The Application Of LC-HRMS In Herb And Food Health Fields

Liquid chromatography coupled to high resolution mass spectrometry（LC-HRMS）,caused by its high sensitivity,high resolution,rapid,reliable,and repeatable characteristics,has been more and more important for the structural annotation of chemical compositions in complex components.However,due to the huge differences of the chemical compositions in complex matrices,such as herbal medicines or foods,it is necessary to establish feasible strategies or methods to obtain reliable LC and MS information to ensure the effective annotation of target molecules“from spectrum to structure”.Therefore,the thesis focused on the construction of LC-HRMS based strategies（methods）for systematical and effective annotation of chemical compositions in complex matrices and further application in the related issues,such as development of new medicine,herb and food resource utilization,food health,etc.1.LC-HRMS based annotation of monoterpene indole alkaloids in the alkaloids from leaf of Alstonia scholaris.Monoterpene indole alkaloids（MIAs）show diverse in chemical structures and biological actives.It is still great challenging to annotate MIAs in complicated matrixes of herbal medicines relied on LC-HRMS.In this chapter,LC-HRMS workflow for annotation of MIAs in the alkaloids from leaf of Alstonia scholaris（ALAS）was conducted,which mainly included three parts:（1）Fragmentation patterns of ALAS-MIAs.Multiple collision energies based MS² capture strategy（MCEs/MS²）was proposed for the first time based on the traditional LC-HRMS information of retention time（RT）,accurate mass（AM）and general MS².The fragmentation patterns of 48 reference MIAs with different structure types were elucidated in detail by using this strategy,including the key mass spectrometry information of diagnostic product ions（DPIs）,characteristic cleavages（CCs）,neutral losses（NLs）and radical losses（RLs）.（2）FBMN elucidated the chemical compositions of ALAS-MIAs.Feature-based molecular networking（FBMN）of ALAS-MIAs was successfully constructed based on the MZmine 2 processing.Meanwhile,the potential structures of MIAs based on biosynthetic pathways（BPs）of Alstonia genus were analyzed.And then,combining with the fragmentation characteristics of each subtype-MIAs（DPIs,CCs and NLs/RLs）,229known and unknown MIAs were interactively annotated and efficiently classified in FBMN（nodes）.（3）LC-HRMS guided isolation of novel MIAs and anti-inflammatory activities.A pair epimers of MIA 1（node 110）and 2 were LC-HRMS guided isolation from FBMN of ALAS-MIAs,and the absolute configuration of both compounds were further identified by spectral data.MIA 1 and 2 showed good anti-inflammatory activities at cellular level by comparing with dexamethasone,which further confirmed that MIAs were the phytochemical basis for anti-inflammatory and analgesic activities of ALAS.The structures of the isolated MIAs further confirmed that the MCEs/MS²-FBMN/BPs workflow can effectively annotate chemical structures in complex components.The study systematically illustrated the chemical compositions of ALAS-MIAs relied on LC-HRMS,to exhibit a reliable material basis for further clinical investigations,and provided strategies and clues for the structural annotation of MIAs in complex components of herbal medicines.2.LC-HRMS identification of FGs in Citrus herbs and quality evaluation of citrus fruit at different harvest periods.Flavanone glycosides（FGs）are the major representative compounds of Citrus genus herbs,FGs generally included four common flavanone aglycones（FAs）including naringenin,hesperetin,eriodictyol and isosakuranetin.C-7 at A-ring of FAs was usually connected with neohesperidoside or rutinoside,led it easily to generate isomeric FGs.Therefore,fast and effective annotation of FGs is an important basis for quality evaluation of citrus related medicinal and food resources.This chapter mainly focused on two parts:（1）Identification of FGs in Citrus herbs.An integrated approach merging RT,AM,MS² and ion ratio（IR）was established successfully（RT-AM-MS²-IR）to improve the identification of four pairs FGs isomers,including eriocitrin/neoeriocitrin,narirutin/naringin,hesperidin/neohesperidin and didymin/poncirin.The feasibility of this approach was validated confidently in biological samples with RSD of ion ratio range from 3.91 to 12.28%.The fragmentation patterns of FAs were studied in negative mode.Naringenin and eriodictyol were prone to generate[^1,3A]^–cleavage and produced the characteristic fragment ion of m/z 151.However,due to the methoxyl substitution of C-3’at B-ring,hesperetin and isosakuranetin were prone to generate[^1,2A]^–cleavage and produced the characteristic radical ion of m/z 164.