Metabolites In Rat Urine Research Articles

Screening and analysis of metabolites in rat urine after oral administration ofApocynum venetumL. extracts using HPLC-TOF-MS

HPLC with diode array detection and ESI-TOF-MS was used for the study of the constituents in Apocynum venetum L. extracts and the metabolites in rat urine after oral administration of A. venetum L. extracts. A formula database of the known constituents in A. venetum L. was established, and 21 constituents were rapidly identified by accurately matching their molecular masses with the formulae of the compounds in the database. Furthermore, 34 metabolites were detected and elucidated in the rat urine. The scientific and plausible biotransformation pathways of the flavonoid components in A. venetum L. were also proposed together with the presentation of clues for potential mechanisms of bioactivity. This specific and sensitive HPLC-ESI-TOF-MS method can be used to identify the chemical components in the extracts of A. venetum L. and their metabolites in rat urine. This method can also be used to reveal the possible metabolic mechanisms of action of the extract components invivo.

Relative determination of the alkaloid metabolites of Er Miao San in rat urine by LC–MS/MS and its application to pharmacokinetics

In the present study, five metabolites of Cortex Phellodendri Chinensis, an important herbal drug, were identified using liquid chromatography multi-stage tandem mass spectrometric techniques (LC-MS(n)). A sensitive and rapid high-performance liquid chromatographic tandem mass spectrometry (LC-MS/MS) method was developed for the quantitation of the five metabolites, utilizing chlorobenzylidine as the internal standard in rat urine. Urine samples were precipitated with acetonitrile. Chromatographic separation was achieved on a Waters C18 analytical column. Detection was performed by a multiple reaction monitoring (MRM) mode via an electrospray ionization (ESI) source operating in the positive ionization mode. The method was linear over the concentration range of 0.05-1.00c for all components. The intra- and inter-day precision values were less than 14.6% and the deviations ranged from -4.4 to 13.8%. The recoveries at three levels were more than 73.7%. The fully validated method was used to determine the metabolites amount in rat urine to investigate the changes caused by coupling with Atractylodes lancea in Er Miao San preparation on metabolism.

Metabolites characterization of chamaechromone in vivo and in vitro by using ultra-performance liquid chromatography/Xevo G2 quadrupole time-of-flight tandem mass spectrometry

Ethnopharmacological relevanceStellera chamaejasme L. (Thymelaeaceae) was a toxic perennial herb and widely used as pesticide and dermatological agents in China. Chamaechromone was a major component in the dried roots of Stellera chamaejasme with anti-HBV and insecticidal activity. Analysis of metabolic profile in vivo and in vitro plays a pivotal role to unravel how TCM works. And the metabolites of chamaechromone might influence the effects and toxicity of Stellera chamaejasme. Moreover, the metabolic routes of chamaechromone provide an important basis for toxicological safety evaluation. Until now, little is known about the metabolism of chamaechromone. The current study was designed to characterize the whole metabolic pathways of chamaechromone in vitro and in vivo. Materials and methodsTwenty-four rats were randomly divided into four groups, including two oral administration groups (100mgkg−1), one intravenous injection group (5mgkg−1), and one control group. The metabolites in rat urine and feces and bile were identified by UPLC/Q-TOF MS analysis and β-glucuronidase hydrolysis. Moreover, the possible metabolic mechanism was further confirmed by Phase I and Phase II metabolism and catechol-O-methyltransferase methylation in rat liver S9 fraction and degradation in rat intestinal bacteria. ResultsA total of 24 metabolites from chamaechromone were detected and identified in vivo and in vitro, 20 of which were novel. And the major metabolic processes were hydroxylation, methylation, glucuronation, acetylation, dehydroxylation and degradation. ConclusionsThe present study revealed the whole metabolic pathways of chamaechromone in rat through both in vitro and in vivo experiments for the first time. And chamaechromone could undergo extensive phase I and phase II metabolism in rat. These findings would provide an important basis for the further study and clinical application of chamaechromone. In addition, the results of this work have showed the feasibility of the UPLC/Q-TOF-MS approach for rapid and reliable characterization of metabolites.

