitivity was about one order reduced, two compounds were excluded from the method due to strong carry-over and one due to insufficient retention relative to LC. The workflow applicability was demonstrated by time-course profiling in wounded Arabidopsis thaliana leaves.