Metabolites In Rat Urine Research Articles

Metabolic fate of the natural anticancer agent cucurbitacin B: an LC-MS/MS-enabled profiling of its major phase I and II conjugates in vivo.

Cucurbitacin B (CuB) is a natural triterpenoid with diverse pharmacological effects including potent anticancer activity. However, its oral bioavailability is hampered by limited metabolism in vivo. We characterized CuB's in vivo metabolism in rats to uncover bioactive metabolites retaining therapeutic potential, using a robust UHPLC-Q-TOF-MS/MS workflow. This workflow combined molecular networking, fragmentation filtering, and mass defect filtering to identify CuB metabolites in rat urine, plasma, and feces following oral administration. Thirteen metabolites were identified and seven were confirmed. Major phase I transformations involved hydrolysis, reduction, epoxidation, and amination. Phase II conjugation included cysteine, glutathione, glucuronide, and gluconic acid conjugates. Notably, one of the main metabolites formed was the cysteine conjugate CuB-Cys. CuB-Cys maintained similar in vitro antiproliferative activity to CuB on HepG2, MCF-7, and PANC-1 cancer cell lines. However, it demonstrated lower cytotoxicity towards non-cancerous L02 cells, highlighting improved therapeutic selectivity. Mechanistically, CuB-Cys induced greater apoptotic signaling in HepG2 cells than CuB via enhanced caspase activation and disrupted BAX-Bcl-2 balance. This represents the first systematic characterization of CuB's in vivo metabolic pathway. The identification and confirmation of CuB-Cys provide insight for drug development efforts aiming to maintain therapeutic efficacy while reducing toxicity, via metabolite-based approaches. Our findings shed light on strategies for improving CuB's clinical potential.

Identification of EA-3167, a Structural Analogue of the BZ Incapacitant, in Objects Delivered from the Zone of a Special Military Operation and Detection of Its Metabolites in Urine during Exposure of Rats

Earlier, we found that two alcohol-containing samples delivered from the zone of special military operation contain a rare psychoactive substance EA-3167, which is a structural analogue of the well-known incapacitant BZ, included in the Schedule 1 of the Chemical Weapons Convention. In addition to EA-3167, additives of methadone, α-pyrrolidinovalerophenone and tetrahydrocannabinol were found in liquids.The aim of the work is to establish the analytical characteristics of EA-3167, as well as its metabolites in rat urine. Research methods. Structural identification of the detected substance by nuclear magnetic resonance, gas and liquid chromatomass spectrometry of delivered objects and rat urine obtained by exposing laboratory animals to the detected substance.The results of the study. Concentrations of EA-3167 in liquids were found to be 910 and 3.9 μg/mL, respectively. It has been established that EA-3167 is subject to intensive metabolism in rats. More than 30 metabolites, representing products of mono- and dihydroxylation of aliphatic moieties of the molecule, ketone formation, and hydrolysis, have been tentatively identified in the urine of laboratory animals. Conclusion. The most convenient markers for confirming poisoning of EA-3167 by gas and liquid chromatography-mass spectrometry were the parent substance, the products of its monohydroxylation and the formation of carbonyl group on the cyclopentyl moiety.

Quantification of 11 metabolites in rat urine after exposure to organophosphates

BackgroundThe aim of the study was to develop a technique for quantitative determination of rat urine metabolites by HPLC–MS/MS, which can be used to search for biomarkers of acute intoxication with organophosphates (OPs).ResultsThe content of metabolites in the urine of rats exposed to a single dose of paraoxon (POX1x); interval, twice daily administration of paraoxon (POX2x); exposure to 2-(o-cresyl)-4H-1, 3, 2-benzodioxaphosphorin-2-oxide and paraoxon (CBPOX) was investigated. New data were obtained on the content in the urine of intact rats as well as rats in 3 models of OP poisoning: 3-methylhistidine, threonine, creatine, creatinine, lactic acid, acetylcarnitine, inosine, hypoxanthine, adenine, 3-hydroxymethyl-butyrate and 2-hydroxymethyl-butyrate.ConclusionsThe proposed assay procedure is a simple and reliable tool for urine metabolomic studies. Within 1–3 days after OP exposure in all three models of acute intoxication, the concentration of metabolites in rat urine, with the exception of adenine, changes similarly and symmetrically, regardless of the method of poisoning modeling, in all three models of acute intoxication. Further studies are needed to determine the specificity and reliability of using urinary metabolite concentration changes as potential biomarkers of acute organophosphate intoxication.Graphical