Metabolism and Excretion of Kakkalide and Its Metabolites in Rat Urine, Bile, and Feces as Determined by HPLC/UV and LC/MS/MS

This study investigated the metabolic fate of kakkalide (irisolidone 7-xylosylglucoside), a major isoflavone found in extracts of Pueraria lobata flowers, and in rat urine, bile, and feces. Using HPLC/UV or LC/MS/MS methods, seven metabolites, tectorigenin-7-O-glucuronide, tectorigenin-7-O-sulfate, tectorigenin-4'-O-sulfate, 6-OH biochanin A-glucuronide, irisolidone-7-O-glucuronide, tectorigenin, and irisolidone were identified in rat urine after oral administration of kakkalide. Furthermore, irisolidone-7-O-glucuronide was found in bile, and irisolidone and kakkalide were found in feces. An HPLC/UV method for simultaneous quantification of all the metabolites and kakkalide in urine, bile, and feces was developed using daidzein or apigenin as the internal standard. Over a 72-h period, 13.2 ± 2.8 % of the kakkalide was excreted as seven metabolites in urine. Over the same time period, irisolidone-7-O-glucuronide excretion in bile accounted for 3.8 ± 1.1 % of the dose, while kakkalide and irisolidone excretion in feces accounted for 2.1 ± 0.7 % and 0.7 ± 0.1 % of the dose, respectively. The results indicate that urine is the primary route of kakkalide elimination in vivo and that extensive metabolism may be one of the reasons for the low bioavailability of kakkalide.

Identification of acteoside and its major metabolites in rat urine by ultra-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry

In this study, metabolites in the urine samples of rats orally administered with acteoside, a phenylethanoid glycoside compound, were detected and identified using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC/ESI-QTOF-MS) combined with an automated MS(E) technique. Up to 35 metabolites (19 metabolites of the parent drug and 16 metabolites of the degradation products) were observed, including processes of oxidization, glucuronidation, sulfation, and methyl conjugation. According to the metabolic pathways, acteoside mainly functioned as a prodrug and underwent hydrolysis before being absorbed into the blood. The degradation products, especially caffeic acid and hydroxytyrosol, were involved in further metabolism which was responsible for the low oral bioavailability but obvious pharmacological activities of acteoside. In summary, this work provided valuable information on acteoside metabolism through the rapid and reliable UPLC/ESI-QTOF-MS technique, which could be widely used for the investigation of natural product metabolites.

Pharmacokinetic study of rutin and quercetin in rats after oral administration of total flavones of mulberry leaf extract

Mulberry leaves, a traditional Chinese medicine, are effective in the treatment of diabetes mellitus. Rutin and quercetin are the main components of total flavones of mulberry leaf extract. To study the pharmacokinetics of rutin and quercetin in rat plasma and their metabolites in rat urine and feces after oral administration of total flavones of mulberry leaf extract. At different timepoints after oral administration of total flavones of mulberry leaf extract in rats, plasma concentrations of rutin and quercetin were determined by RP-HPLC. The main pharmacokinetic parameters were estimated using 3P97 software. The metabolites in rat urine and feces were determined by using UPLC-ESI-QTOF/MS and estimated MetaboLynx™ software. The plasma concentration-time curves of rutin and quercetin both were best fitted with a two-compartment model. Rutin and quercetin were absorbed rapidly and then slowly decreased. Two prototype compounds and seven metabolites were identified. The pharmacokinetic and metabolic results may be useful for further studies of the bioactive mechanism of mulberry leaf flavones and potential development of a new TCM.

Identification of the Major Components of Resina Draconis Extract and Their Metabolites in Rat Urine by LC-ESI-MS n

The aim of our study was to employ a liquid chromatography coupled with electrospray ionization multistage tandem mass spectrometry (LC-ESI-MSn) method for the identification of the major components of Resina Draconis extract (RDE) and their metabolites in rat urine. Based on the above, 18 compounds were tentatively identified from the RDE. Among them, 4 compounds were unambiguously characterized by the comparison of the retention time and mass spectra with those of reference compounds and 14 compounds were tentatively identified by comparing the mass spectra with those of literature. In vivo, 21 compounds, including 13 parent compounds and 8 metabolites, were detected in rat urine after oral administration of RDE. The results may be helpful for future research on traditional Chinese medicine.

