The Development Of An Integrated Method For Screening And Quantification Of Heating Endpoint Temperature Markers Of Pork And Based On Q-Exactive Mass Spectrometry

Objective:In this study,an integrated method was established for detecting the high heating end point temperature of roasted pork based on high resolution mass spectrometry and chemometrics.1.Screening of heat-related markers based on orbittrap mass spectrometry and chemometrics;2.Quantitative method of selected markers using Qu ECHERS-UPLC-Q-Orbitrap HRMS was established;3.The relationship between the heating temperature and the content of selected markers was established for the judgment of the end point temperature of high temperature heating based on the established quantitative method.Methods:1.In this experiment,Q-Orbitrap mass spectrometry was used to obtain sample information,and the data was further filtered according to the p value<0.05 and fold change>2 or fold change<-2.Compounds with statistical differences were submitted to construct OPLS-DA model to calculate variable importance for projection values.Marker were selected according to the variable importance for projection value<0.9;2.Pretreatment method of selected markers was performed using Qu ECh ERS method.In this experiment,the extraction effects of ethyl acetate,acetonitrile and methanol were studied,and the effects of different kinds and doses of adsorbents were investigated based on the recoveries.According to the recovery rate,ethyl acetate and 50mg C18 were selected.In addition,the chromatographic and mass spectrometry conditions were optimized.Finally,acetonitrile and 10m M ammonium acetate were selected as mobile phase,and targeted single ion monitoring scan scanning mode was selected.Results:1.Five characteristic markers were screened out,and four compounds were confirmed by comparison with standard compounds.The four compounds were creatine,creatinine,2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine(PhIP),and toxopyrimidine(TMP).2.A method for quantification of selected biomarkers based on UPLC-quadrupole/orbitrap mass spectrometry was established.This method has a good linear relationship in the concentration range of 0.5～200 ng/mL(r~2>0.999).The recovery rates were 88.1%～94.2%,and the intra-day precision and inter-day precision did not exceed6.5%;the limit of detections of the instrument were 0.1～0.2μg/kg,the limit of quantitations were 0.3～0.5μg/kg;3.We established a method to judge the high heating end point temperature based on the content of selected markers.The results showed that the concentration of Ph IP was 0.25±0.005～0.58±0.02 ng/g and the concentration of TMP was 0.52±0.02～1.05±0.02 ng/g when the heating temperature was 150～180°C.When the heating temperature was 180～200°C,the concentration of Ph IP was 0.58±0.02～2.06±0.12 ng/g,and the concentration of TMP was 1.05±0.02～8.04±0.29 ng/g.The concentration of Ph IP was 2.06±0.12～14.95±0.63ng/g and the concentration of TMP was 8.04±0.29～25.02±1.25 ng/g when the heating temperature was 200～230°C.Conclusion:The combination of orbitrap mass spectrometry and chemometrics is a feasible method for heating endpoint temperature determination.The marker selection process is simple and reliable.Quantitative method is and sensitive and accurate.The results can be used in high heating end point temperature determination and provide a reference for the sold baked food regulation.In addition,this integrated method can be used to select markers to indicate food production,processing,storage,transportation and other process.