Determination of Ketamine, Fluoroketamine, Norketamine, and 2-Norfluoro-ketamine in Urine using Ultra-performance Liquid Chromatography-tandem Mass Spectrometry

Objective:: This study aimed to establish a simple, reliable, and sensitive method for detecting ketamine, fluoroketamine, and their metabolites in urine using UPLC-MS/MS. Methods:: The chromatographic separation was performed on UPLC BEH C18 (50 mm × 2.1 mm, 1.7 μm) at a column temperature of 40°C. The mobile phase consisted of 0.1% formic acid aqueous solution and acetonitrile, with a flow rate set at 0.4 mL/min, following a specific elution procedure. A urine sample was treated with acetonitrile, and midazolam was used as an internal standard. Multiple reaction monitoring was used for quantitative analysis. Results:: Ketamine, fluoroketamine, norketamine, and 2-norfluoro-ketamine exhibited linearity in urine (r>0.99) within the concentration range of 5–2000 ng/mL. Intra-day and inter-day precisions were 9% or less and 12% or less, respectively. The accuracy ranged from 92 to 107%. Mean recoveries were above 76%. The measured matrix effect was between 85 and 104%. Conclusion:: This simple, reliable, and sensitive PLC-MS/MS method was successfully developed to determine ketamine, fluoroketamine, and their metabolite in rat urine.

Analytical toxicology of the semi-synthetic cannabinoid hexahydrocannabinol studied in human samples, pooled human liver S9 fraction, rat samples, and drug product using HPLC-HRMS/MS.

Hexahydrocannabinol (HHC) is an emerging semi-synthetic cannabinoid, that is obtained from cyclization of cannabidiol and subsequent hydrogenation. As a potentially legal alternative of Δ9-tetrahydrocannabinol (Δ9-THC), it is increasingly seized in the United States and Europe. The aims of this study were to investigate the metabolism of HHC in pooled human liver S9 fraction (pHLS9), in rat, and human samples. Additionally, a locally obtained low-THC cannabis product was investigated that was advertised with elevated concentration of HHC. Overall, HHC formed an 11-HO metabolite, as well as a carboxy metabolite. While only the parent compound was detected in rat urine and feces, the hydroxy metabolite was additionally detected in pHLS9 and human plasma. The carboxy metabolite was only detectable in human plasma. The metabolism corresponded well to that of Δ9-THC, although glucuronidation and the formation of an 8-HO metabolite was not observed. Detectability of HHC and its carboxy metabolite in rat urine was investigated using GC-MS, but neither the parent compound, nor the metabolite were detectable. The investigated low-THC cannabis product appeared to be an actual cannabis product, since in addition to HHC, cannabinol, cannabidiol, and Δ9-THC were detected after qualitative analysis. Estimation of its content revealed 30.6 % HHC but also 4 % of Δ9-THC.

Comprehensive Metabolite Identification of Genipin in Rats Using Ultra-High-Performance Liquid Chromatography Coupled with High Resolution Mass Spectrometry.

Genipin, an aglycone of geniposide, is a rich iridoid component in the fruit of Gardenia jasminoides Ellis and has numerous biological activities. However, its metabolic profiles in vivo and vitro remain unclear. In this study, an effective analytical strategy based on ultra-high-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) in positive and negative ion modes was developed to analyze and identify genipin metabolites in rat urine, blood, feces, and fecal fermentation in combination with many methods including post-collection data mining methods, high-resolution extracted ion chromatography (HREIC), and multiple mass defect filtering (MMDF). Simultaneously, the metabolites of genipin in vivo were verified by fecal fermentation of SD rats at different times. Finally, based on information such as reference substances, chromatographic retention behavior, and accurate mass determination, a total of 50 metabolites (including prototypes) were identified in vivo. Among them, 7, 31 and 28 metabolites in vivo were identified in blood, urine, and feces, respectively. Our results showed that genipin could generate different metabolites that underwent multiple metabolic reactions in vivo including methylation, hydroxylation, dehydroxylation, hydrogenation, sulfonation, glucuronidation, demethylation, and their superimposed reactions. Forty-six metabolites were verified in vitro. Meanwhile, 2 and 19 metabolites identified in blood and urine were also verified in fecal fermentation at different times. These results demonstrated that metabolites were produced in feces and reabsorbed into the body. In conclusion, the newly discovered metabolites of genipin can provide a new perspective for understanding its pharmacological effects and build the foundation for thee toxicity and safety evaluations of genipin.