Characterization of major flavonoids, triterpenoid, dipeptide and their metabolites in rat urine after oral administration of Radix Astragali decoction

To characterize of the constituents in rat urine after oral administration of Radix Astragali decoction, a HPLC-DAD-MS/MS technique had been developed. Urine collected from 0 to 24 h, after administration, was purified using a C18 solid-phase extraction cartridge, and then detected by an on-line MS/MS detector. By comparing the retention times and MS/MS data with those obtained from authentic compounds and the published data, a total of 11 compounds including six flavonoids, one dipeptide, one triterpenoid and three of their metabolites in urine were identified. Compounds daidzein, genistein, quercetin, L-asparamide-D-phenylalanine, 4,2′,4′-trihydroxychalcone, Astragaloside-IV, daidzein sulfate, and kaempferol sulfate were for the first time detected by HPLC-MS/MS technique in urine. The present research results success-fully narrowed the range of effective constituents to be found in Astragalus membranaceus and would be useful for the following action mechanism research of this traditional Chinese medicine in treating various diseases.

Simultaneous determination of trantinterol and its metabolites in rat urine and feces by liquid chromatography–tandem mass spectrometry

A highly selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of trantinterol (SPFF) and its major metabolites for the first time. The analytes were extracted from rat urine and feces samples by liquid-liquid extraction (LLE) and determined in multiple reaction monitoring (MRM) mode with clenbuterol as the internal standard. Chromatographic separation was achieved on a Venusil ASB C8 column (2.1mm×100mm, 3μm), with the mobile phase consisted of methanol-0.2% formic acid (30:70, v/v) at the flow rate of 0.2mL/min. Each sample was chromatographed within 5min. This method has a lower limit of quantification (LLOQ) of 0.450, 1.05, 1.35, 0.904 and 1.36ng/mL for trantinterol (SPFF), arylhydroxylamine trantinterol (N-OH-SPFF), tert-butyl hydroxylated trantinterol (Tert-OH-SPFF), 1-carbonyl trantinterol (SPFF-COOH) and 3-methyl sulfone-dechloro-trantinterol (SPFF-SO2CH3) in rat urine, and 0.450, 1.35 and 0.904ng/mL for SPFF, Tert-OH-SPFF and SPFF-COOH in rat feces, respectively. The linear correlation coefficients were greater than 0.990. The intra- and inter-day precision (relative standard deviation, RSD) values were below 15% and the accuracy (relative error, RE) was -9.9% to 11% at three quality control levels. The method has been successfully applied to the excretion study following an oral administration of 1mg/kg trantinterol to rats.

Use of the Local False Discovery Rate for Identification of Metabolic Biomarkers in Rat Urine Following Genkwa Flos-Induced Hepatotoxicity

Metabolomics is concerned with characterizing the large number of metabolites present in a biological system using nuclear magnetic resonance (NMR) and HPLC/MS (high-performance liquid chromatography with mass spectrometry). Multivariate analysis is one of the most important tools for metabolic biomarker identification in metabolomic studies. However, analyzing the large-scale data sets acquired during metabolic fingerprinting is a major challenge. As a posterior probability that the features of interest are not affected, the local false discovery rate (LFDR) is a good interpretable measure. However, it is rarely used to when interrogating metabolic data to identify biomarkers. In this study, we employed the LFDR method to analyze HPLC/MS data acquired from a metabolomic study of metabolic changes in rat urine during hepatotoxicity induced by Genkwa flos (GF) treatment. The LFDR approach was successfully used to identify important rat urine metabolites altered by GF-stimulated hepatotoxicity. Compared with principle component analysis (PCA), LFDR is an interpretable measure and discovers more important metabolites in an HPLC/MS-based metabolomic study.

