Research Of Non-Targeted Metabolomics Based On High Resolution Mass Spectrometry For Identification Of Apple Freshness

Objective:In this study,a method was established for detecting the freshness of apple integrate high resolution mass spectrometry with chemometrics.1.A non-targeted metabolomics method based on high resolution mass spectrometry and stoichiometry for freshness judgment model was established;2.The changes of 43 key compounds content were discussed;3.A non-targeted metabolomics method based on high resolution mass spectrometry and chemometrics for the selecting of apple freshness related markers was established and a selected markers quantitative method with UPLC-HRMS was developed.Methods:1.In this experiment,the ratio and type of pretreatment extraction solvent were optimized in order to extract as many compounds as possible in the sample.High-resolution Q-Orbitrap mass spectrometry was used for data acquisition and combined with chemometrics for dimensionality reduction.Firstly,the compounds of CV%<20%,P value<0.05,fold change>1 or<-1 was retained to established OPLS-DA model,and the variable importance for projection values of the compounds were calculated.Compounds with VIP value>1 was screened and analyzed as important differential compounds;2.A prediction model of apple freshness based on 43 differential compounds was established and the prediction efficiency of the model was verified;3.The freshness biomarkers were further screened from 43 differential compounds,quantitative analysis of the selected markers based on ultra-high performance liquid chromatography-Q-Orbitrap mass spectrometry.In this study,methanol:acetonitrile:water=1:1:2,（v/v/v）was selected as the extraction solvent,C₁₈ and HILIC columns were used for analysis different polarity compounds.In order to adapt to the different polarity of the column,different mobile phase systems were designed and optimized.In addition,parallel reaction monitor was selected for quantification by external standard method.Results:1.The apple freshness judgment model was established,and the prediction efficiency of the model was verified.The prediction efficiency of the model was good and there was no over-fitting;2.The changes of 43 compounds that played key roles in the process of decay were discussed and found to be mainly related to the activation of plant defense mechanism,the explosion of reactive oxygen species,the growth of fungi and the changes of energy metabolism;3.Two characteristic markers were selected and compared with standard compounds.The two compounds were DL-citrine and quinic acid;4.A quantification method for two markers based on UPLC-Q-Orbitrap mass spectrometry was established.This method shows a good linear relationship in their concentration range of0.02～300 ng/m L with R²>0.999.The limit of detections was 0.18～5.0μg/kg,the limit of quantitations were 0.54-15.1μg/kg,the recovery rates were 86.1～95.3%.Conclusion:The integration of Orbitrap mass spectrometry and chemometrics is an effective method for apple freshness determination,which has a simple and scientific marker selection process.Quantitative method shows high sensitivity and accuracy.The results can be applied to apple freshness determination and provide a reference for apple and its products.In addition,this method can be applied to the quality control of apple and its products.