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Melia. toosendan Sieb.et Zucc., a potent herbal medicine, boasts diverse therapeutic properties. As the main components, the isotoosendanin blocks protective autophagy in chemotherapy-induced cultured cancer cells and xenograft tumor tissue to significantly enhancing anticancer activity, and the fraxinellone contributes to pesticidal activity, anti-inflammatory and immunomodulatory effects. Until now, the metabolic profiles remained unknown. In the present study, 13 metabolites of isotoosendanin and 13 metabolites of fraxinellone were characterized in the blood, urine, and feces of rats by UPLC/ESI/qTOF-MS analysis. Five metabolites (F-M3, F-M5, I-M1, I-M5, I-M8) were fully characterized. The isotoosendanin and fraxinellone were mainly involved in hydrogenation, acetylation, and methylenation metabolic reactions. This is the first study illuminates the two components in vivo metabolism, laying the foundation for future pharmacodynamic and mechanistic investigations. It is necessary to deeply understand the process of drug activation, and deactivation, rationally design new drugs, and guide the research and development of new drugs. [ABSTRACT FROM AUTHOR]

One‐dimensional (1D) proton‐nuclear magnetic resonance (1H‐NMR) spectroscopy is an established technique for the deconvolution of complex biological sample types via the identification/quantification of small molecules. It is highly reproducible and could be easily automated for small to large‐scale bioanalytical, epidemiological, and in general metabolomics studies. However, chemical shift variability is a serious issue that must still be solved in order to fully automate metabolite identification. Herein, we demonstrate a strategy to increase the confidence in assignments and effectively predict the chemical shifts of various NMR signals based upon the simplest form of statistical models (i.e., linear regression). To build these models, we were guided by chemical homology in serum/plasma metabolites classes (i.e., amino acids and carboxylic acids) and similarity between chemical groups such as methyl protons. Our models, built on 940 serum samples and validated in an independent cohort of 1,052 plasma‐EDTA spectra, were able to successfully predict the 1H NMR chemical shifts of 15 metabolites within ~1.5 linewidths (Δv1/2) error range on average. This pilot study demonstrates the potential of developing an algorithm for the accurate assignment of 1H NMR chemical shifts based solely on chemically defined constraints. [ABSTRACT FROM AUTHOR]

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.

The identification of drug metabolites formed with different in vitro systems by HPLC-MS is a standard step in preclinical research. In vitro systems allow modeling of real metabolic pathways of a drug candidate. Despite the emergence of various software and databases, identification of compounds is still a complex task. Measurement of the accurate mass, correlation of chromatographic retention times and fragmentation spectra are often insufficient for identification of compounds especially in the absence of reference materials. Metabolites can "slip under the nose", since it is often not possible to reliably confirm that a signal belongs to a metabolite and not to other compounds in complex systems. Isotope labeling has proved to be a tool that aids in small molecule identification. The introduction of heavy isotopes is done with isotope exchange reactions or with complicated synthetic schemes. Here, we present an approach based on the biocatalytic insertion of oxygen-18 isotope under the action of liver microsomes enzymes in the presence of 18O2. Using the local anesthetic bupivacaine as an example, more than 20 previously unknown metabolites were reliably discovered and annotated in the absence of the reference materials. In combination with high-resolution mass spectrometry and modern methods of mass spectrometric metabolism data processing, we demonstrated the ability of the proposed approach to increase the degree of confidence in interpretating metabolism data. [ABSTRACT FROM AUTHOR]

Hirsuteine is one of the major bioactive tetracyclic indole alkaloids found in Uncaria rhynchophylla (Miq.) Jacks, possessing a wide range of pharmacological activities including neuroprotective, anticonvulsant, antihypertensive, sedative and hypnotic, and so forth. The present study was undertaken to assess the metabolism and plasma pharmacokinetics of hirsuteine in rats. After oral administration of hirsuteine at the dose of 30 mg/kg, 13, 21, and 8 metabolites were detected in rat plasma, urine, and bile by ultra‐high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry, respectively. Furthermore, plasma concentrations of hirsuteine and its four metabolites, 4‐hirsuteine N‐oxide, 3,4‐dehydrohirsuteine, 11‐hydroxyhirsuteine, and 11‐hydroxyhirsuteine‐11‐O‐glucuronide were simultaneously quantified in rat plasma, using carbamazepine as the internal standard. The linear calibration curve of hirsuteine was in the concentration range of 0.005–5.0 μg/ml. The lower limit of quantitation in the rat plasma was 5 ng/ml for hirsuteine. This study is the first to comprehensively investigate the metabolism process of hirsuteine and the pharmacokinetic profiles of hirsuteine and its major metabolite, and will provide a scientific basis to further elucidate the pharmacodynamic material basis and therapeutic mechanism of Uncaria prescriptions. [ABSTRACT FROM AUTHOR]

Identification and evaluations of pesticide metabolites are necessary for risk assessment and toxicological research. In this study, the metabolites of penflufen (a widely used chiral pesticide) in rat liver microsomes were identified using liquid chromatography Q-Exactive Plus mass spectrometry. In total, 17 penflufen metabolites were identified, and most of them were hydroxylation products, which were generated by oxygenation at different candidate sites of penflufen. The relative abundance of metabolite M12 (penflufen-3-hydroxy-butyl, 32 %) was the largest, followed by M8 (15.6 %) and M2 (12.8 %). The major metabolite penflufen-3-hydroxy-butyl was first synthesized by 11 reactions with a 99.73 % purity. The absolute configuration of M12 enantiomers were confirmed after preparing enantiomers, and establishing the enantioseparation method. The M12 enantiomers toxicity to Danio rerio (LC50, >10 mg/L) and four kinds of phytopathogens (EC50, 148–34969 mg/L) were significantly lower than parents (LC50, 0.449–24.3 mg/L; EC50, 0.027–92.0 mg/L). In rat liver microsomes, approximately 40–47 % of the penflufen enantiomers were metabolized to M12 enantiomers, and R-penflufen was preferentially metabolized. The generation concentrations of S-M12 were higher than R-M12 after 10 min, and the metabolic half-lives of R-M12 (29.0–32.5 min) were shorter than S-M12 (35.2–38.1 min), and were approximately 4 times longer than parent penflufen enantiomers (4.5–9.5 min). Simultaneously, the generated contents (relative contents) of M8 (27.1–57 %) and M10 (2.22–8.36 %) from S-penflufen were lower than those from R-penflufen (M8, 24.7–92.4 %; M10, 27.4–69.5 %). The enantioselective evaluations of M12, M10 and M8 deserve further study. These findings were helpful in understanding the fate and risks of chiral penflufen.

