Unveiling the Diversity and Modifications of Short Peptides in Buthus martensii Scorpion Venom through Liquid Chromatography-High Resolution Mass Spectrometry.

More recently, short peptides in scorpion venom have received much attention because of their potential for drug discovery. Although various biological effects of these short peptides have been found, their studies have been hindered by the lack of structural information especially in modifications. In this study, small peptides from scorpion venom were investigated using high-performance liquid chromatography high-resolution mass spectrometry followed by de novo sequencing. A total of 156 sequences consisting of 2~12 amino acids were temporarily identified from Buthus martensii scorpion venom. The identified peptides exhibited various post-translational modifications including N-terminal and C-terminal modifications, in which the N-benzoyl modification was first found in scorpion venom. Moreover, a short peptide Bz-ARF-NH2 demonstrated both N-terminal and C-terminal modifications simultaneously, which is extremely rare in natural peptides. In conclusion, this study provides a comprehensive insight into the diversity, modifications, and potential bioactivities of short peptides in scorpion venom.

Biotransformation of antioxidant eriocitrin into characteristic metabolites by the gut microbiota.

Eriocitrin is a flavonoid glycoside with strong antioxidant capacity that has a variety of pharmacological activities, such as hypolipidemic, anticancer and anti-inflammatory effects. We found that the gut microbiota could rapidly metabolize eriocitrin. By using LC/MSn-IT-TOF, we identified three metabolites of eriocitrin metabolized in the intestinal microbiota: eriodictyol-7-O-glucoside, eriodictyol, and dihydrocaffeic acid. By comparing these two metabolic pathways of eriocitrin (the gut microbiota and liver microsomes), the intestinal microbiota may be the primary metabolic site of eriocitrin metabolism. These findings provide a theoretical foundation for the study of pharmacologically active substances.

Metabolic Transformation of Gentiopicrin, a Liver Protective Active Ingredient, Based on Intestinal Bacteria.

Gentiopicrin, the main component of the famous Chinese patent medicine Long Dan Xie Gan Wan, has the characteristics of fast absorption in vivo and low bioavailability. Intestinal bacteria play an important role in the absorption and pharmacokinetics of oral drugs. In this study, the metabolic transformation of gentiopicrin by intestinal bacteria was examined. High-performance liquid chromatography coupled with ion trap time-of-flight mass spectrometry (LC/MSn-IT-TOF) and nuclear magnetic resonance (NMR) were used, and six metabolites were identified, including reduction products (G-M1, G-M2, G-M4, and G-M6), a hydrolytic product (G-M3), and a dehydration product (G-M5) of gentiopicrin aglycone after hydrolysis, reduction, and dehydration reactions were performed by the intestinal flora. This is the first time that chiral metabolites of gentiopicrin (G-M1 and G-M2) were found in this study. In addition, the precursors of glucuronic acid conjugates previously reported in vivo may have come from the intestinal bacterial metabolites G-M1, G-M2, and G-M3. In addition, the metabolic transformation of gentiopicrin in liver microsomes was studied in vitro, and it was found that gentiopicrin did not undergo metabolic transformation under the action of liver microsomes. It is suggested that gentiopicroside may be metabolized in the intestine. This study provides both new insight regarding the investigation of effective substances and an exploration of the pharmacodynamic and toxicological properties of gentiopicrin.

Chemical profiling of Sanjin tablets and exploration of their effective substances and mechanism in the treatment of urinary tract infections.

