Pretreatment And Detection Methods Of Microalgal Intercellular Hormones By Solid-Phase Extraction And High-Resolution Mass Spectrometry

Plant hormones play an important role in improving microalgae biomass and biomass resource synthesis.Plant hormones can also regulate the transboundary intercellular communication between algae and other microorganisms,which has an important impact on the ecological characteristics of microalgae.In-depth understanding of the role of plant hormones requires the establishment of accurate detection methods of plant hormones.However,the extraction and detection of plant hormones are still faced with severe challenges due to the characteristics of various plant hormones,complex properties and trace occurrence.In order to solve this problem,this paper established and optimized the solid phase extraction method of multiple plant hormones in algae liquid,and based on Ultra-high Performance Liquid Chromatography Quadrupole/Electrostatic Field High Resolution Mass Spectrometry(UPLC-Q-OrbitrapHRMS),it realizes the sensitive,accurate and rapid detection of six plant hormones such as kinetin,N,N’-Diphenylurea,Methyl Jasmonate,indole-3-acetic acid,Indole-3-butyric acid and Gibberellin.The established Solid-phase Extraction-High-Resolution Mass Spectrometry was successfully applied to the detection of inter-cellular plant hormones in microalgae,which was of great significance to explore the secretion and action law of inter-cellular plant hormones in microalgae and the mechanism of inter-cellular communication in microalgae.The extraction conditions of intercellular Plant hormone from microalgae on a solid phase extraction column were established and optimized.The extraction and recovery effects of plant hormone from kinetin,N,N’-Diphenylurea,Methyl Jasmonate,indole-3-acetic acid,Indole-3-butyric acid and Gibberellin on Oasis HLB SPE column,Oasis Max SPE column and Proelut PXC SPE column were compared respectively.The pH value of algal solution and eluent pH value were optimized.Firstly,ammonia was used to adjust the pH of the algal solution after centrifugation to 8.Second,methanol and deionized water activated Proelut PXC SPE column was used for extraction(flow rate=0.8 m L/min;Time=37 min);After eluting with water solution of formic acid at pH=2,it was eluted with methanol solution of formic acid at pH=6.After purification,the recoveries of kinetin,N,N’-Diphenylurea and Methyl Jasmonate reached104.08%,129.79%and 84.63%respectively.In addition,using the same method,indole-3-acetic acid,Indole-3-butyric acid and Gibberellin could be successfully purified from the algal solution by using the activated Oasis HLB solid phase extraction column sample,deionized water leaching,and methanol elution,with the recovery rates of 96.97%,106.88%,85.17%,respectively.A UPLC-Q-OrbitrapHRMS method was established for the determination of six plant hormones.The column selection,column temperature and mobile phase conditions were optimized.Acquity HSS T3(2.1 mm×100 mm,1.8μm)column temperature had no significant effect on the determination of plant hormones.When mobile phase is acetonitrile and pH=3formic acid,kinetin,N,N’-Diphenylurea and Methyl Jasmonate have the best detection effect.Indole-3-acetic acid,Indole-3-butyric acid and Gibberellin were the best detection results when the mobile phase was methanol and pH=7.There was a linear relationship between the six plant hormones(0.05-50 ng/m L),and the correlation coefficient R~2 was greater than 0.997.The limits of qualitative detection and quantitative detection were in the range of 0.0005-0.05 ng/m L and0.001-0.1 ng/m L,respectively,which were lower than the existing methods.RSDs of intra-day and inter-day precision tests with low,medium and high quality control concentrations were all less than 10%.In summary,this study aimed at the shortcomings of large sample size,cumbersome steps and low accuracy in the pretreatment and detection of traditional plant hormone,combined with efficient and simple solid phase extraction technology and high sensitivity and high accuracy UPLC-Q-OrbitrapHRMS technology,established and optimized a comprehensive and rapid detection method of plant hormone,which provided an effective technical means for the study of trace plant hormone in complex algal environments.