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. [Display omitted] • An LC-HRMS assay for PEP06, a promising drug targeted to the tumor, was developed. • The in vitro metabolic stability of PEP06 was incubated with living cells. • Forty-three metabolites of PEP06 were characterized in vitro for the first time. [ABSTRACT FROM AUTHOR]

Codonopsis Radix (CR), a traditional tonic medicinal material in China, has been proven to possess a variety of bioactive functions. However, its chemical composition and in vivo metabolic pattern have not been fully elucidated. In this study, AB-8 macroporous resin column chromatography was employed for the enrichment of small molecular components in CR. Furthermore, a method combining ultra-performance liquid chromatography-quadrupole-orbitrap mass spectrometry with Acquire X intelligent data acquisition technology software was developed for the preliminary screening and identification of the chemical composition of CR in vitro and their metabolites in vivo. As a result, a total of 116 components were preliminarily characterized in the CR extract, including 28 polyacetylenes, 33 organic acids, 4 amino acids, 23 alkaloids, 9 phenylpropanoids, 6 terpenoids, 2 nucleosides, and 11 others. Additionally, a total of 84 compounds, including 37 prototype components and 47 metabolites, were identified in the plasma, urine, and feces of rats after oral administration of CR. Specifically, 11, 24, 19, 32, and 25 constituents were identified in the heart, liver, spleen, lung, and kidney, respectively. Of note, the lung and spleen are the organs with the highest distribution of CR compounds. These findings will serve as valuable data for future research on the correlation between the chemical composition and pharmacological effects of CR. [Display omitted] [ABSTRACT FROM AUTHOR]

Pinocembrin-7-O-β-D-glucoside (PCBG) isolated from Penthorum chinense Pursh was proven to display a wide range of pharmacological effects including hepatoprotection, anti-hepatoma and antifungal activities, etc. The research aims to qualitatively analyze the metabolites of PCBG in rat plasma, urine, bile and feces, and further perform the excretion study of PCBG and its major metabolite pinocembrin (PCB). Fifteen rats were divided into three groups (n=5 for each group) for blood, bile, urine and feces collection, respectively. After PCBG suspension was intragastrically administered to rats at 50 mg/kg, biological samples were collected and processed. The metabolites in each matrix were detected by UHPLC-Q-Exactive-MS/MS. A total of 111 metabolites were observed in plasma, urine, bile and feces, which include hydroxylated, sulfated and glucuronized metabolites, etc. In addition, an UHPLC-MS/MS method was established and applied for the excretion quantification of PCBG and PCB in rat urine, bile, and feces samples. Studies on excretion have shown that PCBG is mainly excreted through feces. The cumulative excretion rates of PCBG and PCB in rat urine, bile and feces were (4.5±2.4)%, (0.2±0.1)% and (18.4±10.5)%, respectively. After hydrolysis by β-glucuronidase/sulfatase, the excretion rates of PCB in urine and bile were (5.7±2.8)% and (8.9±4.2)%. This study contributes to preclinical research on PCBG and explains its pharmacological effects. • 111 metabolites of PCBG were observed in plasma, urine, bile and feces. • An UPLC-MS/MS method was applied to determine the contents of PCBG and PCB in rat samples. • This study contributes to preclinical research on PCBG. [ABSTRACT FROM AUTHOR]

Oroxylin A, the main component of Scutellaria baicalensis Georigi has been widely studied due to its well-known pharmacological effects. According to previous studies, Oroxylin A with low bioavailability was converted into glucuronidation and sulphonated metabolites, which had high exposure in plasma and generated certain activities. It is necessary to study the metabolites and metabolic pathways of Oroxylin A. This study aimed to explore the metabolites of Oroxylin A in liver microsomes, primary hepatocyte incubation samples of five different species (human, monkey, dog, mouse, rat), and in bile, urine and faeces of rats. It would provide a systematic description of metabolic pathway of Oroxylin A. Also, a method of high-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry detector (HPLC-Q-TOF-MS/MS) for identification of each metabolite in various biological matrices was developed. This experiment illustrated that phase II metabolites were the main form of Oroxylin A in vitro and in excretion of rats, accompanied with a small amount of phase I metabolites. Furthermore, there were obvious species differences among the metabolism in vitro, especially in phase II. Monkeys and rats may be more suitable for preclinical research than dogs and mice as non-rodent or rodent species. [ABSTRACT FROM AUTHOR]

Datura metel L. has been frequently used in Chinese traditional medicine. However, little is known on the chemical composition and in vivo metabolism of its seeds. In this study, using the strategy “chemical analysis, metabolism of single representative compounds, and metabolism of extract at clinical dosage” that we propose here, 42 constituents were characterized from D. metel seeds water extract. Furthermore, the metabolic pathways of 13 representative bioactive compounds of D. metel seeds were studied in rats after the oral administration of D. metel seeds water extract at a clinical dosage (0.15 g/kg). These included three withanolides, two withanolide glucosides, four amides, one indole, one triterpenoid, one steroid, and one sesquiterpenoid, and with regard to phase II metabolism, hydroxylation, (de)methylation, and dehydrogenation reactions were dominant. Furthermore, the metabolism of D. metel seeds water extract provided to rats at a clinical dosage was investigated by liquid chromatography-tandem mass spectrometry based on the above metabolic pathways. Sixty-one compounds were detected in plasma, 83 in urine, and 76 in fecal samples. Among them, withanolides exhibited higher plasma exposure than the other types. To our knowledge, this is the first systematic study on the chemical profiling and metabolite identification of D. metel seeds, including all compounds instead of single constituents.

Fucoidan, a macromolecule, a type of polysaccharide and contains high percentage of fucose and sulfate ester groups and other compounds in low percentage. In the last decades, a number of interesting biological activities have been studied so fucoidan has been major focus of interest for exploring new drugs against the leading human diseases. It is known in relation to biological pathways and function that it activates the pathways which mediate apoptosis. Using LC–MS and interdisciplincary approach, we developed our goal to elucidate the potential difference between the crude and pure fucoidan and the interaction of fucoidan with potential signaling molecules STAT4 and IL12RB2. Based on our analysis, we conclude that there is major difference between crude and pure fucoidan in terms of identified metabolites and their functional relevance. It leads to the conclusion that there are 18 functions which are common between crude and pure fucoidans, 47 functions pure fucoidan specific, and 84 functions crude fucoidan specific which leads to the conclusion that even the fucoidans obtained from the same organism behave functionally completely different between the processing states (with/without processing) while docking study with STAT4 and IL12RB2 display very strong binding between fucoidan and both these signaling molecules. [ABSTRACT FROM AUTHOR]