Introduction: Sanjin tablets (SJT) are a well-known Chinese patent drug that have been used to treat urinary tract infections (UTIs) for the last 40 years. The drug consists of five herbs, but only 32 compounds have been identified, which hinders the clarification of its effective substances and mechanism. Methods: The chemical constituents of SJT and their effective substances and functional mechanism involved in the treatment of UTIs were investigated by using high performance liquid chromatography-electrospray ionization-ion trap-time of flight-mass spectrometry (HPLC-ESI-IT-TOF-MSn), network pharmacology, and molecular docking. Results: A total of 196 compounds of SJT (SJT-MS) were identified, and 44 of them were unequivocally identified by comparison with the reference compounds. Among 196 compounds, 13 were potential new compounds and 183 were known compounds. Among the 183 known compounds, 169 were newly discovered constituents of SJT, and 93 compounds were not reported in the five constituent herbs. Through the network pharmacology method, 119 targets related to UTIs of 183 known compounds were predicted, and 20 core targets were screened out. Based on the "compound-target" relationship analysis, 94 compounds were found to act on the 20 core targets and were therefore regarded as potential effective compounds. According to the literature, 27 of the 183 known compounds were found to possess antimicrobial and anti-inflammatory activities and were verified as effective substances, of which 20 were first discovered in SJT. Twelve of the 27 effective substances overlapped with the 94 potential effective compounds and were determined as key effective substances of SJT. The molecular docking results showed that the 12 key effective substances and 10 selected targets of the core targets have good affinity for each other. Discussion: These results provide a solid foundation for understanding the effective substances and mechanism of SJT.

Metabolites Analysis of Anti-Myocardial Ischemia Active Components of Saussurea involucrata Based on Gut Microbiota-Drug Interaction.

Saussurea involucrata has been reported to have potential therapeutic effects against myocardial ischemia. The pharmacological effects of oral natural medicines may be influenced by the participation of gut microbiota. In this study, we aimed to investigate the bidirectional regulation of gut microbiota and the main components of Saussurea involucrata. We first established a quantitative method for the four main components (chlorogenic acid, syringin, acanthoside B, rutin) which were chosen by fingerprint using liquid chromatography tandem mass spectrometry (LC-MS/MS), and found that gut microbiota has a strong metabolic effect on them. Meanwhile, we identified five major rat gut microbiota metabolites (M1-M5) using liquid chromatography tandem time-of-flight mass spectrometry (LC/MSn-IT-TOF). The metabolic properties of metabolites in vitro were preliminarily elucidated by LC-MS/MS for the first time. These five metabolites of Saussurea involucrata may all have potential contributions to the treatment of myocardial ischemia. Furthermore, the four main components (10 µg/mL) can significantly stimulate intestinal bacteria to produce short chain fatty acids in vitro, respectively, which can further contribute to the effect in myocardial ischemia. In this study, the therapeutic effect against myocardial ischemia of Saussurea involucrata was first reported to be related to the intestinal flora, which can be useful in understanding the effective substances of Saussurea involucrata.

Chemical compositions of Commiphora opobalsamum stem bark to alleviate liver complications in streptozotocin-induced diabetes in rats: Role of oxidative stress and DNA damage.

CONTEXT: Hyperglycaemia plays an important role in the development of non-alcoholic fatty liver disease, which is a common complication in diabetics. OBJECTIVE: The present study aimed to investigate the chemical composition and the efficacy of Commiphora opobalsamum stem bark butanol fraction in ameliorating liver injury associated with diabetes induced by streptozotocin (STZ) in rats. MATERIALS AND METHODS: The butanol fraction was applied to high-performance liquid chromatography-mass spectrometry (HPLC/MSn) to identify the most bioactive metabolites. Diabetes was induced by a single intraperitoneal injection of STZ (60 mg/kg body weight), while treatment with the plant extract was performed (100 mg/kg body weight) for three weeks after diabetic induction for one month. RESULTS: Thirty eight metabolites were tentatively identified from the butanol fraction of C. opobalsamum stem bark. Insulin, glutathione, superoxide dismutase, and high density lipoprotein levels in diabetic rats were significantly low (p < 0.05), while glucose, α-amylase, malondialdehyde, aspartate and alanine aminotransferases, cholesterol, triglycerides, low density lipoprotein, tumour necrosis factor-α, interleukin-6, and DNA fragmentation levels were significantly high. Treatment with the plant extract showed improvements in the seleced parameters by variable degrees. Conclusion: The plant extract is considered as a promising natural therapeutic agent against liver injury, hyperglycemia, oxidative stress, inflammation, hyperlipidaemia, and DNA damage.

