| Glycerol ester(GE)is the main component of butterfat,which is closely related to the nutrition of milk.The establishment of the analytical method for GEs can evaluate and utilize the nutrition of different dairy better.At present researches,the identification analyses mostly stayed at the“Sum Composition”level,while the quantification analyses were usually limited by the quantification performance of instruments and inter-instrumental errors.Therefore,the sample treatment methods were analyzed and compared in this study.Also,the identification methods and data acquisition strategies were explored.Based on these researches,a liquid chromatography-mass spectrometry(LC-MS)method was developed,aiming at providing a more targeted and accurate method for the research of GE in dairy products.The main research contents and conclusions are as follows:(1)The sample treatment of GE was compared,analyzed and optimized.After optimization,ultrasonic was adopted to assist the extraction of Folch method,while vortex was selected for IPA method and Matyash method.The treatment time was 2.5 min.Comparing these three methods with Mojonnier method,the results showed that all four methods had satisfying repeatability.In terms of recovery,Folch method scored the highest(96.37),followed by IPA method(92.62),and Mojonnier method was the worst(74.22).Folch method and IPA method had the best coverage of triglycerides,while Mojonnier method had higher response in diglycerides and monoglycerides.So,Folch method was selected for GE extraction.The redissolution of GE in chloroform-methanol(2:1,V/V)mixed reagent and IPA were compared,and IPA was selected as redissolution solvent in further research.Finally,the linearity(R~2>0.99),accuracy(recovery between 78.03%and 109.93%),sensitivity(LOD between 0.05μg/mL and 0.2μg/mL;LOQ between 0.25μg/mL and 1μg/mL),matrix effect(-9.98%～8.43%)and precision(intra-day RSD<13%and inter-day RSD<18%)of the treatment method were verified,and the results showed that the treatment could meet the requirements of GE analysis.(2)An identification method of GEs based on retention time and IDA-MS/MS was established.Based on the identification results of MS-DIAL,the mathematical relationships between the retention time(RT)of GE and the number of acyl carbon(CN)and acyl double bonds(DB)were explored.The results showed that the RT of GEs had a logarithmic function relationship with the CN and a linear function relationship with DB.Furthermore,the binary function model of RT with CN and DB was fitted,and it was compared with the retention time prediction model based on equivalent carbon number.The results showed that the R~2(TG 0.9984 vs 0.9967;DG 0.9724 vs 0.9443)and MSE(TG 0.0102 vs 0.0214;DG 0.1685 vs0.3200)of binary function model were both better,and linear-chain GEs could be distinguished by binary function model.The fatty acid composition of GE was identified according to the MS/MS spectra,and the identification efficiency was improved by using R programming,which promoted the identification depth of GE from“Sum Composition”to“Molecular Lipid”.(3)A new data acquisition strategy for semi-quantification of GE was proposed.Aiming at the shortcomings of pseudotargeted strategy,the quantification potential of data independent acquisition was fully explored,and the SWATH-MS2&1 strategy was proposed.The new semi-quantification strategy was compared with the untargeted strategy based on SWATH-MS1,the pseudotargeted strategy based on SWATH-MS/MS and the recognized pseudotargeted strategy based on MRM(P-MRM).The results showed that the new strategy inherited the coverage and sensitivity from SWATH-MS1 and the linearity and clustering effect from SWATH-MS/MS.Compared with P-MRM,the coverage of SWATH-MS2&1 was improved by about 7.3%,and the clustering effect was obviously better.At the same time,the sensitivity(GEs detected after 4096-fold dilution,141 vs 143),linearity(GEs whose R~2>0.8,132 vs 126)and repeatability(GEs whose RSD<0.3,98.2%vs 98.7%)of SWATH-MS2&1and P-MRM were very close,which fully proved the superiority of the new strategy.(4)The new GE analysis method was applied to detect different dairy samples,including5 kinds of animal milk,4 kinds of plant milk and 3 kinds of food for special medical purpose.A total of 254 GEs in“Sum Composition”level and 982 possible in“Molecular Lipid”level were detected.According to the semi-quantification analysis,the main difference between GEs in 12 kinds of milk were linear-chain triglyceride.The average chain length and unsaturation of GEs in animal milk were less than those in plant milk,and the composition of GEs in milk had a great relationship with the geography origin and production environment,followed by the biological origin.Based on the SWATH-MS1 area of glycerides,the concentrations of GEs were further estimated,and the algorithm of similarity score was proposed.The analysis results of similarity scores among dairy products were similar to those of semi-quantification,which indicated that both of them had great potential in the counterfeiting prevention and origin tracing of dairy. |
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