Research On Chlorogenic Acids Functional Components In Lonicera Flos And Origin Traceability

Objective:Under the platform of ultra high performance liquid chromatography tandem mass spectrometry,a precise screening and quantitative method for chlorogenic acid analogs in Lonicera Flos was established,which provided a basis for the quality classification of Lonicera japonica and Lonicera Flos.Methods:1.Establish a simultaneous and rapid determination method for the separation and quantification of 8 chlorogenic acid isomers in Lonicera Flos.Acetonitrile and 0.1%formic acid-water were used as the mobile phase,the chromatographic conditions were optimized to separate 8 chlorogenic acid standards.Under the negative ion mode,the Full MS-dd MS~2 scan mode was used for data collection via Q-orbitrap fusion to establish a standard curve.Every 0.1 g of Lonicera Flos samples was extracted by 7 m L 60%methanol aqueous solution,followed by ultrasound for 40 minutes at room temperature.The quantification step was carried out by external standard method.2.Summarize the fragmentation behaviors of chlorogenic acid standards.6 characteristic neutral loss/product ion marker fragments were identified.A triple quadrupole(QQQ)was used to perform multi-channel neutral loss/precursor ion scans,and then,the data of each channel were integrated to obtain a list of potential precursor ions of CGAs.With the aid of HRMS and peak align,the second fragmentation of the preset precursor ions(Full MS-precursor list-dd MS2)were performed using Q-orbitrap fusion to tentatively deduce the structure of the newly found CGAs.3.With the aid of iknife-REIMS method,40 samples of Lonicera japonica and Lonicera Flos from 8 origins were analyzed.The data was collected and combined with chemometrics method which included principal component analysis(PCA)and orthogonal partial least squares-discriminant analysis(OPLS-DA)to build the statistical model.The models were applied to achieve quick identification of the origins and varieties of unknown LF samples.Results:1.An ultra-high performance liquid chromatography-high resolution mass spectrometry(UHPLC-HRMS)analytical method for simultaneous determination of 8chlorogenic acid isomers was developed.The 8 chlorogenic acid isomers were effectively separated within 14 minutes,and the linear relationship of each was good within the linear range.The correlation coefficient was all greater than 0.99.The detection limit(LOD)range from 0.3to1.5μg/kg,and the limit of quantification(LOQ)ranged from 1 to 4.9μg/kg.The additive amount at three level was set as 100,200,500μg/kg respectively,the average recovery rate of the 3 additive levels ranged from 77.6 to 107.8%,the intra-day precision is 0.9-5.1%(n=5),and the inter-day precision is 1.6～6.2%(n=5).2.A method for non-targeted screening of CGAs in LFs by multi-marker fragment(neutral loss/signature product ion)scans combined with high-resolution mass spectrometry was established.Six characteristic marker fragments were used to perform multiple NL-PI scans,and the secondary mass spectrograms were used to identify the structure of the newly found CGAs.Finally,51 CGAs were found in the extract of LFs,belonging to 16categories.3.With the aid of iknife-REIMS combined with chemometric methods,a statistical model was built up to achieve quickly identification the origin and quality classification of Lonicera japonica and related products.The result indicated that the statistical model was reliable and valid.Conclusion:1.The detection method established by UHPLC-Q-orbitrap can efficiently separate and qutify 8 chlorogenic acid isomers in Lonicera Flos within 14 minutes with low detection limit and high recovery rate.The method is suitable for the content analysis of functional components of different varieties of Lonicera Flos;2.Multiple marker scans combined with high-resolution mass spectrometry technology were successfully used to comprehensively screen CGAs in Lonicera japonica.The method is sensitive and accurate in screening and identification,it provides a new idea for the profiling of unknown compounds containing the similar structures in complex matrices.3.The statistical model of the origin and variety of Lonicera Flos is reliable.The method,REIMS technology combined with chemometric methods,provides a new solution for analysis of similar species and their products,as well as origin traceability and further quality classification.