Characterization of Polyester Coatings Intended for Food Contact by Different Analytical Techniques and Migration Testing by LC-MSn.

Polymeric coating formulations may contain different components such as cross-linking agents, resins, lubricants, and solvents, among others. If the reaction process or curing conditions are not applied in a proper way, these components may remain unreacted in the polymeric network and could be released and migrate into foods. In this study, several polyester coatings intended for food contact were investigated. Firstly, Fourier-transform infrared spectroscopy with an attenuated total reflectance (ATR-FTIR) spectrometer and confocal Raman microscopy were used to identify the type of coating. Then, different techniques, including gas chromatography coupled to mass spectrometry (GC-MS) and analysis by matrix-assisted laser desorption coupled to time-of-flight mass spectrometry (MALDI-TOF-MS), among others, were used to investigate the potential volatile and non-volatile migrants. Moreover, migration assays were carried out to evaluate the presence of monomers and to tentatively identify possible oligomers below 1000 Da. The analyses were performed by liquid chromatography coupled to ion trap mass spectrometry (LC-MSn). Using the information collected from each analytical technique, it was possible to elucidate some of the starting substances used in the formulation of the polyester coatings analyzed in this study. In migration tests, several polyester oligomers were tentatively identified for which there is not toxicological data available and, therefore, no migration limits established to date.

Characterization of the metabolites of trans-resveratrol-3-O-glucoside in monkeys and dogs.

In this study, a liquid chromatography-tandem multi-stage mass spectrometry (LC/MSn) method was established to characterize the metabolites of TRG in monkeys and dogs. A total of seven metabolites of TRG besides the prototype were found, which were identified as TR (M1), TRN (M2), trans-resveratrol-4'-O-glucuronide (M2'), trans-resveratrol-3-O-glucoside-4'-O-glucuronide (M3), trans-resveratrol-3-O-glucoside-5-O-glucuronide (M3'), trans-resveratrol-3-sulfate (M4) and trans-resveratrol-4'-sulfate (M4'). Additionally, the metabolic pathways of TRG in monkeys and dogs were proposed. There were also species differences of metabolism of TRG between monkeys and dogs.

LC-MS Based Analysis and Biological Properties of Pseudocedrela kotschyi (Schweinf.) Harms Extracts: A Valuable Source of Antioxidant, Antifungal, and Antibacterial Compounds.

The impact of two extraction solvents on the phenolic composition, antioxidant, and enzymes inhibitory and antimicrobial activities of two parts (leaves and stem bark) of P. kotschyi was studied. Two different LC-DAD-MSn approaches were used to identify and quantify the bioactive compounds in the different extracts. A total of thirty-two compounds were quantified, being the procyanidin the most abundant in stem bark while catechin and flavonoids are most abundant in leaves. Overall, the stem bark extraction using methanol showed higher amounts of total phenolic (131.83 ± 1.81 mg GAE/g) and flavanol (14.14 ± 0.11 mg CE/g) while the leaves extraction using water exhibited stronger levels of total flavonoid (44.95 ± 0.38 mg RE/g) and phenolic acid (63.58 ± 2.00 mg CAE/g). As regards the antioxidant assays, methanol stem bark extracts were characterized by the highest antioxidant activities (DPPH: 1.94 ± 0.01 mmol TE/g, ABTS: 3.31 ± 0.01 mmol TE/g, FRAP: 2.86 ± 0.02 mmol TE/g, CUPRAC: 5.09 ± 0.08 mmol TE/g, phosphomolybdenum: 5.16 ± 0.23 mmol TE/g and metal chelating: 17.12 ± 0.46 mg EDTAE/g). In addition, the methanolic extracts of stem bark had highest impact on acetylcholinesterase (2.54 mg GALAE/g), butyrylcholinesterase (5.48 mg GALAE/g). In contrast, the methanolic extracts of leaves was potent against tyrosinase (77.39 ± 0.21 mg KAE/g) and α-glucosidase (0.97 ± 0.01 mmol ACAE/g), while a higher anti-α-amylase (0.97 ± 0.01 mmol ACAE/g) was observed for water extracts of the same part. All of the tested extracts showed inhibitory effects on elastase, except methanolic leaves extracts. Additionally, the extracts exhibited appreciable antifungal toward A. ochraceus, A. fumigatus, P. ochrochloron, T. viride, and P. funiculosum and promising antibacterial activity against M. flavus, S. aureus, L. monocytogenes, E. coli, P. aeruginosa, E. cloacae, and S. typhimurium. Taken together, the outcomes demonstrated P. kotschyi as a novel source of bioactive molecules of interest with an evident therapeutic value.