Metabolite quantification: A fluorescence-based method for urine sample normalization prior to 1H-NMR analysis.

Metabolomics is a multi-discipline approach to systems biology that provides a snapshot of the metabolic status of a cell, tissue, or organism. Metabolomics uses mass spectroscopy (MS) and nuclear magnetic resonance (NMR) to analyze biological samples for low molecular weight metabolites. Normalize urine sample pre-acquisition to perform a targeted quantitative analysis of selected metabolites in rat urine. Urine samples were provided from rats on a control diet (n = 10) and moderate sucrose diet (n = 8) collected in a metabolic cage during an eight hour fast. Urine from each sample was prepared by two different methods. One sample was a non-normalized sample of 1200µL and the second sample was a variable volume-normalized to the concentration of urobilin in a standard sample of urine. The urobilin concentration in all samples was determined by fluorescence. Ten metabolites for each non-normalized and normalized urine sample were quantified by integration to an internal standard of DSS. Both groups showed an improvement in pH range going from non-normalized to normalized samples. In the group on the control diet, eight metabolites had significant improvement in range, while the remaining two metabolites had insignificant improvement in range comparing the non-normalized sample to the normalized sample. In the group on the moderate sucrose diet all ten metabolites showed significant improvement in range going from non-normalized to normalized samples. These findings describe a pre-acquisition method of urine normalization to adjust for differences in hydration state of each organism. This results in a narrower concentration range in a targeted analysis.

Comprehensive Analysis of Pterostilbene Metabolites In Vivo and In Vitro Using a UHPLC-Q-Exactive Plus Mass Spectrometer with Multiple Data-Mining Methods

Pterostilbene, a stilbene phytoalexin, is mainly obtained from blueberries and grape vines; however, its metabolic mechanisms were unclear in vivo. In the present study, three different methods were used to prepare biological samples, and then, an efficient strategy based on ultrahigh-performance liquid chromatography coupled with mass spectrometry was developed to screen and identify pterostilbene metabolites in rat urine, plasma, liver, and feces. In order to elucidate pterostilbene or its metabolites involved in vitro, this study was assessed by the liver microsome system. As a result, a total of 88 pterostilbene metabolites were characterized. Among them, 77 metabolites in vivo and 14 metabolites in vitro were found; 50 and 38 metabolites were observed in rat plasma and urine, while only 4 and 12 metabolites were detected in rat feces and liver, inferring that plasma and urine possessed more diverse types of pterostilbene metabolites; 41 metabolic products were obtained by solid-phase extraction, and 9 and 10 metabolites were screened by methanol precipitation and acetonitrile precipitation, respectively, indicating that solid-phase extraction could be adopted as the most acceptable method for pterostilbene metabolism. The results also demonstrated that pterostilbene mainly underwent glucosylation, dehydrogenation, hydrogenation, demethoxylation, sulfation, NAC binding, methylene ketogenic, acetylation, and methylation. In summary, this research provides an idea for the further study of drug metabolism.