（2）Quality evaluation of citrus fruit at different harvest periods.The immature fruit of Citrus aurantium L.,also named Aurantii fructus indexed in the Pharmacopoeia,is a raw material of traditional Chinese medicine health food.The contents of FGs and other ingredients in citrus fruit collected at different harvest periods were changed significantly,which affected the pharmacological activities.The contents of FGs,polymethoxyflavones,coumarins,synephrine and limonin in citrus fruits at different harvest periods were analyzed.The relative contents of the major compounds reached at higher contents in June and then decreased gradually,while the total accumulation showed increasing tendency along the harvest times.H₂O₂-induced RIN-m5F cells were employed to evaluate their antioxidant capacity.The citrus fruits harvested from mid-June to early July possessed excellent antioxidant capacities by inhibiting intracellular reactive oxygen species（ROS）intensity（p<0.01）,improving superoxide dismutase（SOD）,catalase（CAT）,and glutathione peroxidase（GSH）activities（p<0.01）.In addition,the chemical compositions and antioxidant capacities of citrus leaves,stems and roots were also evaluated.This study developed a LC-HRMS strategy for the identification of FGs in citrus herbs,and provided reference for the appropriate harvest period range of citrus fruit and the utilization of other parts of citrus resources.3.Chemical migrants in microwavable plastic food containers and potential health risks.Microwavable plastic food containers（MPFCs）are mainly made of polypropylene（PP）,which are extensively used by people in daily life due to its characteristics of low cost,convenient storage,rapid heating,et al.,especially exhibited huge consumption in take-out fast food industries.No evidence has been previously reported to indicate that MPFCs may release chemical migrants that have potential safety risks to human health.In this chapter,the hot-selling MPFCs in the global consumer markets were collected to carry out migration experiments of chemicals according to the EU regulation（No 10/2011）.（1）Migration of small molecular chemicals in MPFCs.A LC-HRMS based strategy integrating non-target,accurate-target and extensive-target（NT-AT-ET）was established to efficiently annotate 42 intentionally added substances（IAS）and more than 100 non-intentionally added substances（NIAS）migrated from MPFCs.The migration levels of major IAS/NIAS was higher in 95%ethanol compared to other simulants,the contents of N,N-bis（2-hydroxyethyl）alkyl（C8-C18）amines PEG oligomers,Irgafos 168 OXO,etc.,in some MPFCs products exceeded the Cramer’s threshold for class III toxic compounds（0.09 mg/kg）.However,the migration abundance of IAS/NIAS decreased significantly after repeated use.When test in acidic food simulants,migration level of diisobutyl phthalate（DIBP）and dibutyl phthalate（DBP）were significantly enhanced,on the contrary,NX8000 level was significantly decreased.（2）Migration of microplastics in MPFCs.Furthermore,microplastics（MPs）were detected with high values of over one million particles/L in some MPFCs in a single test（1～5μm,5～50μm and>50μm）.MPs showed irregular migration in most of MPFC reuses that were different from IAS/NIAS.This rigorous migration might result in an annual intake of IAS/NIAS and MPs up to 55.15 mg and 150 million particles,respectively,if take-out food was consumed once a day.The effective LC-HRMS strategies were individually established for different complicated matrices,and various organic molecules were systematically annotated.In short,MIAs in ALAS were effectively illustrated,FGs in Citrus herbs were accurately identified,and chemical migrants in MPFCs were comprehensively annotated.The further studies on guided separation,activity evaluation,quantitative analysis and risk assessment were carried out based on these works.This thesis effectively expanded the application of LC-HRMS techniques in herb and food health fields,and provided references for related studies.