Levels of Pesticides and Their Metabolites in Wistar Rat Amniotic Fluids and Maternal Urine upon Gestational Exposure

Concentrations of pesticides and selected metabolites in rat urine and amniotic fluid were determined as biomarker upon oral administration of Wistar rats to two pesticide mixtures consisting of three to five pesticides (bitertanol, propiconazole, cypermethrin, malathion, and terbuthylazine). The pesticides and their metabolites were found in rat amniotic fluid and urine, generally in dose-response concentrations in relation to dosage. The measurement of the substances in the amniotic fluid indicated that the fetus was exposed to the pesticides as well as their metabolites. Moreover, the pesticides detected in urine demonstrated the exposure as well as the ability of the rat to excrete these compounds.

Identification of Sinensetin Metabolites in Rat Urine by an Isotope-Labeling Method and Ultrahigh-Performance Liquid Chromatography–Electrospray Ionization Mass Spectrometry

Sinensetin (SIN), one of the major polymethoxyflavones (PMFs) contained mainly in the citrus peels, has been reported to possess various bioactivities, including antifungal, antimutagenic, anticancer, and anti-inflammatory activities. Although the biotransformation of SIN in fungi and insects has been reported, the information about the metabolism of SIN in mammals is still unclear. In this study, formation of SIN metabolites in rats was investigated. Four isotope-labeled SINs ([4'-D3]SIN, [3'-D3]SIN, [5-D3]SIN, and [6-D3]SIN) were synthesized and administered to rat. The urine samples were collected and main metabolites were monitored by ultrahigh-performance liquid chromatography-electrospray ionization mass spectrometry. The administered compound and four SIN metabolites were detected in rat urine. These metabolites were identified as 4'-hydroxy-5,6,7,3'-tetramethoxyflavone, 5-hydroxy-6,7,3',4'-tetramethoxyflavone, 6-hydroxy-5,7,3',4'-tetramethoxyflavone, and 7-hydroxy-5,6,3',4'-tetramethoxyflavone sulfate.

A urinary metabonomics study on biochemical changes in yeast-induced pyrexia rats: A new approach to elucidating the biochemical basis of the febrile response

Fever is a prominent feature of many diseases, such as infection, inflammation and trauma. In the clinic, fever can be easily judged by measuring the body temperature; however, the pathogenesis of fever is still not fully understood. A febrile response is a systemic pathological process that can cause metabolic disorders. Metabonomics can provide powerful tools to reveal the pathological mechanisms for such a systemic disease. Thus, to reveal subtle metabolic changes under the condition of fever and to explore its mechanism, an ultra performance liquid chromatography coupled with a quadrupole time-of-flight mass spectrometry metabonomics approach was employed to investigate the urine biochemical characteristics of yeast-induced pyrexia rats. The acquired data were subjected to principal component analysis for differentiating the pyrexia rats from the control rats. Potential biomarkers were screened by using orthogonal partial least-squares-discriminant analysis and were identified by accurate mass, database, and MS/MS fragment information obtained from the MSE technique. Sixteen metabolites in rat urine were identified as potential biomarkers. The relative intensities of the 15 potential biomarkers were calculated. The thermoregulatory circuitry of “endogenous pyrogen (EP) ↑-hypothalamus Na+/Ca2+-cAMP↑” was partially confirmed in this study. The results suggested that UPLC/MS-based metabolic profiling of rat urine identifies impaired tryptophan metabolism as the mechanism of yeast-induced fever. This research provided informative data that the impaired tryptophan metabolism might be one of the important reasons in elucidating the biochemical basis of the febrile response.

Simultaneous determination of arctiin and its metabolites in rat urine and feces by HPLC

Arctiin, an important lignan compound in Fructus Arctii, has been reported to possess various kinds of bioactivities. Previous studies on the pharmacokinetic of arctiin after oral administration showed that it had a rapid absorption phase followed by a sharp but lasting disappearance. To gain deep insight into the action mechanism of arctiin, the excretion and metabolism of arctiin in vivo should be further studied. In this paper, three metabolites were isolated and identified in rat feces as (−)-enterolactone (M-1), (−)-arctigenin (M-2) and [(2R,3R)-2-(3′-hydroxybenzyl)-3-(3″,4″-dimethoxybenzyl)-butyrolactone] (M-3). Based on the structures of three metabolites, possible metabolic pathways of arctiin in rats are proposed. At the same time, the cumulative excretion rate of arctiin and its metabolites in rat urine and feces were determined, indicating that arctiin was excreted 19.84% in urine and 1.80% in feces, respectively, enterolactone, the most main metabolite, was excreted 35.80% in feces. These results provide very important information for understanding the metabolism and excretion of arctiin in vivo and speculating its action mechanism, they can provide useful information and reference for further metabolic investigations on arctiin in humans.