Characterization of Phytochemicals in Berry Fruit Wines Analyzed by Liquid Chromatography Coupled to Photodiode-Array Detection and Electrospray Ionization/Ion Trap Mass Spectrometry (LC-DAD-ESI-MSn) and Their Antioxidant and Antimicrobial Activity.

Fruits are a valuable source of phytochemicals. However, there is little detailed information about the compounds contained in fruit wines. In this study, wines from six different berries were analyzed using HPLC-DAD-ESI-MSn. About 150 compounds were identified, including anthocyanins (34), hydroxycinnamic acids (12) and flavonols (36). Some of the compounds were identified for the first time in berry wines. The blackberry wines were found to contain the largest number of bioactive compounds (59). Elderberry wines where the richest source of polyphenols (over 1000 mg/L) and contained the largest amounts of all of the analyzed groups of compounds (hydroxycinnamic acids, anthocyanins and flavonols). The lowest concentration of polyphenols was observed in the wines made from cranberries and bilberries (less than 500 mg/L). The antioxidant activity was determined in relation to ABTS+, DPPH, and FRAP. The highest values were observed in the blackberry wines, and the lowest for the cranberry wines. The wines were analyzed to test their antimicrobial activity. Five of the six wines (with the exception of elderberry wine) inhibited Bacillus cereus growth and two (blackberry and cranberry wines) were active against Listeria monocytogenes.

Extraction, Enrichment, and LC-MSn-Based Characterization of Phlorotannins and Related Phenolics from the Brown Seaweed, Ascophyllum nodosum.

Phenolic components from the edible brown seaweed, Ascophyllum nodosum, have been associated with considerable antioxidant activity but also bioactivities related to human health. This study aims to select and identify the main phlorotannin components from this seaweed which have been previously associated with potential health benefits. Methods to enrich phenolic components then further select phlorotannin components from ethanolic extracts of Ascophyllum nodosum were applied. The composition and phenolic diversity of these extracts were defined using data dependent liquid chromatography mass spectroscopic (LC-MSn) techniques. A series of phlorotannin oligomers with apparent degree of polymerization (DP) from 10 to 31 were enriched by solid phase extraction and could be selected by fractionation on Sephadex LH-20. Evidence was also obtained for the presence of dibenzodioxin linked phlorotannins as well as sulphated phlorotannins and phenolic acids. As well as diversity in molecular size, there was evidence for potential isomers at each DP. MS2 fragmentation analyses strongly suggested that the phlorotannins contained ether linked phloroglucinol units and were most likely fucophlorethols and MS3 data suggested that the isomers may result from branching within the chain. Therefore, application of these LC-MSn techniques provided further information on the structural diversity of the phlorotannins from Ascophyllum, which could be correlated against their reported bioactivities and could be further applied to phlorotannins from different seaweed species.

Transforming of Triptolide into Characteristic Metabolites by the Gut Microbiota.