Datura metel L. has been frequently used in Chinese traditional medicine. However, little is known on the chemical composition and in vivo metabolism of its seeds. In this study, using the strategy "chemical analysis, metabolism of single representative compounds, and metabolism of extract at clinical dosage" that we propose here, 42 constituents were characterized from D. metel seeds water extract. Furthermore, the metabolic pathways of 13 representative bioactive compounds of D. metel seeds were studied in rats after the oral administration of D. metel seeds water extract at a clinical dosage (0.15 g/kg). These included three withanolides, two withanolide glucosides, four amides, one indole, one triterpenoid, one steroid, and one sesquiterpenoid, and with regard to phase II metabolism, hydroxylation, (de)methylation, and dehydrogenation reactions were dominant. Furthermore, the metabolism of D. metel seeds water extract provided to rats at a clinical dosage was investigated by liquid chromatography-tandem mass spectrometry based on the above metabolic pathways. Sixty-one compounds were detected in plasma, 83 in urine, and 76 in fecal samples. Among them, withanolides exhibited higher plasma exposure than the other types. To our knowledge, this is the first systematic study on the chemical profiling and metabolite identification of D. metel seeds, including all compounds instead of single constituents. [ABSTRACT FROM AUTHOR]

Datura metel L. is a widely used traditional herbal medicine, and withanolides and amides are the two groups of main bioactive constituents in Datura metel seeds. This study aimed to elucidate the metabolism of four representative bioactive compositions containing daturataturin A (1), daturametelin I (2), N-trans-feruloyltyramine (3), and cannabisin F (4) in rats. After separately oral administration of 20 mg/kg withanolides (1, 2) and amides (3, 4) to rats, a total of 12, 24, and 21 metabolites were detected in the plasma, urine, and fecal samples, respectively. Among them, three hydroxylated metabolites, 1-M3, 2-M2, and 3-M5, were detected in plasma and rat liver microsome incubation system in high abundance. Two metabolites of 1 and 2 were unambiguously identified by comparing with reference standards. Particularly, the methylated metabolite 27α-methoxy-(22R)-22,26-epoxy-27-[(β-D-glucopyranosyl)oxy]ergosta-2,4,6,24-tetraene-1,26-dione (daturametelin L) is a new compound. The withanolides could readily get hydroxylation or methylation metabolism. Meanwhile, the phase II metabolism (glucuronidation or sulfation) was the major reaction for the amides. This is the first study on in vivo metabolism of these active compounds in seeds of Datura metel.

Microbial endophytes are known as versatile producers of useful metabolites, which have extensive applications in pharmacy, fragrance, agriculture and food. This study aims to screen sclareol-biotransforming microorganisms from Salvia sclarea, an untapped source of diverse endophytes. In this study, 50 culturable endophytes were isolated from S. sclarea grown in Xinjiang using sclareol as the sole carbon source and screened for their potential to transform sclareol into analogues. A fungal endophyte, identified as the generally recognized as safe (GRAS) strain Aspergillus tubingensis, can produce labd-14-ene-3β,8α,13β-triol and 8α,13β-dihydroxylabd-14-en-3-one from sclareol, involving hydroxylation and carbonylation at the C3 site. Structures of the two metabolites were elucidated by HR-ESI-MS and NMR analysis. S. sclarea was proven to be a good source of endophytes that are prospective producers of secondary metabolites with valuable chemical and biological properties. This study is the first report regarding the isolation of endophytes from S. sclarea., (© 2023 Wiley-VHCA AG, Zurich, Switzerland.)

Abstract The compound 2′,3′,5′‑tri‑ O ‑acetyl‑ N 6 ‑(3‑hydroxyphenyl) adenosine (also known as IMM-H007) is a new adenosine analogue that displays anti-hyperlipidaemic activity in many preliminary studies. To clarify its biotransformation process, in vitro and in vivo metabolic patterns of IMM-H007 in rat liver microsomes (RLMs), urine, feces, serum, and various tissues were investigated using high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD), off-line cryogenically cooled probe nuclear magnetic resonance (cryoNMR), and high-performance liquid chromatography quadrupole TOF MS (HPLC-QTOFMS) measurements. A total of 21 metabolites were detected and identified based on accurate mass measurements, diagnostic product ions, and 1D and 2D NMR data. All of the 21 metabolites were detected in vivo besides the 7 ones (LM1–3, LM4a-b, LM5, LM6 (M8)) in vitro. Among them, eight metabolites were phase I metabolites composed of the hydrolysis products LM1–3, LM4a, LM4b, LM5 and M7–8, and hydrolysis and hydroxylation products M6. Others were phase II metabolites including glucuronidation products M2, M4, M9, M11a-c, and M12a-c; and sulfation products M3, M5, and M10. Notably, 14 metabolites (LM1–3, LM4a-b, LM5, M9–10, M11a-c, M12a-c) were unreported before and the distribution of IMM-H007 and its all metabolites was reported for the first time. The results revealed IMM-H007 was metabolized mainly in the small intestine and serum, kidney, stomach, small and large intestines were important samples for metabolites presence. This work improves understanding of the metabolism, distribution, and excretion of IMM-H007, and demonstrates the HPLC/HPLC-MS/off-line cryoNMR approach can be applied for detection and identification of metabolites in complex biological matrices. Highlights • In vitro and in vivo metabolite patterns of IMM-H007 are comprehensive profiled. • Off-line coupling system is used for detection and identification of metabolites. • 21 metabolites are characterized by the HPLC-DAD, HPLC-QTOFMS and NMR techniques. • Absorption, distribution, metabolism and excretion of metabolites are studied. [ABSTRACT FROM AUTHOR]

Datura metel L. is a widely used traditional herbal medicine, and withanolides and amides are the two groups of main bioactive constituents in Datura metel seeds. This study aimed to elucidate the metabolism of four representative bioactive compositions containing daturataturin A (1), daturametelin I (2), N-trans-feruloyltyramine (3), and cannabisin F (4) in rats. After separately oral administration of 20 mg/kg withanolides (1, 2) and amides (3, 4) to rats, a total of 12, 24, and 21 metabolites were detected in the plasma, urine, and fecal samples, respectively. Among them, three hydroxylated metabolites, 1-M3, 2-M2, and 3-M5, were detected in plasma and rat liver microsome incubation system in high abundance. Two metabolites of 1 and 2 were unambiguously identified by comparing with reference standards. Particularly, the methylated metabolite 27α-methoxy-(22R)-22,26-epoxy-27-[(β-D-glucopyranosyl)oxy]ergosta-2,4,6,24-tetraene-1,26-dione (daturametelin L) is a new compound. The withanolides could readily get hydroxylation or methylation metabolism. Meanwhile, the phase II metabolism (glucuronidation or sulfation) was the major reaction for the amides. This is the first study on in vivo metabolism of these active compounds in seeds of Datura metel. [ABSTRACT FROM AUTHOR]

