| With the development of metabolism, the analysis of low molecular weight metabolites has aroused wide attention. As the end products of gene expression, metabolites especially the polar metabolites play a key role in biological system. Respiration is in a key position in the entire metabolic networks, the metabolic process of which is complex and diverse. The respiration mainly consists of glycolysis, pentose phosphate pathway and tri-carboxylic acid cycle, and the metabolites such as sugar phosphates, carboxylic acids, nucleotides and reduced coenzyme etc. play an important physiological role in plant. The plant respiration can be completely understood by analyzing the metabolites.Because of extremely high polarity and similar chemical structure, the respiratory metabolites are not retained on conventional reversed phase chromatography columns. In recent years, some analytical methods such as anion-exchange chromatography and ion-pair reversed phase liquid chromatography were dominantly used for research of these metabolites and could separate the metabolites somewhat, however, the present methods were not incompatible with electrospray mass spectrometer and confined by low sensitivity, most of which were mainly applied to simple material such as bacteria, yeasts and cultured cells, there were few researches on complex plant material. Moreover, only a small number of compounds were detected utilizing these methods, and the comprehensive and effective ways of detecting metabolites under complex substrate were absent.HILIC (hydrophilic interaction chromatography) is a chromatographic technique used to improve the retention behavior of highly polar compounds and has a good compatibility with mass spectrometry. Here we established a high throughput analytical method for qualitative and quantitative determination of 29 metabolites relating to respiration in rice at the same time, in which Shodex Asahipak NH2P-504E was used as stationary phase and hydrophilic interaction chromatography was coupled to tandem mass spectrometry of highly sensitivity and selectivity. Results of the research are as follows:1) Established the best conditions of mass spectrum.The best scan mode for 29 target metabolites was selected and each metabolite standard was scanned by Q1 mass in positive ion mode and negative ion mode respectively, the response intensity of parent ions was compared in different mode. The result shows that the compounds like carboxylic acid, sugars phosphate, and nucleotides such as AMP,ADP,ATP,cGMP,GTP were monitored in negative mode while the compounds like NAD+,NADH,NADP,NADPH,FAD,acetyl coenzyme A, succinyl coenzyme A were monitored in positive mode. The parent ion of every metabolite was scanned under Q2 mass for characteristic daughter ions. The qualitative and quantitative analysis was operated in selected reaction monitoring.2) The appropriate chromatographic column was selected. The influence of two chromatographic columns (ZIC-HILIC and Shodex Asahipak NH2P-504E ) on retention behavior of target metabolites were investigated, and Shodex Asahipak NH2P-504E was better.3) The effects of pH, kinds and concentration of buffers on separation were obtained.The pH and buffers were important factors affecting the retention behavior of target metabolites, and the point was as follows: As the increase of pH value and concentration of buffer, the elution capacity of mobile phase was enhanced and the numbers of appeared peak were increased, while the sensitivity and shape of peaks was improved. The tailing problem of citric acid and isocitric acid as well as the difficulty of elution about phosphate compounds could not be solved with ammonium formate and ammonium acetate ranged at 0～20mM ,however, the peaks of all targeted compound were appeared by utilizing 15mM ammonium carbonate or ammonium hydrogen carbonate, the difficulties of tailing and adsorption were solved successfully. There were no significant differences between the two buffers. The best combination of buffer and pH was optimized, in which the concentration of buffer was 15mM and pH value was 10.3.4)The best conditions for extracting respiratory metabolites were obtained.In the study, the extraction efficiency of different extraction solvents such as boiling water, 80% acetonitrile, (750:200:50,v/v/v) acetonitrile/water/formic acid,(3:7,v/v)chloroform/acetonitrile plus water were compared and the result shows that the last extraction solvents was best. In addition, two extraction modes of shaking and sonicating were compared and shaking was proved efficiently to extract target metabolites.5) Established the determination method for metabolites relating to respiration in rice. In this study, a series of parameters were determined, which contained standard curve, range of linearity, limit of detection, limit of quantification, spiked recovery and precision. The result shows good linearity over the range of 0.01~10mg/kg for 29 metabolites relating to respiration with correlation coefficient of 0.99. Meanwhile, the limit of detection was 0.001～0.25mg/kg and the spiked recovery was 63.3～133.5% with the relative standard deviations under 20%.6) The 29 metabolites relating to respiration in leaf and root of rice (Zhenshan97B) were detected by the established extraction method and HILIC-MS/MS analytical method. Using the standard addition method for quantitative analysis, we detected 22 target compounds form leaf and 8 target compounds in root at one time.HILIC-MS/MS analytical method established in this study had the characters of highly sensitivity, highly throughput and accuracy in qualitative and quantitative analysis, which be applicable to quantitative of metabolites relating to respiration in plants and corroborate analysis. |
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