Metabolite identification and the development of a simultaneous quantification method for wogonin and wogonin-7-O-glucuronide:application to a distribution study in mice liver

Wogonin possesses potent inhibitory activities against cancer cell growth in vitro and in vivo and has attractive safety profiles.A highly sensitive liquid chromatography coupled with tandem mass spectrometry method was developed for the identification of major metabolites in mice liver after intravenous administration of wogonin.Five metabolites were identified and biotransformation pathways were elucidated as well.Furthermore,a method was developed and validated for the simultaneous quantitatively determination of wogonin and wogonin-7-O-glucuronide in mice liver.After liquid-liquid extraction by ethyl acetate,the analytes were separated on a C_（18）column with a mobile phase of methanol-10 mM ammonium acetate water （80：20,v/v）.The detection was operated with negative selected reaction monitoring mode using electrospray ionization technique. The linear response range was 0.2-40μg/g for both wogonin and wogonin-7-O-glucuronide in mice liver.The developed quantification method was suitable for distribution study after intravenous infusion of wogonin injection in animals.

Pharmacokinetic study and metabolite identification of the bidesmosidic triterpenoid saponin BTS-1 in rat plasma

Assays based on high-performance liquid chromatography(HPLC)and liquid chromatography tandem mass spectrometry(LC–MSn)have been developed and validated for the determination and metabolite identification of the bidesmosidic triterpenoid saponin,BTS-1(3-O-β-D-galactopyranosyl-(1-2)-[β-D-xylopyranosyl-(1-3)]-β-D-glucuronopyranosyl gypsogenin 28-O-α-L-arabinopyranosyl-(1-3)-βD-xylopyranosyl-(1-4)-α-L-rhamnopyranosyl-(1-2)-β-D-fucopyranoside),in rat plasma.The assay was successfully applied to a pharmacokinetic study in rats given a single oral dose of BTS-1(400 mg/kg).The results indicated that the compound was rapidly absorbed(Tmax=1.2870.29 h,Cmax=37.475.6 mg/mL)and slowly eliminated(t1/2=13.276.6 h).In addition,secondary glycosides and aglycones of BTS-1 were detected and identified.Since these metabolites are known to be activeα-glucosidase inhibitors,they probably play an important role in mediating the pharmacological effects of the saponin.

Detecting and Identifying in vivo Metabolites of Brodimoprim via LC/ESI-MS with Data-dependent Scanning

The present article covers a simple approach to detect and subsequently identify in vivo metabolites of brodimoprim, using high performance liquid chromatography coupled to ion trap mass spectrometer（LC/ESI-MS）, which is based on a data-dependent acquisition of isotope ions and result verified by full scan mass spectrum. The distinguished advantage of data-dependent scan is rapidness because it requires minimum sample preparation, and all the necessary data can be obtained in one chromatographic run. In addition, it is highly sensitive and selective, allowing detection of trace metabolites even in the presence of complex biomatrix. As a result, four phase-Ⅰ（M1--M4） and four Phase-Ⅱ（M5--M8） metabolites of brodimoprim were identified in urine after the oral administration of hrodimoprim to Wistar rats. Their chemical structures were proposed based on the interpretation of their CID fragmentation characterizations and the metabolic pathway was exhibited in this article.

Tentative Identification of Phytochemicals and Antioxidant Activities during Fruit-Ripening on Chamaedorea radicalis Mart.

This work aims to determine the phytochemical characterization of the pericarp of Chamaedorea radicalis Mart.fruit as a non-timber product with potential to obtain phytochemicals with potential applications in the industry.Fruit from C.radicalis were grouped in four ripening stages named as S1,S2,S3 and S4,according to maturity;S1 the most unripe stage and S4 the completely ripe stage.Determinations of total phenolic compounds,free radical scavenging activities and total flavonoid contents using spectrophotometric methods were done.Also,the tentative identification of phytochemicals during fruit ripening was done using UPLC-MS-MS.Total phenolic compound(TPC)content ranged from 7.24 to 12.53 mg gallic acid equivalents per gram of fresh weight(mg GAE/g FW).Total flavonoids(TF)contents ranged from 0.163 to 0.23 mg of quercetin equivalents per g FW(mg QE/g FW).Free radical scavenging activity against DPPH and ABTS radicals varied from 40.80 to 53.68 and from 22.29 to 37.76 mmol Trolox equivalents g FW(mmol TE/g FW),respectively.Antioxidant assay in vitro by FRAP(ferric reducing antioxidant power)method showed that S3 was the highest level with antioxidant power while S4 was the lowest with Red ripeness stage showed the lowest contents for all determinations.Mass spectrometry allowed detection of 26 compounds,including phenolics,alkaloids and saponins.Afzelin,Kaempferol 3-neohesperidoside and the four saponins identified were present in all ripeness stages.Preliminary phytochemical identification and the spectrophotometric determinations showed that the pericarp of C.radicalis presented antioxidants and compounds related to alkaloids,phenolics and saponins.The presence and abundance of each phytochemical regarding each ripeness stage should be considered.