Abstract: Forsythoside A (FTA), the main active constituent isolated from Fructus Forsythiae, has various biological functions including anti‐oxidant, anti‐viral and anti‐microbial activities. However, while research on FTA has been mainly focused on the treatment of diseases on a material basis, FTA metabolites in vivo have not been comprehensively evaluated. Here, a rapid and sensitive method using a UHPLC‐LTQ‐Orbitrap mass spectrometer with multiple data processing techniques including high‐resolution extracted ion chromatograms, multiple mass defect filters and diagnostic product ions was developed for the screening and identification of FTA metabolites in rats. As the result, a total of 43 metabolites were identified in biological samples including 42 metabolites in urine, 22 metabolites in plasma and 15 metabolites in feces. These results demonstrated that FTA underwent a series of in vivo metabolic reactions including methylation, dimethylation, sulfation, glucuronidation, diglucuronidation, cysteine conjugation and their composite reactions. The research enhanced our understanding of FTA metabolism and built a foundation for further toxicity and safety studies. [ABSTRACT FROM AUTHOR]

In Saudi Arabia, halophytes occupy tidal and intertidal forest ecosystems. They and their associated microflora have immense potential to yield novel and important useful natural products. Three halophytes (Avicennia marina,Halocnemum strobilaceum,Zygophyllum qatarense) were targeted for the isolation and identification of populations of endophytic and rhizospheric bacteria having antimicrobial potential. A total 554 bacterial isolates were initially screened against oomycetes fungal pathogens,Phytophthora capsiciandPythium ultimum. Of these, only 57 rhizospheric and endophytic bacteria exhibited inhibition against the targeted bioassay oomycetes.Tentative identification of the bacteria was on the basis of 16S rRNA gene sequences which revealed 92–100% sequence identity to type strains of related species and placed these organisms in six major classes:Actinobacteria, γ-Proteobacteria, Firmicutes,α-Proteobacteria,Flavobacteriiaandβ-Proteobacteria. When checked for lytic enzyme production, mostly the isolates ofActinobacteriaandFirmicuteswere potential enzyme producers. Detection of secondary metabolite biosynthetic genes – type I polyketide synthases, type II polyketide synthases and nonribosomal peptide synthetases – confirmed that 21 (35.5%) isolates were positive for at least one type of the biosynthetic gene. In order to identify metabolites, three isolates,Alteromonas australica(EA73),Aidingimonas halophila(EA105) andHalomonas zincidurans(EA127), were selected and subjected to chemical analyses using liquid chromatography–mass spectrometry and gas chromatography–mass spectrometry. Both analyses showed the presence of different bioactive compounds in the culture extracts of isolates some of which are already reported for their diverse biological activities such as 2, 4-Diacetylphloroglucinol. Our results demonstrated that halophytes represent an important source of potentially active bacteria producing antifungal metabolites of medical significance. [ABSTRACT FROM AUTHOR]

Traditional Chinese medicine (TCM) exerts its therapeutic effect in a holistic fashion with the synergistic function of multiple characteristic constituents. The holism philosophy of TCM is coincident with global and systematic theories of metabolomics. The proposed pseudotargeted metabolomics methodologies were employed for the establishment of reliable quality control markers for use in the screening strategy of TCMs. Pseudotargeted metabolomics integrates the advantages of both targeted and untargeted methods. In the present study, targeted metabolomics equipped with the gold standard RRLC-QqQ-MS method was employed for in vivo quantitative plasma pharmacochemistry study of characteristic prototypic constituents. Meanwhile, untargeted metabolomics using UHPLC-QE Orbitrap HRMS with better specificity and selectivity was employed for identification of untargeted metabolites in the complex plasma matrix. In all, 32 prototypic metabolites were quantitatively determined, and 66 biotransformed metabolites were convincingly identified after being orally administered with standard extracts of four labeled Citrus TCMs. The global absorption and metabolism process of complex TCMs was depicted in a systematic manner. [ABSTRACT FROM AUTHOR]

Wu-tou Decoction (WTD) is a famous traditional Chinese medicine (TCM) formula which is applied to treat arthritis and pain of joints. In this study, a sensitive and rapid method was developed for the separation and identification of the absorbed constituents and metabolites of WTD in the rats plasma by ultra high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). A total of 22 absorbed prototype constituents and 49 metabolites were identified or tentatively characterized in dosed plasma. The possible metabolic pathways of these constituents involved sulfation, glucuronidation, demethylation, hydroxylation and so on. What’s more, we optimized the conventional process ways of network pharmacology and proposed a new concept called target-network pharmacology (T-NP). T-NP method used the absorbed constituents and the corresponding target proteins to generate compound-target network, and compare to the conventional method indifferent using the compounds collected from herbs, it could reduce the false positive results. We found that the following proteins were related to the WTD therapeutic effects, such as PTGS2, PTGS1, MAPK3, PPARG, TNF, IL4 and IL6. On the whole, the proposed method clearly presented the metabolic processes of WTD and the results gave a comprehensive metabolic profile of WTD in vivo for the first time. The combining use of the T-NP method could discover potential drug targets and disclose the biological processes of WTD, which will open up a new approach in the study of TCM in future. [ABSTRACT FROM AUTHOR]

Modified quantitative structure retention relationships (QSRRs) are proposed and applied to describe two retention data sets: A set of 94 metabolites studied by a hydrophilic interaction chromatography system under organic content gradient conditions and a set of tryptophan and its major metabolites analyzed by a reversed-phase chromatographic system under isocratic as well as pH and/or simultaneous pH and organic content gradient conditions. According to the proposed modification, an additional descriptor is added to a conventional QSRR expression, which is the analyte retention time, tR(R), measured under the same elution conditions, but in a second chromatographic column considered as a reference one. The 94 metabolites were studied on an Amide column using a Bare Silica column as a reference. For the second dataset, a Kinetex EVO C18 and a Gemini-NX column were used, where each of them was served as a reference column of the other. We found in all cases a significant improvement of the performance of the QSRR models when the descriptor tR(R) was considered. [ABSTRACT FROM AUTHOR]

Alstonia scholaris has been used in “Dai” ethnic medicine to treat chronic respiratory diseases for a long history, and the major bioactive constituents are alkaloids. An alkaloidal extract of A. scholaris leaves (AAS) has been developed into an investigational new drug, and has been approved for phase I/II clinical trials by China Food and Drug Administration. However, little is known on the chemical composition and in vivo metabolism of AAS, thus far. In this study, an ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC/qTOF-MS) method was established to characterize the chemical constituents of AAS. Samples were separated on an ACQUITY UPLC CSH column (2.1 × 100 mm, 1.7 μm) with acetonitrile and water containing 0.3% formic acid as the mobile phase. On the basis of high-accuracy mass spectral analysis, a total of 35 alkaloids were characterized from AAS, including 11 scholaricine-type, 9 vallesamine-type, 12 picrinine-type, and 3 tubotaiwine-type alkaloids. Furthermore, the metabolic pathways of 4 representative alkaloids in rats were studied. They mainly undertook hydroxylation and glucuronidation reactions. Based on the above metabolic pathways, the metabolism of AAS (10 mg/kg) in rats after oral administration was studied by LC/MS. A total of 33 compounds in plasma, 40 compounds in urine, and 38 compounds in feces were characterized. The results indicated that scholaricine-type alkaloids could get into circulation more readily than the other types. This is the first systematic study on chemical profiling and metabolites identification of AAS. [ABSTRACT FROM AUTHOR]