Extension of the Temporal Window for the Determination of Alpha-Methylthiofentanyl and Thiofentanyl in Rat Urine by Monitoring the Metabolite Norfentanyl Using Online Solid-Phase Extraction (SPE) Coupled with Ultra-High Performance Liquid Chromatography–Tandem Mass Spectrometry (UHPLC–MS/MS)

Alpha-methylthiofentanyl and thiofentanyl are psychoactive substances that have been abused in recent years, strongly threatening social and public security. Because they are rapidly metabolized in vivo, their determination presents a great challenge for forensic science. An automated method of online solid-phase extraction coupled with ultra-high performance liquid chromatography—tandem mass spectrometry was developed for the determination of alpha-methylthiofentanyl, thiofentanyl, and the metabolite norfentanyl in urine. The analytes were first enriched and purified by an Oasis HLB online SPE column and eluted on a Hypersil GOLD C18 analytical column. Chromatographic separation was achieved with mobile phase components of 2 mM ammonium formate and 0.1% formic acid in water and same composition in methanol in gradient elution mode. The limit of detection and lower limit of quantitation of the method for the three analytes were 0.2 and 2 pg/ml, respectively. Acceptable linearity was obtained from 2 to 800 pg/ml. The accuracy was in the range of 87.98–108.95%; and precision, calculated as the relative standard deviation, was less than 14.63%. The method recoveries were from 65.56 to 113.72%. This highly sensitive method was applied to study the detection window of the precursor compounds and metabolites in rat urine. Both alpha-methylthiofentanyl and thiofentanyl are metabolized to norfentanyl, which was first revealed in this study. With the aid of norfentanyl, the detection windows of alpha-methylthiofentanyl and thiofentanyl were extended to 18–26 days and 20–27 days, respectively. This study provides important auxiliary tools for the forensic identification of fentanyl analogs.

Metabolism and Mass Balance in Rats Following Oral Administration of the Novel Antifibrotic Drug Fluorofenidone.

ObjectiveFluorofenidone (AKF-PD) is a novel antifibrotic small-molecule compound. The purpose of this study was to investigate the metabolic and excretory pathways of AKF-PD in rats.MethodsHigh-performance liquid chromatography with mass spectrometric (HPLC-MS) detection was used to analyze the metabolites in rat urine. The metabolites were separated by chromatography and their structure was confirmed. HPLC was used to determine the contents of the parent compound and its metabolites in feces and urine after quantitative administration to study the excretion pathway.ResultsAKF-PD was mainly oxidized to the carboxyl group after methyl hydroxylation. After oral administration, the total amount of the prototype drug and its hydroxylated metabolites and carboxylated metabolites excreted from the urine and feces of rats was 87%. However, most of them are excreted in urine and feces in the form of carboxylated metabolites, and rarely excreted in the form of prototype drugs and hydroxylated metabolites. Which is that the urinary discharge of hydroxylated metabolites, fluorine ketones, and carboxylated metabolites were 0.2%, 1.1%, and 75.2%, respectively, while the fecal discharge were 0.2%, 0.3%, and 10.1%, respectively.ConclusionAKF-PD is mainly oxidized into 2-hydroxymethyl and 5-carboxyl AKF-PD through the Phase I metabolic reaction in rats. AKF-PD is a highly permeable compound classified by biopharmaceutics and is mainly excreted from the urine in the form of metabolites.

Metabolite Identification of Huangqi Jianzhong Tang in Rat Urine and Feces after Oral Administration Based on UHPLC-Q-Exactive-MS.

Huangqi Jianzhong Tang (HQJZ), a famous traditional Chinese medicine formula, is widely used in the treatment of gastrointestinal diseases in China. In this study, an ultra-high-performance liquid chromatography coupled with quadrupole-Exactive mass spectrometry was used to identify the metabolites in rat urine and feces after oral administration of HQJZ coupled with Compound Discover 3.0 software. The possible metabolic pathways were calculated and deduced based on 71 previous detected prototype compounds. As results, 88 compounds were identified in urine and feces, respectively. In urine sample, we identified 20 prototype compounds and 68 related metabolites. Meanwhile, a total of 21 prototype compounds and 67 related metabolites were identified in feces. Among them, flavonoids and saponins were the main ingredients in vivo. Further, we also speculated that HQJZ experienced oxidation, reduction, acetylation, methylation and glucuronic acid reaction in urine and feces. This study established a reliable method for the detection of prototype components and metabolites of traditional Chinese medicines, which would provide helpful information for further research into the active substances of HQJZ.

