Quantitative Aanalysis Of 37 Phytohormones In Oryza Sativa By High-PerformanceLiquid Chromatography-Mass Spectrometry

Phytohormones were a series of trace organic compounds synthesized in plants, which played an important role in regulation on plant growth and development. Phytohormones rarely act alone, and for any processes, many of these regulators have interacted in order to produce the final efffect. Therefore, it is necessary to focus on the pretreatment and determination method with regard to multi-phytohormones at the organ level, which is the foundation work for biological effects of phytohormones.It is usually rather difficult to determine phytohormones owing to the complexity of rice matrix and to the their presence in trace amount and in dynamic range. Therefore, it is very important to how to separate and purify phytohormones effectively from rice. The present work is majorly premised on the technology of mix-mode solid phase extraction, choosing organic solvent such as 80% methanol-water mixture as extraction solvent and optimizing the conditions for the mix-mode solid phase extraction. Through grouping the conditions of extraction, seperation and purification, and combining the technology of solid-phase extraction, we established a method to anaysis the phytohormones in rice by liquid chromatography tandem mass spectrometry and evaluated the matrix effect in detection. Results of the present work are as follows:1.A qualitative and quantitative method was researched and established for 37 phytohormones by liquid chromatography tandem mass spectrometry. In this work, we researched the variety of organic acids, oven temperature and liquid phase programme,which had significant impact on capacity factor and resolution in chromatographic separation to resolve the seperation of geometric isomer. The results showed that when any acids was not added into the aqueous phase, only ammonium formate, cytokinin glucosides(Z7G-Z9G-ZOG,DZ7G-DZ9G-DZOG) was seperated on baseline with the resolution of 2; Comparied with oven temperature, liquid phase programme had a remarkable effect on chromatographic separation of gibberellin homologue(GA1-GA3,GA4-GA7,GA9-GA53), and the resolution increased by 50%-130% approximately. When the condition of oven temperature and liquid phase programme were 40℃and 4%/min, the gibberellin homologue could separate, but the resolution of GA1-GA3 was still less than 0.5; At the same time, we researched the factors which influenced the method sensitivity such as the component of mobile phase and the MS/MS conditions. The results showed that in dynamic MRM acquisition mode, methanol- ammonium formate(5mM pH=6) not only made sure the seperation of geometric isomer, but also showed favourable mass spectrometry response in the positive mode, which significantly enhanced method sensitivity 5-10 times; Adding different level standard materials (5μg/l,25μg/l,50μg/l) for the test of recovery(n=3), the results showed that the recovery range was 61%-109% with relative standard deviation lower than 16%; The limit of detection(S/N=3) were 0.0005-25μg/l.2.The present work established a preparion method that extracted by 80% methanol-water mixture, cleaned-up by mix-mode solid phase extraction. In this research, we probed into the effect on the extraction efficiency, seperation and purification by extract solvent, extraction times, sample solvent composition and eluent solvent composition of solid-phase extraction. The results showed that 80% methanol-water mixture is suitable extraction solvent, which need 12 hours to keep extraction amounts of Z9G maximum that is 2 times more than the direct method online. Through diluted extraction solution to make the percentage of methanol less than 33%, Mixed anion/cation exchange reversed-phase solid-phase extraction can retain phytohormones to a maximum extent. Comparied with the regular method, this method was simple, efficient and simplified the enrichment step.3.In this research, we evaluated the matrix effect in detection, and we use the established mix-mode solid phase extraction method to eliminate matrix effect. The results showed that the matrix effect would be related to the characteristic of compound, different marix medium and different level standard materials. Different compound presented matrix effect to varying degrees in different matrix, and the concentration remarkably affected the matrix effect, the smaller the concentration, the bigger the matrix effect, expecially gibberellin, auxin and abscisic acid. We can reduce the precence of coeluting substances in matrix by mix-mode solid phace extraction that had a good selectivity for the 37 phytohormones separation and the recovery range was 77%-109%. But when the mix standard solution was added to the matrix medium after solid phase extration on the concentration of 1μg/l, the recovery of ZROG was 63%, and the recovery of GA3, GA20 was less than 40%, this is because that the level approached limit of quantitation and made the result distortion.