Establishment And Application Of The Screening And Quantitative Method Of Bioactive Compounds In Lemons By UPLC-Q-TOF-HRMS

As one of the world’s four major economic citrus,lemon is rich in a variety of bioactive compounds,and is favored by the public for its unique flavor,good nutrition and health functions.Lemons are rarely used for fresh food like other citrus fruits directly,it usually needs to be processed into food or medicinal materials,such as lemon tea,lemon oil,lemon jam and so on.Bioactive compounds are one of the main reasons for promoting human health in lemons,and it mainly includes flavonoids,phenolic acids,limonoids and alkaloids.Therefore,it is very necessary to conduct a systematic study on the main bioactive compounds in lemons to guide the consumption of fresh food,the research and development of medicine and health products.This research also provide theoretical basis and technical support for the deep processing of lemons,and provide references for the selection and processing of lemon varieties.In this paper,ultra-high performance liquid chromatography-quadrupole-time-of-flight high-resolution mass spectrometry（UPLC-Q-TOF-HRMS）technology was used to establish a mass spectrum database of many types of bioactive compounds.At the same time,this article used lemons as the research object,using UPLC-Q-TOF-HRMS technology to combine information-dependent acquisition（IDA）and sequential window acquisition of all theoretical mass spectra（SWATH）mass spectrometry data acquisition modes,respectively.Then,this research conducted qualitative and quantitative analysis of the biologically active ingredients in lemons.All of the above provide a theoretical basis for further research and development of lemon functional foods.The content of the paper was divided into five chapters,of which the second to the fourth chapters were the research parts.The main research reports were as follows:The edible and medicinal value,bioactive compounds and the analysis methods of lemon were reviewed.At the same time,the problems to be solved,research contents and research programs of this paper were introduced in the review.Based on UPLC-Q-TOF-HRMS technology,this study established a mass spectrometry database of many types of bioactive compounds commonly found in citrus.By using standard references for bioactive compounds,the mass spectrometry database including 46 flavonoids,29 coumarins,10 phenolic acids,7 alkaloids and 3 limonoids have been created with UPLC-Q-TOF-HRMS technology and Library View 1.3.1database management software,and characteristic secondary mass spectrometry fragment ion information of multi-type bioactive compounds were analyzed.This mass spectrometry database includes the structural,molecular,CAS,precise mass,collision energy,scanning ion patterns and MS² fragment ion high-resolution spectra for each compound,which laid a method and theoretical basis for the screening and identification of non-targeting bioactive compounds.Based on the bioactive ingredient mass spectrometum database constructed in Chapter 2,this study established an ultra-performance liquid chromatography-quadrupole-time-of-flight high-resolution mass spectrometry method for simultaneous screening and accurate identification of many types of bioactive compounds,such as flavonoids,coumarins,phenolic acids,alkaloids and limonoids,which was further applied for rapid and accurate screening of bioactive compounds of six different parts of lemons（peel,pulp and juice）without standard references.This screening method uses Acquity UPLC HSS T3（2.1×100 mm,1.8μm,Waters）as a chromatography separation column and the column temperature was 40°C,the injection volume was 1.0μL and the flow rate was 0.3 m L·min^-1.The mobile phases consisted of 0.1%formic acid in water（A）and acetonitrile（B）.In positive and negative ion mode,information-dependent scanning（IDA）mode was used to collect mass spectrometry data of the primary precursor ion and the secondary fragment product ion in the full mass-to-charge ratio range（100-800）.Then,it can achieve rapid screening and accurate identification without relying on standard references of bioactive compounds by comparing with the retention time of the target compound in this method and the mass spectrometry database constructed in Chapter 2.Through actual sample analysis this method can be widely used in the identification of bioactive compounds in lemon and other citrus fruits.The results show as follows:（1）In this screening method,78 compounds（including 19 pairs of isomers）could be effectively separated in 11 minutes in positive ion mode,39 compounds（including 6 pairs of isomers）could be effectively separated in 6 minutes in negative ion mode,and 22 of which could be screened and identified in both positive and negative ion modes.（2）In the actual sample screening,40 flavonoids,27 coumarins,9 phenolic acids,7 alkaloids and 3 limonoids were detected in the six kinds of lemon samples.Specifically,72,71,70,66,64 and 63 kinds of bioactive compounds were identified in the screening of Bianhongningmeng,Beijingningmeng,Keerticuningmeng,Youlikeningmeng,and Lisibenningmeng,and Meiguocuningmeng,respectively.The number of bioactive compounds in different parts showed the following order:peel≥pulp≥juice.Moreover,this study established an UPLC-Q-TOF-HRMS method for a quantitative analysis of 27 compounds（14 flavonoids,8 coumarins,3 limonoids and 3phenolic acids）in lemon samples.In the positive and negative electrospray ion mode,this study used sequential window acquisition of all theoretical mass spectra（SWATH）combined with the characteristic secondary fragment product ion peak area technology to quantify the target compounds by external standard method.The method verification results showed that the method had good sensitivity,accuracy and precision,and it’s suitable for the quantitative analysis of many types of bioactive compounds in lemons.The results show as follows:（1）The LOQ was as low as 0.001 ng/m L,and all 27 standards showed good linearity with correlation coefficients>0.99.The accuracy of the method is measured by recovery experiments,and the result ranged from 84.27±3.02%to114.25±1.77%.Furthermore,this method showed good repeatability with interday relative standard deviations（RSDs）≤6.11%,and intraday RSDs≤5.38%.（2）It was found that the content of bioactive compounds in different parts and different varieties of lemon samples was quite different,when analyzing the content of bioactive compounds in different parts of the six lemons（peel,pulp and juice）.The content of biologically active ingredients in lemon peel significantly was higher than pulp and juice,and the content of 27 bioactive compounds in the 6 lemon samples showed the following order:Keerticuningmeng>Lisibenningmeng>Youlikeningmeng>Beijingningmeng>Meiguocuningmeng>Bianhongningmeng.In addition,for many types of bioactive compounds,the content of flavonoids was the highest among the 27 bioactive compounds,and is one of the main sources of bioactive compounds in lemons,followed by limonoids.