The importance of the gut microbiota in drug metabolism, especially in that of nonabsorbable drugs, has become known. The aim of this study was to explore the metabolites of triptolide by the gut microbiota. With high-performance liquid chromatography coupled with tandem mass spectrometry and ion trap time-of-flight multistage mass spectrometry (LC-MS/MS and LC/MSn-IT-TOF), four metabolites of triptolide (M1, M2, M3, and M4) were found in the intestinal contents of rats. M1 and M2, were isomeric monocarbonyl-hydroxyl-substituted metabolites with molecular weights of 390. M3 and M4 were isomeric dehydrogenated metabolites with molecular weights of 356. Among the four metabolites, the dehydrogenated metabolites (M3 and M4) were reported in the gut microbiota for the first time. The metabolic behaviors of triptolide in the gut microbiota and liver microsomes of rats were further compared. The monocarbonyl-hydroxyl-substituted metabolites (M1 and M2) were generated in both systems, and another monohydroxylated metabolite (M5) was found only in the liver microsomes. The combined results suggested that the metabolism of triptolide in the gut microbiota was specific, with two characteristic, dehydrogenated metabolites. This investigation might provide a theoretical basis for the elucidation of the metabolism mechanism of triptolide and guide its proper application in clinical administration.

Analysis of polymer impurities in cefixime raw materials and preparations / 药学学报

To establish a method for the determination of polymer impurities in cefixime raw materials and preparations, a cefixime degradation solution containing polymer impurities was prepared by forced polymerization. Polymer impurities in the degradation solution were separated and identified by high performance gel chromatography and the column switching-LC-MSn method. A new RP-HPLC method for cefixime polymer was established and validated with a Phenomenex Gemini-C18 column using a mobile phase gradient elution of 0.5% formic acid-water solution and 0.5% formic acid-acetonitrile solution. The results showed that when using this high performance gel chromatography method some small molecular weight impurities were co-eluted with the polymers, resulting in a poor specificity and poor quantitative accuracy. But when using the RP-HPLC method, three polymer impurities were detected with good specificity, sensitivity and robustness, including two cefixime dimers, and dehydrate dimer. Therefore, the described RP-HPLC method is suitable for the quality control of polymer impurities in cefixime, and cefixime degradation solution can be used as suitable solution for analysis of cefixime polymers.

Analysis of polymer impurities in ceftazidime raw materials and preparations / 药学学报

To establish a method for the determination of polymer impurities in ceftazidime raw materials and preparations, a ceftazidime degradation solution containing polymer impurities was prepared by forced polymerization. Polymer impurities in the degradation solution were separated and identified by high performance gel chromatography and the column switching-LC-MSn method. A new RP-HPLC method for ceftazidime polymer was established and validated with a Phenomenex Gemini-C18 column using a mobile phase gradient elution of 0.02 mol·L-1 phosphate buffer, methanol and acetonitrile. The results showed that when using this high performance gel chromatography method some small molecular weight impurities were co-eluted with the polymers, resulting in a poor specificity and poor quantitative accuracy. But when using the RP-HPLC method, four polymer impurities were detected in the 25-45 min time range with good specificity, sensitivity and robustness, including two ceftazidime dimers, trimers, and derivatives. Therefore, the described RP-HPLC method is suitable for the quality control of polymer impurities in ceftazidime, and ceftazidime degradation solution can be used as suitable solution for analysis of ceftazidime polymers.

