Determination of cytokinins in plant samples by polymer monolith microextraction coupled with hydrophilic interaction chromatography-tandem mass spectrometry

In this study, a sensitive assay of cytokinins was developed using polymer monolith microextraction/hydrophilic interaction chromatography/electrospray ionization-tandem mass spectrometry (PMME/HILIC/ESI-MS/MS). The extraction was realized on a poly(2-acrylamido-2-methyl-1 -propanesulfonic acid-co-ethylene dimethacrylate) (poly(AMPS-co-EDMA)) capillary monolith and the subsequent separation was carried out on a Luna silica column. Several parameters of PMME and HILIC were optimized to obtain the optimum results. After optimizing the extraction conditions, 10 mM ammonium formate of pH 2.5 was chosen as the matrix solution to obtain the highest extraction efficiency. The MS sensitivity for cytokinins investigated could be enhanced 3-fold by the use of hydrophilic interaction chromatography with the mobile phase of 85% acetonitrile with 0.01% (v/v) formic acid and 15% water with 0.01% (v/v) formic acid, when compared to the use of conventional reversed phase liquid chromatography (RPLC). Good linearities were obtained for five cytokinins with correlation coefficients (R-2) > 0.9962. The LODs (S/N = 3) for the targets were found to be 0.0028-0.068 ng mL-(t). Reproducibility of the method was obtained with intra-day and inter-day relative standard deviations (RSDs) less than 12.7% and the recoveries in plant samples ranged from 70.3% to 113.3%. The method was applied to the determination of cytokinins in Oryza saliva, Arabidopsis thaliana and oil seed rape tissues.