Determination Of Plant Hormones By Matrix Solid-phase Dispersion-high Performance Liquid Chromatography-tandem Mass Spectrometry

Plant hormones are natural products with small molecular that regulate all plant physiological and developmental processes at low concentrations. Accurate determination of plant hormones is increasingly important for in-depth study of their biosynthesis, transport, metabolism and molecular regulatory mechanisms. Owing to the complexity of plant matrix and the trace concentrations of plant hormones, pretreatment method remains a bottleneck in the botany research area. Thus it is important to develop highly selective, sensitive and reliable methods for qualitative and quantitative analysis of plant hormones, which is a key to promote the intensive study of botany.The traditional analysis of plant hormones usually adopted complicated sample preparation methods, which containing overnight extraction, solvent extraction, solid phase extraction (SPE) or chromatographic clean-up, and purification by preparative liquid chromatography and so on. These methods are time-consuming (usually takes a few days) with low extraction efficiency. In view of this, this thesis was focused on developing new sample pretreatment methods, coupled with chromatography-mass spectrometry technology, for the analysis of gibberellins, brassinosteroids and cytokinins in real plant samples with high sensitivity and selectivity. The main conclusions are as followings.(1) A method for the analysis of three gibberellins (GA1, GA3and GA4) in Arabidopsis thaliana by matrix solid-phase dispersion extraction (MSPD) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed. The conditions of sample pretreatment, chromatography and mass spectrometry detection were optimized. The limits of detection (LODs) of three GAs were1.1-1.4ng/g. Finally, the proposed method was applied for the quantitative determination of GA1, GA3and GA4in Arabidopsis thaliana. The detected concentrations of GA1, GA3and GA4in three plant samples were11.20-15.14ng/g,1.15-1.53ng/g and2.08-3.43ng/g, respectively. This method is simple, selective, and efficient.(2) Based on the method above, the MSPD process was improved by pre-filling dispersant in SPE column and extraction with static equilibration for selective extraction of trace gibberellins in rice plant. The static equilibration time, the ratio of the sample to solid phase, clean-up material and elution solvent were optimized to reduce the matrix effects and to obtain high extraction efficiency. The MSPD was coupled with LC-MS/MS for separation and detection of eight GAs with LODs in the range of0.03-0.67ng/g. The relative recoveries of eight GAs at low concentration vary rather wide (46.6%to117.9%), with relative standard deviation (RSD) values below39.3%; while at higher concentrations, however, the relative recoveries were in the range of69.3%to122.5%(25ng/g) and85.7%to119.0%(35ng/g), with RSD values below11.3%and4.6%, respectively. The results showed that the addition of static equilibration process in MSPD increased the extraction recovery of GAs; while the pre-filled SPE sorbent further purified the extracts and reduced the matrix effect, indicating that the present method is an effective mean to improve the detection sensitivity.(3) An in-line MSPD-tandem MAX-MCX method for selective extraction and purification of brassinosteroids (BRs) was established. Reversed-phase sorbents were preferred for the MSPD process, which could effectively remove the neutral and hydrophobic interferences in plant. While the following tandem MAX-MCX process could facilitate the elimination of ionizable compounds, reducing its negative impact on MS detection. Since the same solvent was used for elution, the MAX-MCX columns could be in-line placed under the MSPD cartridge for convenience. Extraction and purification of BRs from plant samples were completed in one step, which reduced the time cost from days to a few hours. Solvent extraction of plant tissues overnight was totally avoided. The method was coupled easily with LC-MS/MS to achieve high sensitivity and selectivity for trace analysis of endogenous BRs (24-epiBL,24-epiCS, DS, d-epiCS, TE and TY) in sub-gram rice plant samples. The LODs were found to be in the range of0.008-0.04ng/mL. The minimal detectable amount of BRs in single injection was0.04-0.2pg. The new method was applied to measure endogenous BRs in rice plants at two important growth stages (booting stage and maturity stage). The results showed an obvious difference in BR concentrations at different growth stages of plant.(4) A new dispersive matrix solid-phase extraction (DMSPE) method was developed based on the conjunction of MSPD and dispersive solid-phase extraction (d-SPE). First, plant sample was blended with solid dispersant, and then the homogeneous mixture was transferred into a centrifuge tube. After adding the extraction solvent, the mixture was centrifuged, during which the extraction and purification of target analytes were accomplished. This method was validated to extract and purify exogenous and endogenous cytokinins (CKs) in0.02g of plant sample. The supernatant was separated and detected by LC-MS/MS. The LODs were found to be in the range of0.01-0.06ng/mL. The minimal detectable amount of CKs in single injection was0.05-0.30pg. This method was simple, reliable, and avoided overnight solvent extraction. The time cost was reduced from more than12h to about0.5h. The proposed DMSPE-HPLC-MS/MS method was suitable for the highly sensitive and selective detection of CKs in sub-gram plant samples.