Rapid Characterization of Impurity Profiles in Vancomycin Raw Material by Column-Switching LC/MSn / 中国药学杂志

OBJECTIVE: To characterize the impurity structures in vancomycin raw material. METHODS: Impurities were eluted gradiently on a Chromasil 100-5 C18(4.6 mm×250 mm, 5 μm) column, with triethylamine buffer solution (pH 3.2)-acetonitrile-tetrahydrofuran (92∶7∶1, V/V/V) as mobile phase A, and triethylamine buffer solution (pH 3.2)-acetonitrile-tetrahydrofuran (70∶29∶1, V/V/V) as mobile phase B. Stress tests were performed on the raw material to specify those impurities. By application of on-line LC/MSn method, the impurities were analyzed in positive mode and their structures were characterized based on the degradation mechanism and mass fragmentation regularity of sugar-lipopeptides. RESULTS: Totally 14 impurities were characterized in the raw material, seven of which were reported for the first time. The relationships between the chemical structures and chromatographic behaviors of vancomycin, demethylvancomycin and methylated vancomycin with their two β-isomers were summarized. CONCLUSION: The structures of related impurities in vancomycin raw material can be rapidly identified by on-line LC/MSn method together with stress degradation experiments.

Induction of signal molecules and expression of functional genes after Pichia pastoris stimulation in Glycyrrhiza uralensis Fisch adventitious roots.

Glycyrrhiza uralensis Fisch is threatened by over-development and consumption, and therefore, in urgent need of protection. Elicitation is considered to be an effective strategy to enhance the secondary metabolites in plant cell and organ cultures. Secondary metabolite, signal molecules, and gene expression in adventitious roots were studied by HPLC-ESI-MSn , commercially available kits and qRT-PCR method, respectively. In the present study, with the addition of linolenic acid, linoleic acid, and Pichia pastoris, the highest concentration of metabolites was achieved by P. pastoris treatment. The contents of total flavonoids (7.16 mg/g) and polysaccharide (149.76 mg/g) peaked at 100 mg/L of P. pastoris, which increased by 3.09-fold and 3.28-fold compared with the control, respectively. However, the highest concentration of glycyrrhizic acid (0.62 mg/g) and glycyrrhetinic acid (0.29 mg/g) were obtained in 200 mg/L of P. pastoris and which were 3.89-fold and 2.42-fold more than the control group, respectively. ESI-MSn analysis indicated that licoricesaponine B2, licoricesapoine G2, licoricesaponine J2, ononin, uralenin, gancaonin C were only identified in the P. pastoris treatment group. Furthermore, P. pastoris also enhanced accumulation of salicylic acid, jasmonic acid, nitric oxide and activities of antioxidant enzymes involved in the plant defense response. In addition, the transcriptional activity of genes involved in glycyrrhizic acid biosynthesis was significantly increased under the treatment of P. pastoris. The results provided a scientific evidence for the further exploitation of G. uralensis adventitious roots and clinical medication. PRACTICAL APPLICATIONS: This study provided an effective strategy to enhance metabolites by Pichia pastoris treatment in adventitious roots of G. uralensis. The data provide a scientific evidence for the further exploitation of G. uralensis adventitious roots and clinical medication.

Rapid structure identification of nineteen metabolites of ondansetron in human urine using LC/MSn.

Ondansetron, a 5-hydroxytryptamine type 3 (5-HT3 ) receptor antagonist, is regarded as an excellent candidate to treat chemotherapy- and radiotherapy-induced nausea and vomiting. To better understand the metabolic profiles of ondansetron in human urine, the metabolites were analyzed using liquid chromatography/mass spectrometry (LC/MSn ). Urine samples were collected after oral administration of 8 mg ondansetron to healthy volunteers. Then samples were treated by solid-phase extraction and detected with LC/MSn . Besides ondansetron, in human urine, a total of 19 metabolites including 13 new metabolites were detected and identified via comparing the retention time and product ion spectra with those of reference standards isolated and characterized. The results showed that ondansetron was metabolized via hydroxylation, glucuronidation, sulfation and minor N-demethylation in human. LC/MSn was demonstrated to be useful and sensitive in the metabolic study of ondansetron.

Blood plasma level determination using an automated LC-MSn screening system and electronically stored calibrations exemplified for 22 drugs and two active metabolites often requested in emergency toxicology.