Identification and evaluations of pesticide metabolites are necessary for risk assessment and toxicological research. In this study, the metabolites of penflufen (a widely used chiral pesticide) in rat liver microsomes were identified using liquid chromatography Q-Exactive Plus mass spectrometry. In total, 17 penflufen metabolites were identified, and most of them were hydroxylation products, which were generated by oxygenation at different candidate sites of penflufen. The relative abundance of metabolite M12 (penflufen-3-hydroxy-butyl, 32 %) was the largest, followed by M8 (15.6 %) and M2 (12.8 %). The major metabolite penflufen-3-hydroxy-butyl was first synthesized by 11 reactions with a 99.73 % purity. The absolute configuration of M12 enantiomers were confirmed after preparing enantiomers, and establishing the enantioseparation method. The M12 enantiomers toxicity to Danio rerio (LC 50 , >10 mg/L) and four kinds of phytopathogens (EC 50 , 148–34969 mg/L) were significantly lower than parents (LC 50 , 0.449–24.3 mg/L; EC 50 , 0.027–92.0 mg/L). In rat liver microsomes, approximately 40–47 % of the penflufen enantiomers were metabolized to M12 enantiomers, and R -penflufen was preferentially metabolized. The generation concentrations of S -M12 were higher than R -M12 after 10 min, and the metabolic half-lives of R -M12 (29.0–32.5 min) were shorter than S -M12 (35.2–38.1 min), and were approximately 4 times longer than parent penflufen enantiomers (4.5–9.5 min). Simultaneously, the generated contents (relative contents) of M8 (27.1–57 %) and M10 (2.22–8.36 %) from S -penflufen were lower than those from R -penflufen (M8, 24.7–92.4 %; M10, 27.4–69.5 %). The enantioselective evaluations of M12, M10 and M8 deserve further study. These findings were helpful in understanding the fate and risks of chiral penflufen. [Display omitted] • Penflufen-3-hydroxy-butyl was major metabolite of penflufen in rat liver microsomes. • Major metabolite penflufen-3-hydroxy-butyl was first synthesized with 99.73 % purity. • Confirming absolute configuration and building separation method of M12 enantiomers. • The racemate and enantiomers of M12 were low-toxic to zebrafish and phytopathogens. • R -penflufen and R -M12 were preferentially metabolized in rat liver microsomes. [ABSTRACT FROM AUTHOR]

Licorice ( Glycyrrhiza uralensis Fisch.) is one of the most popular herbal medicines worldwide. This study aims to identify the metabolites of seven representative bioactive licorice compounds in rats. These compounds include 22β-acetoxyl glycyrrhizin ( 1 ), licoflavonol ( 2 ), licoricidin ( 3 ), licoisoflavanone ( 4 ), isoglycycoumarin ( 5 ), semilicoisoflavone B ( 6 ), and 3-methoxy-9-hydroxy-pterocarpan ( 7 ). After oral administration of 250 mg/kg of 1 or 40 mg/kg of 2 – 7 to rats, a total of 16, 43 and 31 metabolites were detected in the plasma, urine and fecal samples, respectively. The metabolites were characterized by HPLC/DAD/ESI–MS n and LC/IT-TOF-MS analyses. Particularly, two metabolites of 1 were unambiguously identified by comparing with reference standards, and 22β-acetoxyl glycyrrhizin-6″-methyl ester ( 1 - M2 ) is a new compound. Compound 1 could be readily hydrolyzed to eliminate the glucuronic acid residue. The phenolic compounds ( 4 – 7 ) mainly undertook phase II metabolism (glucuronidation or sulfation). Most phenolic compounds with an isoprenyl group (chain or cyclized, 2–5 ) could also undertake hydroxylation reaction. This is the first study on in vivo metabolism of these licorice compounds. [ABSTRACT FROM AUTHOR]

Antrodia cinnamomea is a precious medicinal mushroom popularly used for adjuvant cancer therapy in Taiwan. Its major bioactive constituents are ergostane and lanostane triterpenoids. Although clinical trials for A. cinnamomea have been recently initiated, its metabolism remains unclear. The present study aims to elucidate the metabolism and pharmacokinetics of A. cinnamomea in rats. After oral administration of an ethanol extract, 18 triterpenoids and 8 biotransformed metabolites were detected in rats plasma by UHPLC/qTOF-MS. Four of the metabolites were prepared by semi-synthesis and fully identified by NMR, while the others were tentatively characterized by comparing with the metabolites of single compounds (antcins B, C, H and K). Furthermore, a multi-component pharmacokinetic study of A. cinnamomea was carried out to monitor the plasma concentrations of 14 triterpenoids (ergostanes 1 – 3 , 5 – 8 , 14 – 16 ; lanostanes 9 , 10 , 17 , 19 ) and 2 metabolites ( M5 , M6 ) by LC/MS/MS in rats after oral administration of the ethanol extract (1.0 g/kg). The results showed that ergostanes and Δ 7,9(11) lanostanes, but not Δ 8 lanostanes, could get into circulation. The low-polarity ergostanes (antcins B and C) undertook hydrogenation (C-3 or C-7 carbonyl groups) or hydroxylation to produce polar metabolites. High-polarity ergostanes (antcins H and K) and Δ 7,9(11) lanostanes were metabolically stable. We also discovered that ergostanes and lanostanes showed remarkably different pharmacokinetic patterns. The ergostanes were generally absorbed and eliminated rapidly, whereas the lanostanes remained in the plasma at a low concentration for a relatively long time. The results indicate that high-polarity ergostanes are the major plasma-exposed components of A. cinnamomea , and may play an important role in its therapeutic effects. [ABSTRACT FROM AUTHOR]

Coumarins are an important group of bioactive constituents in licorice ( Glycyrrhiza uralensis ), a worldwide popular herbal medicine. This study aims to elucidate the metabolism of two major licorice coumarins, glycyrin and glycyrol in rats. After oral administration of 40 mg/kg glycyrin, neither the parent compound nor its metabolites could be detected in rats plasma or urine samples, indicating that glycyrin had poor oral bioavailability. Two hydroxylated metabolites, 4″-hydroxyl glycyrin and 5″-hydroxyl glycyrin, were detected in rat liver microsome incubation system. Among them, the major metabolite 4″-hydroxyl glycyrin, which is a new compound, was obtained by microbial transformation of Syncephalastrum racemosum AS 3.264. Its structure was fully identified by 1D and 2D NMR. Meanwhile, glycyrol, together with three metabolites, were detected in rats urine and fecal samples after oral administration (40 mg/kg). Their structures were tentatively characterized by LC/MS. Glycyrol mainly undertakes hydroxylation metabolism, accompanied by hydration and dehydrogenation as minor reactions. This is the first systematic study on metabolism of glycyrin and glycyrol. The results could be valuable to evaluate druggability of these bioactive natural products. [ABSTRACT FROM AUTHOR]

Modified quantitative structure retention relationships (QSRRs) are proposed and applied to describe two retention data sets: A set of 94 metabolites studied by a hydrophilic interaction chromatography system under organic content gradient conditions and a set of tryptophan and its major metabolites analyzed by a reversed-phase chromatographic system under isocratic as well as pH and/or simultaneous pH and organic content gradient conditions. According to the proposed modification, an additional descriptor is added to a conventional QSRR expression, which is the analyte retention time, tR(R), measured under the same elution conditions, but in a second chromatographic column considered as a reference one. The 94 metabolites were studied on an Amide column using a Bare Silica column as a reference. For the second dataset, a Kinetex EVO C18 and a Gemini-NX column were used, where each of them was served as a reference column of the other. We found in all cases a significant improvement of the performance of the QSRR models when the descriptor tR(R) was considered.