High-Throughput Method for the Simultaneous Determination of Doxorubicin Metabolites in Rat Urine after Treatment with Different Drug Nanoformulations.

Doxorubicin (DOX) is one of the most effective cytotoxic agents against malignant diseases. However, the clinical application of DOX is limited, due to dose-related toxicity. The development of DOX nanoformulations that significantly reduce its toxicity and affect the metabolic pathway of the drug requires improved methods for the quantitative determination of DOX metabolites with high specificity and sensitivity. This study aimed to develop a high-throughput method based on high-performance liquid chromatography with fluorescence detection (HPLC-FD) for the quantification of DOX and its metabolites in the urine of laboratory animals after treatment with different DOX nanoformulations. The developed method was validated by examining its specificity and selectivity, linearity, accuracy, precision, limit of detection, and limit of quantification. The DOX and its metabolites, doxorubicinol (DOXol) and doxorubicinone (DOXon), were successfully separated and quantified using idarubicin (IDA) as an internal standard (IS). The linearity was obtained over a concentration range of 0.05–1.6 μg/mL. The lowest limit of detection and limit of quantitation were obtained for DOXon at 5.0 ng/mL and 15.0 ng/mL, respectively. For each level of quality control (QC) samples, the inter- and intra-assay precision was less than 5%. The accuracy was in the range of 95.08–104.69%, indicating acceptable accuracy and precision of the developed method. The method was applied to the quantitative determination of DOX and its metabolites in the urine of rats treated by novel nanoformulated poly(lactic-co-glycolic acid) (DOX-PLGA), and compared with a commercially available DOX solution for injection (DOX-IN) and liposomal-DOX (DOX-MY).

Study of the metabolism of myricetin in rat urine, plasma and feces by ultra-high-performance liquid chromatography.

Myricetin is a common natural flavonoid compound with various pharmacological activities. However, the metabolite characterization of this substance remains inadequate. In this study, a simple and rapid system strategy based on UHPLC-Q-Exactive Orbitrap mass spectrometry combining parallel reaction monitoring mode was established to screen and identify myricetin metabolites in rat urine, plasma and feces after oral administration. A total of 38 metabolites were fully or partially characterized based on their accurate mass, characteristic fragment ions, retention times, corresponding cLogP values, etc. These metabolites were presumed to be generated through glucuronidation, glucosylation, sulfation, dihydroxylation, acetylation, hydrogenation, hydroxylation and their composite reactions. In addition, the characteristic fragmentation pathways of flavonoids with more metabolites were summarized for the subsequent metabolite identification. The study provides an overall metabolic profile of myricetin, which would be of great help in predicting the in vivo pharmacokinetic profiles and understanding the action mechanism of this active ingredient.

2C-B-Fly-NBOMe Metabolites in Rat Urine, Human Liver Microsomes and C. elegans: Confirmation with Synthesized Analytical Standards.

Compounds from the N-benzylphenethylamine (NBPEA) class of novel psychoactive substances are being increasingly utilized in neurobiological and clinical research, as diagnostic tools, or for recreational purposes. To understand the pharmacology, safety, or potential toxicity of these substances, elucidating their metabolic fate is therefore of the utmost interest. Several studies on NBPEA metabolism have emerged, but scarce information about substances with a tetrahydrobenzodifuran (“Fly”) moiety is available. Here, we investigated the metabolism of 2-(8-bromo-2,3,6,7-tetrahydrobenzo[1,2-b:4,5-b’]difuran-4-yl)-N-(2-methoxybenzyl)ethan-1-amine (2C-B-Fly-NBOMe) in three different systems: isolated human liver microsomes, Cunninghamella elegans mycelium, and in rats in vivo. Phase I and II metabolites of 2C-B-Fly-NBOMe were first detected in an untargeted screening and identified by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Several hypothesized metabolites were then synthesized as reference standards; knowledge of their fragmentation patterns was utilized for confirmation or tentative identification of isomers. Altogether, thirty-five phase I and nine phase II 2C-B-Fly-NBOMe metabolites were detected. Major detected metabolic pathways were mono- and poly-hydroxylation, O-demethylation, oxidative debromination, and to a lesser extent also N-demethoxybenzylation, followed by glucuronidation and/or N-acetylation. Differences were observed for the three used media. The highest number of metabolites and at highest concentration were found in human liver microsomes. In vivo metabolites detected from rat urine included two poly-hydroxylated metabolites found only in this media. Mycelium matrix contained several dehydrogenated, N-oxygenated, and dibrominated metabolites.