Fast and comprehensive qualitative and quantitative methods preferably by gas chromatography-mass spectrometry (GC-MS) and/or liquid chromatography-mass spectrometry (LC-MS) are needed to support the (differential) diagnosis of acute poisonings in emergency toxicology. One option is a commercially available qualitative screening solution based on LC-MSn (Bruker Daltonik Toxtyper™, TT). Identified and toxicologically relevant compounds should be quantified to assess severity of poisonings. The aim of the present study was to test the TT system for quantification simultaneous with the screening process in blood plasma exemplified for 22 relevant drugs and two active metabolites. A standard liquid-liquid extraction was used for sample work-up followed by 1:5 dilution of the final extracts. They were analyzed using the TT system consisting of a Bruker amaZon speed ion trap and a Thermo Fisher Dionex Ultimate 3000 LC system. Plasma levels were assessed using full-scan data and an electronically stored five-point calibration. The calibration model was linear for the studied ranges and could be used for at least two months. The method was validated according to international guidelines. The acceptance criteria recommended for emergency toxicology for accuracy and precision were fulfilled for all tested compounds, but bromazepam, lorazepam, oxycodone, and prothipendyl could reliably be determined only above the therapeutic range. In conclusion, the presented procedure allowed the combination of a comprehensive LC-MSn screening with fast automated assessment of plasma levels for emergency toxicology of tested compounds.

Qualitative and quantitative analyses of Chrysanthemum morifolium ‘Fubaiju’ by LC-MSn and HPLC-UV / 中国药科大学学报

@#A qualitative analytical method of liquid chromatography coupled with mass spectrometry(LC-MSn)was developed for the identification of main constituents in Chrysanthemum morifolium ‘Fubaiju’. High-performance liquid chromatography(HPLC)was developed for the quantification of five active components, including chlorogenic acid(1), luteolin-7-O-β-D-glucopyranoside(2), luteolin-7-O-β-D-glucopyranuronide(3), 3, 5-Di-caffeoylquinic acid(4), and apigenin-7-O-β-D-glucopyranoside(5). A total of 22 compounds, including 13 flavonoids and 9 phenolic acids, were identified based on their retention behaviors, UV profiles and MS fragment information. Furthermore, a validation method with good linearity(r> 0. 999 9), precision, stability, repeatability and recovery was successfully applied for simultaneous determination of five major components in 10 batches of C. morifolium ‘Fubaiju’ by HPLC-UV method. The established method was proved to be a validation strategy for the quality evaluation of C. morifolium ‘Fubaiju’.

A fragmentation study of isoflavones by IT-TOF-MS using biosynthesized isotopes.

To aid in the identification and quantification of biologically and agriculturally significant natural products, tandem mass spectrometry can provide accurate structural information with high selectivity and sensitivity. In this study, diagnostic fragmentation patterns of isoflavonoids were examined by liquid chromatography-ion trap-time of flight-mass spectrometry (LC-IT-TOF-MS). The fragmentation scheme for [M+H-2CO]+ ions derived from isoflavones and [M+H-B-ring-CO]+ ions derived from 5-hydroxyisoflavones, were investigated using different isotopically labeled isoflavones, specifically [1',2',3',4',5',6',2,3,4-13C9] and [2',3',5',6',2-D5] isoflavones. Specific isotopically labeled isoflavones were prepared through the biosynthetic incorporation of pharmacologically applied 13C- and D-labelled L-phenylalanine precursors in soybean plants following the application of insect elicitors. Using this approach, we empirically demonstrate that the [M+H-2CO]+ ion is generated by an intramolecular proton rearrangement during fragmentation. Furthermore, [M+H-B-ring-CO]+ ion is demonstrated to contain a C2H moiety derived from C-ring of 5-hydroxyisoflavones. A mechanistic understanding of characteristic isoflavone fragmentation patterns contributes to the efficacy and confidence in identifying related isoflavones by LC-MSn.