Glycycoumarin is a major bioactive coumarin of licorice (Glycyrrhiza uralensis), one of the most popular herbal medicines worldwide. In this work, the metabolism of glycycoumarin in rats was investigated. After oral administration of 40 mg/kg glycycoumarin, 4 and 10 metabolites were respectively detected in rats plasma and urine samples by liquid chromatography coupled with mass spectrometry (LC/MS). These metabolites were tentatively characterized by analyzing their tandem mass spectra and high-resolution mass spectra, and the structures of glucuronides were confirmed by β-glucuronidase hydrolysis. Glycycoumarin mainly undertakes hydroxylation and glucuronidation metabolism, accompanied by hydrogenation and dehydrogenation as minor reactions. Two hydroxylated metabolites, 4''-hydroxyl glycycoumarin and 5''-hydroxyl glycycoumarin, were obtained by microbial transformation of Syncephalastrum racemosum AS 3.264, and their structures were fully identified by 1D and 2D NMR. Both metabolites are new compounds. Furthermore, they were proved to be catalyzed by P450 enzymes by rat liver microsomes incubation experiments. Finally, a metabolic pathway of glycycoumarin in rats was proposed. This is the first systematic study on metabolites identification of glycycoumarin. [ABSTRACT FROM AUTHOR]

The identification of metabolites in drug discovery is important. At present, radioisotopes and mass spectrometry are both widely used. However, rapid and comprehensive identification is still laborious and difficult. In this study, we developed new analytical software and employed a stable isotope as a tool to identify drug metabolites using mass spectrometry. A deuterium-labeled compound and non-labeled compound were both metabolized in human liver microsomes and analyzed by liquid chromatography/time-of-flight mass spectrometry (LC-TOF-MS). We computationally aligned two different MS data sets and filtered ions having a specific mass-shift equal to masses of labeled isotopes between those data using our own software. For pioglitazone and flurbiprofen, eight and four metabolites, respectively, were identified with calculations of mass and formulas and chemical structural fragmentation analysis. With high resolution MS, the approach became more accurate. The approach detected two unexpected metabolites in pioglitazone, i.e., the hydroxypropanamide form and the aldehyde hydrolysis form, which other approaches such as metabolite-biotransformation list matching and mass defect filtering could not detect. We demonstrated that the approach using computational alignment and stable isotopic mass-shift filtering has the ability to identify drug metabolites and is useful in drug discovery. [ABSTRACT FROM AUTHOR]

Abstract: Ethnopharmacological relevance: TongMai Keli (TM) is a widely used traditional Chinese medicine preparation for the treatment of cardiovascular and cerebrovascular diseases. It is composed of Puerariae Lobatae Radix (roots of Pueraria lobata (Willd.) Ohwi), Salviae Miltiorrhizae Radix (roots of Salvia miltiorrhiza Bge.), and Chuanxiong Rhizoma (rhizomes of Ligusticum chuanxiong Hort.). The aim of this study is to identify the in vivo metabolites of TM, and to elucidate the pharmacokinetics of TM constituents and their metabolites. Materials and methods: For metabolites identification, TM was orally administered to rats (n=3), and the metabolites in plasma were identified by UHPLC/DAD/qTOF-MS analysis and β-glucuronidase hydrolysis. For pharmacokinetic study, rats (n=10) were treated with TM at a clinical dose, and the plasma was analyzed by LC/MS/MS. Results: A total of 25 metabolites from TM were identified in rats plasma. Glucuronide and sulfate conjugations were the major metabolic reactions, and produced 14 metabolites. The analytical method for pharmacokinetic study was fully validated with good linearity (r>0.99), wide dynamic ranges (6–6000ng/mL), and low variations (<14.3%). The plasma concentration–time curves of puerarin and nine metabolites were profiled. Conclusion: Isoflavones from Puerariae Lobatae Radix were the major metabolites in rat plasma after oral administration of TM. Puerarin and other isoflavone glycosides could reach their first C max within 30min, and were then rapidly eliminated, followed by their phase II metabolites. [Copyright &y& Elsevier]

Abstract: Gegen-Qinlian-Wan (GQW) is a popular traditional Chinese patent medicine for the treatment of diarrhea. It is composed of four herbal medicines, Puerariae Radix, Scutellariae Radix, Coptidis Rhizoma, and Glycyrrhizae Radix. In this study, a rapid and sensitive method based on ultra high-performance liquid chromatography coupled with diode-array detection and quadrupole time-of-flight mass spectrometry (UHPLC-DAD-qTOF-MS) was established to characterize the chemical constituents and rats metabolites of GQW. Samples were separated on an Agilent Zorbax Eclipse Plus-C18 column (100mm×2.1mm, 1.8μm) by gradient elution using acetonitrile and water containing 0.1% formic acid as the mobile phase. On the basis of UV and qTOF high-accuracy mass spectral analysis, a total of 62 compounds were identified or tentatively characterized from GQW, including 42 flavonoids, 8 alkaloids, 6 triterpenoids, 3 phenylethanoid glycosides, and 3 other types. Among them, 27 compounds were confirmed by comparing with reference standards. Furthermore, metabolites in rats plasma and urine after oral administration of GQW were also analyzed. A total of 42 compounds were identified, including 29 prototypes and 13 metabolites through metabolic pathways of demethylation, methylation, hydrolysis, sulfate conjugation, and glucuronide conjugation. Glucuronidated flavonoids were the main constituents in the plasma, and were then transformed into aglycones and excreted from urine. This is the first systematic study on the chemical constituents and metabolic profiling of GQW. [Copyright &y& Elsevier]