Nontargeted metabolomics-based mapping urinary metabolic fingerprints after exposure to acrylamide

Acrylamide classified as a probable carcinogen to humans is a high production volume chemical in industrial applications released to aquatic and environmental ecosystems, and also widely found in the thermal processing of starch-rich foods. To gain insight into the urinary metabolomics that may induce physiological responses stimulated by acrylamide, rats were orally administered with a single dose of 13C3-acrylamide (10 mg/kg bw) in the treatment group and urine samples were continuously collected every 2 h during the first 18 h and every 3 h during the period from 18 h to 36 h. A reliable nontargeted screening method for the analysis of urinary metabolomics in rats was developed using ultra-high performance liquid chromatography coupled to quadrupole-Orbitrap high-resolution mass spectrometry. All metabolites in urine of rats receiving isotope-labeled acrylamide were screened by validated orthogonal partial least squares-discriminant analyses compared to the animals in the control group, while exposure biomarkers were further confirmed according to the characteristic fragmentation rules and time-dependent profiles. Here we identified 2 new specific exposure biomarkers, named N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L-cysteine-sulfoxide and N-acetyl-S-(2-carboxyl)-L-cysteine, compared to 4 currently acknowledged mercapturic acid adducts of acrylamide. In addition, our findings on analysis of acrylamide metabolic pathway and identification of exposure biomarkers confirmed that acrylamide could significantly affect energy metabolism and amino acid metabolism by the Kyoto Encyclopedia of Genes and Genomes pathway analysis for key metabolites. Homocysteine thiolactone and hypoxanthine may be potential biomarkers for the cardiotoxicity, while methionine sulfoxide, hippuric acid and melatonin may be specifically related to the neurotoxicity. Thus, the current study provided new evidence on the identification of emerging exposure biomarkers and specific signature metabolites related to the toxicity of acrylamide, and shed light on how acrylamide affected energy and amino acid metabolism by further mapping urinary metabolic fingerprints.

Comprehensive Identification of Astilbin Metabolites in Rats Based on Multiple Metabolite Templates Combined with UHPLC-Q-Exactive Mass Spectrometry.

Astilbin, a dihydroflavonoid compound widely found in plants, exhibits a variety of pharmacological activities and biological effects. However, little is known about the metabolism of this active compound in vivo, which is very helpful for elucidating the pharmacodynamic material basis and application of astilbin. To establish a rapid profiling and identification method for metabolites in rat urine, faeces and plasma using a UHPLC-Q-Exactive mass spectrometer in negative ion mode. In this study, a simple and rapid systematic strategy and 7 metabolite templates, which were established based on previous reports, were utilized to screen and identify astilbin metabolites. As a result, a total of 71 metabolites were detected and characterized, among which 32 metabolites were found in rat urine, while 27 and 38 metabolites were characterized from rat plasma and faeces, respectively. These metabolites were presumed to be generated through ring cleavage, sulfation, dehydrogenation, methylation, hydroxylation, glucuronidation, dehydroxylation and their composite reactions. This study illustrated the capacity of the sensitive UHPLC-Q-Exactive mass spectrometer analytical system combined with the data-mining methods to rapidly elucidate the unknown metabolism. Moreover, the comprehensive metabolism study of astilbin provided an overall metabolic profile, which will be of great help in predicting the in vivo pharmacokinetic profiles and understanding the action mechanism of this active ingredient.

Non-targeted metabonomics to investigate the differences in the properties of ginseng and American ginseng based on rapid resolution liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry.

