Abstract:

A method was developed for the determination of 17 cytokinins in rice by solid phase extraction and liquid chromatography-tandem mass spectrometry. The rice samples were cryogenically ground under liquid nitrogen and extracted with methanol-water (80 : 20, v/v) at 4 ℃ for 16 h. The supernatant was purified on a column packed with polymer cation exchange resin (PCX). The samples were transported and eluted on an analytical column ZORBAX Extend-C18 (100 mm×2.1 mm, 1.8 μm) by methanol and 5 mmol/L ammonium formate aqueous solution. All the analytes were detected in selected reaction monitoring (SRM) mode under positive electrospray ionization (ESI⁺) and quantified by external standard method. The separation conditions were optimized in order to achieve the sufficient separation for the several isomers of cytokinins, such as trans-zeatin-7-glucoside (tZ7G), trans-zeatin-9-glucoside (tZ9G), and trans-zeatin-O-glucoside (tZOG). Moreover, the extraction efficiency of different extraction solvents and clean-up effects of PCX cartridge for each analyte were further investigated. The results showed that the correlation coefficients were not less than 0.9984 in the linear range, and the limits of detection were ranged from 0.01 to 0.05 ng/g. The mean recoveries of the 17 cytokinins at three spiked levels of 0.2, 1 and 5 ng/g were from 60.2% to 125.4% with the relative standard deviations (RSDs) of 5.4%-29.7% (n=6). Finally, five endogenous cytokinins were successfully quantified in real sample, and their contents were between 0.02 and 0.93 ng/g. It means that this method is reliable for quantitative analysis of cytokinins in rice.

Key words: cytokinins, liquid chromatography-tandem mass spectrometry (LC-MS/MS), polymer cation exchange resin (PCX), rice