Abstract: FR429 is an ellagitannin with a potential antitumor activity, isolated and purified from Polygonum capitatum Buch.-Ham.ex D.Don, which is a traditional Miao-nationality herbal medicine in Guizhou and Yunnan of China. Our preliminary result of pharmacology study has indicated that the antitumor activity of FR429. However, the metabolism of FR429 has not been reported yet. In this study, LC–ion trap-time of flight mass spectrometry (LC–IT-TOF/MS) was used to characterize unpredictable metabolites of FR429 biotransformed by intestinal bacteria in vitro. Total thirteen metabolites were detected and characterized via comparisons of their accurate molecular masses and fragment ions of each MS n stage with those of the parent drug, and four of them were also elucidated by NMR. The results demonstrated that FR429 could be transformed by intestinal bacteria in vitro, mainly via hydrolysis and reduction reaction. This work provided a basis for the further study on the biotansformation of FR429 in vivo. [Copyright &y& Elsevier]

ZJM-289, [2-(1-diethylaminoacetoxy)pentyl] benzoic acid-{2-methoxy-4-[2-(4-nitrooxybutoxy carbonyl)-vinyl]}phenyl ester hydrochloride, is a novel nitric oxide–donating derivative of 3-n-butylphthalide synthesised on the hypothesis that it may be hydrolysed in vivo into 3-n-butylphthalide, ferulic acid and nitric oxide in hope that the three components may exert effects on the platelets as well as on central nervous system synergistically. In this study, ZJM-289 was extensively metabolised in rats. Eight major metabolites were identified by liquid chromatography (LC)–mass spectrometry (MS)/MS in rat plasma, bile, urine and faeces after intravenous administration. Metabolites M1, M2, M3, M4 and M5 were hydrolytic products of ZJM-289, M6 and M7 was a hydroxylation product of M5, and M8 was a glucuronide of M1. The pharmacologically active metabolite ferulic acid (M3) was a major metabolite in all the biological matrixes examined. 3-n-Butylphthalide was also present at a moderate level in the circulation. And along with the previous research, the anti-platelet activity of ZJM-289 was more potent than that of 3-n-butylphthalide both in vivo and in vitro. All these findings validated the theory of drug design. [ABSTRACT FROM AUTHOR]

Picroside II is one of the main active constituents of Picrorhiza kurroa, which has hepatoprotective, anticholestatic, antioxidant, and immune-modulating activity. To gain an understanding of the biotransformation of picroside II in vivo, liquid chromatography -electrospray ionization ion-trap mass spectrometry (LC-ESI-IT-MS) was used to investigate the metabolism of picroside II in rats after intravenous administration of a single dose. This method could simultaneously determine picroside II and its metabolites in rat bile. The bile samples were purified by use of a C solid-phase extraction (SPE) cartridge and were separated on a Hypersil ODS2 C analytical column. Two phase II metabolites of picroside II in rat bile were characterized, and elucidation of their structures was performed by comparing changes in molecular masses (Δ M), retention times, and MS spectral patterns of metabolites with those of the parent drug. Two metabolites identified for the first time in this research were glucuronide and sulfate conjugates. [ABSTRACT FROM AUTHOR]

The polyphagous insect Sitophilus oryzae L. (Coleoptera:Curculionidae) has a tremendous adaptability in feeding behaviour, making it a serious invasive pest of stored cereals. The present study identifies the metabolite composition of Sitophilus oryzae (S. oryzae) using Nuclear Magnetic Resonance (NMR) spectroscopy. Assignment of 1D-proton by NMR, 1H-1H COSY, 2D-TOCSY 1H-1H, had been done. Amongst the various biochemically important metabolites isoleucine, valine, leucine, β-hydroxybutyrate, lysine, glutamate, glutamine, proline, lactate, alanine, di-methylamine, α-glucose, β-glucose, choline, glycerophosphorylcholine and tyrosine are present in S. oryzae. In wheat-fed S. oryzae, the presence of threonine and the absence of lactate is observed. In rice-fed S. oryzae, however, the presence of lactate and the absence of threonine were observed. Barley-fed S. oryzae shows presence of both tyrosine and lactate. It is concluded that the pest S. oryzae has adaptability on different stored cereals and grains, depicting the presence of earlier reported metabolites. The present study aims to identify the key metabolic components and associated enzymes in Sitophilus oryzae fed on different cereals. [ABSTRACT FROM AUTHOR]

Abstract: Tanshinone IIA, the major component extracted from Radix salvia miltiorrhiza, has been observed to possess various kinds of pharmacological activities including antioxidant, prevention of angina pectoris and myocardial infarction and anticancer. Tanshinone IIA was incubated with rat liver microsomes and the resulting metabolites were identified by liquid chromatography/tandem mass spectrometry. The results showed the formation of three main hydroxyl metabolites. The three hydroxyl metabolites of tanshinone IIA were proved to be tanshinone IIB, hydroxytanshinone IIA and przewaquinone A by comparing the tandem mass spectra and the chromatographic retention time with that of the respective authentic compounds. Tanshinone IIB, hydroxytanshinone IIA and przewaquinone A are all the chemical components of total tanshinones. It was reasonable to presume that the three hydroxy metabolites of tanshinone IIA were pharmacologically active the same as tanshinone IIA and the total tanshinones. [Copyright &y& Elsevier]

Increasing use of organophosphorus flame retardants (OPFRs) has aroused great concern to their uncertain environment risk, especially to human health risk. In our study, hepatotoxicity screening of six aryl-OPFRs, potential hepatotoxicity mechanism of 2-ethylhexyldiphenyl phosphate (EHDPP) using RNA-sequencing and its metabolites were investigated in human hepatocytes (L02). The toxicity results demonstrated that EHDPP should be prioritized for further research with the highest toxicity. Further RNA-seq results through GO and KEGG enrichment analysis indicated that exposure to 10 mg/L of EHDPP significantly affected energy homeostasis, endoplasmic reticulum (ER) stress, apoptosis, cell cycle, and inflammation response in cells. The top 12 hub genes were validated by RT-qPCR and conformed to be mainly related to glycolysis and ER stress, followed by cell cycle and inflammation response. Western blot, apoptosis detection, glycolysis stress test, and cell cycle analysis were further performed to verify the above main pathways. Additionally, it was found in the metabolism experiment that detoxification of EHDPP by phase I and phase II metabolism in cells wasn't significant until 48 h with a metabolic rate of 6.12%. EHDPP was stable and still dominated the induction of toxicity. Overall, this study provided valuable information regarding the toxicity and potential metabolism pathway of EHDPP. [Display omitted] • EHDPP was the most toxic one among the six aryl-OPFRs towards human hepatocytes. • Energy homeostasis, ER stress, apoptosis, cell cycle and inflammation response were affected in EHDPP-induced toxicity. • EHDPP was stable in human hepatocytes. • The potential metabolism pathway of EHDPP included phase I and phase II metabolism. [ABSTRACT FROM AUTHOR]