Identification of the metabolites of biologically active xanthones isolated from Halenia elliptica D.Don by high performance liquid chromatography coupled to ion trap time-of-flight mass spectrometry

Metabolism study has been carried out on 1-hydroxy-2,3,5-trimethoxyxanthone （HM-1） and 1-hydroxy-2,3,4,7- tetramethoxyxanthone （HM-2）, which are two biologically active ingredients isolated from the Tibetan herb, Halenia elliptica D. Don., in rat liver microsomes in vitro. A method of high performance liquid chromatography coupled to ion trap time-of-flight mass spectrometry （LCMSn-ESI-IT-TOF） was applied to analyze metabolites of HM-1 and HM-2 on line, and five metabolites were identified containing 1,5-dihydroxy-2,3-dimethoxyxanthone （HM-5）, 1,7-dihydroxy-2,3,4-trimethoxyxanthone （HM-9）, 1,4, 7- trihydroxy-2,3-dimethoxyxanthone （HM-10）, 1,4-dihydroxy-2,3,7-trimethoxyxanthone （HM-11） and 1,2-dilaydroxy-3,4,7- trimethoxyxanthone （HM-12）. Among these metabolites, HM-9, HM-11, and HM-12 were isomers mutually. The results indicated that HM-1 and HM-2 occurred Phase I metabolic reaction of demethylation in rat microsomes in vitro.

Evaluation of the metabolism of PEP06,an endostatin-RGDRGD 30-amino-acid polypeptide and a promising novel drug for targeting tumor cells

PEP06 is a novel endostatin-Arg-Gly-Asp-Arg-Gly-Asp(RGDRGD)30-amino-acid polypeptide featuring a terminally fused RGDRGD hexapeptide at the N terminus.The active endostatin fragment of PEP06 directly targets tumor cells and exerts an antitumoral effect.However,little is known about the kinetics and degradation products of PEP06 in vitro or in vivo.In this study,we investigated the in vitro metabolic stability of PEP06 after it was incubated with living cells obtained from animals of different species;we further identified the degradation characteristics of its cleavage products.PEP06 underwent rapid enzymatic degradation in multiple types of living cells,and the liver,kidney,and blood play important roles in the metabolism and clearance of the peptides resulting from the molecular degradation of PEP06.We identified metabolites of PEP06 using full-scan mass spectrometry(MS)and tandem MS(MS2),wherein 43 metabolites were characterized and identified as the degradation metabolites from the parent peptide,formed by successive losses of amino acids.The metabolites were C and N terminal truncated products of PEP06.The structures of 11 metabolites(M6,M7,M16,M17,M21,M25,M33,M34,M39,M40,and M42)were further confirmed by comparing the retention times of similar full MS spectrum and MS2 spectrum information with reference standards for the synthesized metabolites.We have demonstrated the metabolic stability of PEP06 in vitro and identified a series of potentially bioactive downstream metabolites of PEP06,which can support further drug research.