The basic properties of herbal medicines are cold, hot, warm, and cool. The differentiation of these properties is important for the diagnosis and treatment of diseases. Ginseng and American ginseng possess opposite properties of warm and cool, respectively. At present, the mechanisms and the influence of steaming leading to the differences in their properties are not clear and require further investigation. Therefore, nontargeted metabonomics based on liquid chromatography-mass spectrometry was applied to investigate the effects of ginseng, American ginseng, and their variants on the changes in endogenous metabolites in rat urine. A total of 19 potential biomarkers were screened out and identified, of which 17, 7, and 5, were respectively related to warm, cool, and both warm and cool properties with opposite effects. The metabolic pathways corresponded to fatty acids, lipids, glycolysis, and energy metabolisms. The warm and tonic effects of red ginseng are stronger than those of ginseng and consistent with the theory of traditional Chinese medicine. The red American ginseng has cool property; however, the degree of coolness is less than that of American ginseng. This study provides a reference methodology to understand the effects of processing and mechanisms associated with the differences in the properties of herbal medicines.

25CN-NBOMe Metabolites in Rat Urine, Human Liver Microsomes and C.elegans-Structure Determination and Synthesis of the Most Abundant Metabolites.

N-Benzylphenethylamines are novel psychedelic substances increasingly used for research, diagnostic, or recreational purposes. To date, only a few metabolism studies have been conducted for N-2-methoxybenzylated compounds (NBOMes). Thus, the available 2,5-dimethoxy-4-(2-((2-methoxybenzyl)amino)ethyl)benzonitrile (25CN-NBOMe) metabolism data are limited. Herein, we investigated the metabolic profile of 25CN-NBOMe in vivo in rats and in vitro in Cunninghamella elegans (C. elegans) mycelium and human liver microsomes. Phase I and phase II metabolites were first detected in an untargeted screening, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification of the most abundant metabolites by comparison with in-house synthesized reference materials. The major metabolic pathways described within this study (mono- and bis-O-demethylation, hydroxylation at different positions, and combinations thereof, followed by the glucuronidation, sulfation, and/or N-acetylation of primary metabolites) generally correspond to the results of previously reported metabolism of several other NBOMes. The cyano functional group was either hydrolyzed to the respective amide or carboxylic acid or remained untouched. Differences between species should be taken into account in studies of the metabolism of novel substances.

UPLC-Q-TOF/MS-Based Metabolomics Approach to Reveal the Hepatotoxicity of Emodin and Detoxification of Dihydromyricetin.

Dihydromyricetin (DMY), an important flavanone found in Ampelopsis grossedentata, plays a protective role in liver injury. Our previous research found that DMY protected L02 cells against hepatotoxicity caused by emodin. In this study, serum, urine, and liver samples from rats were systematically used for biochemical analysis, pathological observation, and nontargeted metabolomics to evaluate the toxicity of emodin and DMY intervention. After oral administration of DMY, DMY may alleviate liver injury by improving liver metabolism. Approximately, 8 of 15 metabolites in rat urine and serum were significantly regulated by DMY. Metabolic pathway analysis showed that glutathione metabolism, pyrimidine metabolism, and tryptophan metabolism were the most affected pathways, and 18 proteins were predicted to be potential targets of DMY during the alleviation of liver injury induced by emodin. This research is of great significance in confirming the liver-protective effect of DMY, especially during acute liver injury caused by traditional Chinese medicine.

Improved metabolite characterization by liquid chromatography – Tandem mass spectrometry through electron impact type fragments from adduct ions

Using a chimeric collision cell mounted on a quadrupole time-of-flight platform, collision induced dissociation (CID) and electron induced dissociation (EID) were investigated for the LC-MS analysis of low molecular weight compounds including drugs and endogenous metabolites. Compared to CID, EID fragmentation of the [M+H]+ species (10–20 eV) from standard compounds resulted in additional specific and informative fragments, mostly due to neutral losses and, in some cases due to ring openings. Some analytes, for example reserpine and vinpocetine, provided characteristic [M+H]•2+ species. For most analytes for sodium and potassium adducts and multimers a radical cation M•+ and electron impact type fragments were observed in the EID spectra, providing the opportunity to use EI libraries to support metabolite identification. EID opens the possibility to get structural information from adduct ions which is often not the case with CID. EID enabled the putative characterization of two metabolites in rat urine as glucuronides of 5,6-dihydroxyindole based on EID fragmentation of the potassium adducts.