Acetochlor is a chloroacetamide herbicide that has been widely used for weed control in recent decades. The contamination from its residue in the environment has raised major serious concerns. The aerobic degradation of acetochlor has been well studied; however, little is known regarding its anaerobic degradation. In the study, anaerobic sludge with high acetochlor degradation efficiency was obtained by pressure acclimation in a continuous flow anaerobic reactor. The acetochlor degradation dynamics followed a first-order kinetic reaction equation. The acclimated sludge could degrade six chloroacetamide herbicides with the degradation efficiencies observed as alachlor > acetochlor > propisochlor > butachlor > pretilachlor > metolachlor, and the N -alkoxyalkyl structure of these herbicides significantly affected their biodegradability. Five metabolites, 2-ethyl-6-methyl- N -(ethoxymethyl)-acetanilide, N -(2-methyl-6-ethylphenyl) acetamide, N -2-ethylphenyl acetamide, N -2-ethylphenyl formamide and 2-ethyl- N -carboxyl aniline were identified, and a putative anaerobic acetochlor degradation pathway, initiated by dechlorination, was subsequently proposed. During acclimation, the community diversity of both eubacteria and archaea in the anaerobic sludge decreased, while the abundance of microbes belonging to genera Sporomusa , Sporobacterium , Dechloromonas , Azotobacter and Methanobacterium were significantly increased and dominated the acclimated sludge, and showing a positive correlation with the acetochlor degradation capacity. These findings should be valuable to elucidate the mechanisms associated with the anaerobic catabolism of acetochlor and facilitate the engineering application of anaerobic treatment for removing acetochlor from wastewater. Unlabelled Image • Acclimated sludge exhibited a greater anaerobic degradation efficiency of acetochlor. • Acetochlor changed microbial community to acquire acetochlor-degrading capacity. • The predominant microbes in a continuous flow anaerobic reactor were determined. • A putative anaerobic degradation pathway of acetochlor was proposed. • The acclimated sludge could degrade six important chloroacetamide herbicides. [ABSTRACT FROM AUTHOR]

An ultra-performance liquid chromatography hybrid triple quadrupole-linear ion trap mass spectrometry(UPLC-QtrapMS) method was established to identify the metabolites in rat plasma,bile,urine and feces after oral administration of Cinnamomi Cortex(CC) aqueous extract. Several survey experiments,such as enhanced mass spectrum scan(EMS),precursor ion scan(PI),neutral loss scan(NL) and multiple ions monitoring(MIM) were applied to search target components,and two separate enhanced product ion(EPI) scans were triggered via information-dependent acquisition(IDA) method to generate the MS/MS spectra. According to the mass spectrometric data collected from reference standards and reported literature,the structures of metabolites were deduced. A total of76 metabolites and 5 original compounds were tentatively identified in rats after oral administration of CC aqueous extract. Deglycosylation,methylation,sulfonation,and glucuronidation were observed as the primary metabolic pathways for the chemical constituents of CC. These data are able to benefit the clarification of the therapeutic material basis,the clinical usage and further R&D of CC.

Ultra-high-performance liquid chromatography coupled with tandem high-resolution mass spectrometry (UHPLC-HR-MSn ) method was used to analyze the constituents of Fufang Gancao Tablets and its main metabolites in rat plasma. Rat plasma was collected both before and after oral administration of Fufang Gancao Tablets. After solid phase extraction, ACQUITY UHPLC BEH C₁₈ (2.1 mm×100 mm, 1.7 μm) was used with 0.1% formic acid-acetonitrile solution as the mobile phase for gradient elution. The chemical components in Fufang Gancao Tablets and their prototypes and metabolites in plasma samples were analyzed by LTQ-Orbitrap equipped with an ESI ion source in a positive ion mode. Based on the accurate mass measurements, the retention time and mass fragmentation patterns, a total of 55 compounds were tentatively identified from Fufang Gancao Tablets, including 42 flavonoids, 9 triterpenes and 4 alkaloids. Furthermore, metabolites in rat plasma after oral administration of Fufang Gancao Tablets were also analyzed. A total of 26 compounds were identified, including 20 prototypes and 6 metabolites mainly through metabolic pathways of hydroxylation, glucuronide conjugation, and sulfate conjugation, etc. Our results showed that the ultra-high-performance liquid chromatography coupled with linear ion trap quadrupole Orbitrap high resolution mass spectrometry (UHPLC-LTQ-Orbitrap) could comprehensively elucidate the chemical constituents of Fufang Gancao Tablets and their migrating components in rat plasma, providing scientific basis for further studying the metabolism process and pharmacodynamic substance of Fufang Gancao Tablets., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

A rapid and sensitive UHPLC-HR-MSn method was used for the identification of Kudiezi injection and its main metabolites in rat plasma. After the tail intravenous injection of Kudiezi, ACQUITY UHPLC BEH C₁₈ (2.1 mm×100 mm, 1.7 μm) was used, with 0.1% formic acid-acetonitrile solution as the mobile phase for gradient elution. Kudiezi injection and plasma were detected by ESI-LTQ-Orbitrap equipped with an ESI ion source in a negative mode. Based on the accurate mass measurements, the retention time and the mass fragmentation patterns, a total of 53 compounds were tentatively identified and characterized. Furthermore, metabolites in rat plasma after the intravenous administration of Kudiezi injection were also analyzed. A total of 38 compounds were identified, including 27 prototypes and 11 metabolites through metabolic pathways of methylation, glucuronide conjugation, sulfate conjugation and hydrolysis. As a result, UHPLC-LTQ-Orbitrap technique was applied to comprehensively expound Kudiezi injection's chemical components and constituents migrating to rat plasma, and provide scientific basis for further studies on Kudiezi injection's in vivo metabolic process and effective material base., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

The role of non-targeted metabolomics with its discovery power is constantly growing in many different fields of science. However, its biggest advantage of uncovering the unexpected is turning into one of its biggest bottlenecks, particularly in metabolite identification. Among different methods for metabolite identification or ID confirmation, tandem MS analysis plays a very important role. However, this method is limited to only certain types of MS analysers, making for example TOF-MS inaccessible for this type of metabolite identification. To overcome this, in-source fragmentation has been used to fragment molecules and obtain product ions. Since the molecule of interest is not isolated prior to its fragmentation, the acquired spectrum contains many different signals arising from the fragmentation of all compounds present in the sample. Therefore, to assign product ions to their precursors, a novel use of correlation analysis was tested with r ≥0.9 as an assignation of a product ion belonging to the precursor. This method and chosen cut-off was tested on three different sample complexity levels: conducting the analysis on a single standard, mix of co-eluting standards and on a plasma sample. Obtained results clearly proved the effectiveness of the proposed methodology for metabolite ID confirmation. Moreover, the proposed strategy can be successfully applied for semi-quantification of co-eluting molecules with the same monoisotopic mass but that differ in fragmentation pattern. The proposed methodology can greatly improve the robustness and throughput of identification in metabolomics studies by use of TOF-MS, which is crucial to obtain meaningful and trustful results., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)