UPLC-QTOF-MS FOR METABOLITES IDENTIFICATION IN CACO-2 CELLS OF BENZYLISOQUINOLINE ALKALOIDS IN NELUMBINIS PLUMULA

The aim of the study was to identify main metabolites of benzylisoquinoline alkaloids from Nelumbinis Plumula after biotransformation by Caco-2 cells.Caco-2 cells were seeded to a 6-well plate and cultured for a period of time until 80%of each well was filled with cells.Then,cell medium was replaced and the norcoclaurine,liensinine,isoliensinine and neferine were respectively added to

INNOVATIVE BIOLOGICAL AND CHEMICAL STUDIES OF HERBAL MEDICINES APPLICATION OF MASSLYNX AND UNIFI PLATFORM SOLUTION TO ACCELERATE THE IDENTIFICATION OF INGREDIENTS AND THEIR METABOLITES OF TCM

Traditional Chinese Medicine(TCM)has been used in prevention and treatment of disease in clinical practice for thousands of years,with an indispensable role of multiple ingredients.Thus,a rapid and effective chemical ingredients analysis was of necessary to be established for the evaluation of the holistic quality of TCM.As could afford the data with high resolution and high sensitivity,

The Application of Ion Mobility-Mass Spectrometry in Untargeted Metabolomics: from Separation to Identification

Untargeted metabolomics aims to comprehensively profile metabolites as many as possible in biological samples.Recently,ion mobility-mass spectrometry(IM-MS)has emerged as a powerful technology for untargeted metabolomics.The emerging role of IM-MS in untargeted metabolomics enables the separation of metabolite isomers and generation of multidimension data to support the identification of metabolites.In this review,we first introduced the basic principles of IM-MS instruments commonly used for untargeted metabolomics.Then,we demonstrated the application of IM-MS for metabolite separation and identification of both known and unknown metabolites.Finally,we discussed the future developments of IM-MS technology to improve untargeted metabolomics.

Characterization of chemical constituents and in vivo metabolites of Kai-Xin-San prescription by HPLC/DAD/ESI-MS^n

This work aims to elucidate the chemical constituents of Kai-Xin-San（KXS） and its metabolites in rat plasma.KXS extracts were separated on an Agilent HPLC SB-C 18 column,analyzed by ion-trap tandem mass spectrometry and high-accuracy qTOF mass spectrometry in negative ion mode.A total of 39 compounds,including 11 ginsenosides,14 Polygala saponins,5 sucrose esters,8 oligosaccharide esters and 1 xanthone were characterized from KXS.Fifteen of them were confirmed by reference standards.No constituents were detected from Poria or Acori Tatarinowii Rhizoma.After oral administration of KXS（7 g/kg）,10 ginsenosides and 18 Polygala compounds were detected in rat plasma.This study indicates that ginseng saponins,Polygala saponins and saccharide esters could be the major effective components of KXS prescription.

Comprehensive relative quantitative metabolomics analysis of lycopodium alkaloids in different tissues of Huperzia serrata

Qian ceng Ta,the whole plant of Huperzia serrata,is an important landscape and medicinal herbs and contains abundant bioactive lycopodium alkaloids.Although the structures of more than 100 lycopodium alkaloids in Huperzia serrata have been isolated and identified,the content and distribution of these alkaloids in different tissues are still unclear.In current study,an ultra-performance liquid chromatography-mass spectrometry based comprehensive metabolomics strategy was developed,including the extraction,separation,identification,and statistical analysis.The results showed that different types lycopodium alkaloids could be separated at different time-windows,which was helpful for further metabolite identification.Peak4388 and peak3954 were metabolite biomarkers for the different tissues according to the principle component analysis and partial least squares-discriminant analysis model.A computational tool based in-house database was also built up and used for putative identification.Of the 2354 true peaks after four-step filtration,118 peaks were putatively identified as lycopodium alkaloids by using in-house database,and four of which was identified by authentic standards.Alternatively,another computational software was used to predict the fragmentation pattern,to dereplicate the structure of identified peaks,and identified the peak3585 to N-methylhuperzine A.The integration of both computational tools could be used for more metabolites identification.

Multi-channel microfluidic chip-mass spectrometry platform for cell analysis

In this review, we highlight the latest development of multi-channel microfluidic chip-mass spectrometry（chip-MS） in cell analysis and metabolite detection. Following a brief introduction about history and development of multi-channel microchip and MS combination, we will elaborate the key issues of constructing chip-MS platform interface. Then exciting progresses made in this field should be reviewed with well exemplified works, including chip-MS technology for cell introduction, pretreatment of cell secretions and cell metabolite analysis. We will also describe the development of integrated total analysis systems proposed by our group. We hope this brief review will inspire interested readers and provide knowledge about chip-MS platform in the bioanalysis field, particularly in cell analysis and metabolite identifying applications.