Tandem Solid Phase Extraction Followed by Online Trapping–Hydrophilic Interaction Chromatography–Tandem Mass Spectrometry for Sensitive Detection of Endogenous Cytokinins in Plant Tissues

Abstract

Cytokinins (CKs) are a group of plant hormones that play pivotal roles at low concentration in plant growth, development and regulatory pathways. In order to study the function, metabolism and signal transduction of CKs, high performance analytical techniques are required for determination of their endogenous levels. To develop a highly sensitive, selective and reliable method for identification and quantification of CKs by employing a tandem solid phase extraction (SPE)-online trapping-hydrophilic interaction chromatography (HILIC)-MS/MS method. The extraction was performed firstly with tandem SPE containing a C(18) cartridge and a silica@C(8) /SO(3) H cartridge. After CKs were eluted from the silica@C(8)/SO(3) H cartridge, the desorption solvent was concentrated and redissolved in H(2)O and then injected into the online trapping-HILIC-MS/MS system with (Poly(MAA-co-EGDMA)) monolith as the trapping column. Subsequently, trapping, washing, desorption, separation and detection were accomplished automatically on the system. Good linearities were obtained for eight cytokinins with correlation coefficients (R(2)) > 0.9964. The limits of detection (LOD; S:N = 3) for the targets ranged from 0.042 to 1.6 pg/mL. Reproducibility of the method was evaluated with intraday and interday relative standard deviations (RSDs) less than 13.4% and the recoveries ranged from 77.3% to 116.3%. The results showed that the LOD of the analytical method were at least one order of magnitude lower compared with other previously reported methods. Furthermore, only 20 mg of plant tissues were required for the quantitative analysis of the major CKs, which is, to the best of our knowledge, the smallest amount reported so far for the determination of endogenous CKs in plant tissues. The tandem SPE-online trapping-HILIC-MS/MS method developed in current study provides a powerful tool for the convenient and highly sensitive quantification of the major CKs in plant tissue.