Non-targeted Screening Strategy Based On Nanospray High Resolution Mass Spectrometry And Its Application In The Agricultural Products

Animals and plants are important carriers of biogeochemical cycles of elements.Plants utilize inorganic elements to synthesize organic compounds for their growth,and these organic compounds,which serve as nutrients for animals,are released into the environment in the form of mineral elements and organic compounds after biological metabolism processes to complete the material cycle within the ecosystem.Elements play physiological functions in the form of biological molecules in plants and animals,and also transform into some unknown biological molecules.The identification of organic biological molecules,especially the unknown compounds,in animals and plants is of great significance for the study of biochemical processes and biogeochemical cycling of elements.Non-targeted screening based on liquid chromatography high-resolution mass spectrometry（LC-HRMS）is a new kind of unknown compounds identification strategy.In the absence of the reference standard,retention time and accurate quality of the analyte are the important basis for non-targeted screening;however,the complex pretreatment and lengthy chromatographic separation process usually lead to the loss of original information in biological samples.Ambient ionization MS（AMS）is an emerging mass spectrometry that can realize the direct and rapid analysis of various complex matrix samples without pretreatment,including desorption electrospray ionization mass spectrometry（DESI-MS）,dielectric barrier discharge mass spectrometry（DBDI-MS）,nanospray mass spectrometry（nanospray–MS）,etc.Nanospray,one of the simplest AMS techniques based on ESI,is suitable for the direct ionization analysis of small-volume biological samples.However,the existing non-targeted screening strategies are unsuitable for AMS analysis due to the lack of the retention time in AMS methods.Therefore,developing a novel non-targeted screening strategy suitable for AMS analysis is of great importance for the rapid screening of unknown biomolecules in small-volume biological samples and the biogeochemical cycling of elements.In the present study,a new non-targeted screening strategy based on AMS for biomolecular analysis was established and applied for the rapid screening of bioactive compounds in small-volume liquid biological samples,micro-zone plant tissues,and even plant cells.The main research contents and results were as following:（1）A new non-targeted screening strategy suitable for AMS analysis was developed based on nanospray HR-MS.The screening strategry was used successfully for the rapid analysis of multiple bioactive components in different biological samples with the absence of reference standards.The study provides new research ideas for the study of speciation and transformation of elements in plants and animals in biogeochemical studies.（2）The screening strategy was applied to the screening of unknown organic iodine in breast milk samples to examine the feasibility of the screening strategy applied to real samples.The in-tube SPME pretreatment was combined with nanospray HR-MS to reduce matrix interference.The results showed that the method showed good feasibility and anti-interference.Using this method,a novel organic iodine C₁₂H₂₃O₁₁I in breast milk was identified successfully,which was presumably to be a new organic iodine produced by the reaction of iodine with lactose.This finding provides a new evidence for the study of iodine speciation transformation in human body during biogeochemical cycling of iodine.（3）The biological molecules in plant tissues are the main occurrence forms of elements,and the species and relative contents of biomolecules in different regions of tissues are different.The screening strategy was also used for the in situ and micro-zone analysis of plant tissue,in order to obtain the original biomolecular information of different plant tissues and the transformation rules of biomolecules during plant stress response to environment.A quartz capillary was inserted into the plant tissue for in situ sampling and nanospray ionization analysis without sample pretreatment.Using the method,12 bioactive compounds in vegetables,fruits and medicinal plants were identified,including alkaloids,flavonoids,organic acids and glycosides.When radish,garlic and apple are exposed to environmental stresses,some of the biomolecules in the tissues undergo enzymatic transformation.The labile compounds in plant tissues were captured successfully using the methd,including glucosinolates in radish root,alliin in garlic and chlorogenic acid in apples.In addition,the enzymatic reaction intermediates existing in only 10 seconds in injured radish root tissues were captured successfully;what’s more,the conversion processes of glucosinolates were tracked continuously.（4）The in situ method of nanospray HR-MS were applied for the semi-quantitative analysis of two glucosinolates（GLs）in radishes from three producing areas of Wuhan,Shijiazhuang and Xining.The relationship between GLs content in radish and sulfur content in soil was studied by using signal intensity of mass spectrometry as the basis of GLs content.The study showed that there were significant differences in GLs content in radishes from three habitats.The sequence of relative content of GLs was Wuhan<Shijiazhuang<Xining,which is a slight difference in the background values of soil sulfur with three soils.The background value of soil sulfur in Xining area is about 10times of that in Shijiazhuang,but the content of GLs in radish of Shijiazhuang is two times of that in Xining radish,which may be related to the application of sulfur-containing fertilizer.（5）The feasibility of this strategy in the analysis of small-volume samples of single cell was preliminarily discussed.The capillary microsampling technique developed by Professor Akos Vertes was combined with the screening strategy proposed in this study.With the screening strategy,16 organic sulfated compounds in Raphanus sativus root cells and 3 solanen alkaloids in potato sprout cells were identified,14 of which were newly discovered compounds.This technique has great potential in the screening of unknown compounds in single cells.