Characterization of Major Flavonoids, Triterpenoid, Dipeptide and Their Metabolites in Rat Urine after Oral Administration ofRadix AstragaliDecoction

Characterization of Major Flavonoids, Triterpenoid, Dipeptide and Their Metabolites in Rat Urine after Oral Administration of<i>Radix Astragali</i>Decoction

Analytical method for urinary metabolites of the fluorine-containing pyrethroids metofluthrin, profluthrin and transfluthrin by gas chromatography/mass spectrometry

An analytical method was developed for measurement of the major urinary metabolites in rats administered fluorine-containing pyrethroids (metofluthrin, profluthrin and transfluthrin) which are widely used recently as mosquito repellents or mothproof repellents. Eight metabolites, 2,3,5,6-tetrafluorobenzoic acid, 4-methyl-2,3,5,6-tetrafluorobenzoic acid, 2,2-dimethyl-3-(1-propenyl)-cyclopropanecarboxylic acid, 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid (carboxylic metabolites), 2,3,5,6-tetrafluorobenzyl alcohol, 4-methyl-2,3,5,6-tetrafluorobenzyl alcohol, 4-methoxymethyl-2,3,5,6-tetrafluorobenzyl alcohol and 4-hydroxymethyl-2,3,5,6-tetrafluorobenzyl alcohol (alcoholic metabolites), were extracted from enzymatic hydrolyzed urine using toluene and then concentrated. After transformation to their tert-butyldimethylsilyl derivatives for carboxylic metabolites or their trimethylsilyl derivatives for alcoholic metabolites, analysis was conducted by gas chromatography/mass spectrometry in the electron impact ionization mode. The calibration curves for each metabolite were linear over the concentration range of 0–20μg/ml in urine, and the quantification limits were between 0.009 and 0.03μg/ml. The relative errors and the relative standard deviations on replicate assays were less than 6% and 5%, respectively, for all concentrations studied. The measurements were accurate and precise. The collected urine samples could be stored for up to 1 month at −20°C in a freezer. The proposed method was applied to the analysis of several urine samples collected from rats treated with these pyrethroids.

Identification of urinary metabolites in rats administered the fluorine-containing pyrethroids metofluthrin, profluthrin, and transfluthrin

The major metabolites in urine of rats administered the fluorine-containing pyrethroids transfluthrin, profluthrin, and metofluthrin, which are used widely recently as mosquito repellents or mothproof repellents, were identified to establish biological indexes for evaluating the absorption amounts of these pyrethroids in the general population. A single dose of 300 mg kg−1 body weight of each pyrethroid was separately administered intraperitoneally to male Sprague-Dawley rats and urine samples were collected until 24 hours after the administration. The metabolites identified by gas chromatography-mass spectrometry were as follows: 2,3,5,6-tetrafluorobenzyl alcohol, 2,3,5,6-tetrafluorobenzoic acid, and 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid (DCCA) for transfluthrin; 4-methyl-2,3,5,6-tetrafluorobenzyl alcohol, 4-methyl-2,3,5,6-tetrafluorobenzoic acid, 4-hydroxymethyl-2,3,5,6-tetrafluorobenzyl alcohol (HOCH2–FB–Al), and 2,2-dimethyl-3-(1-propenyl)-cyclopropanecarboxylic acid (MCA) for profluthrin; 4-methoxymethyl-2,3,5,6-tetrafluorobenzyl alcohol, HOCH2–FB–Al and MCA for metofluthrin. In addition, several compounds estimated to be metabolites were detected as follows: hydroxylated DCCA and its lactone for transfluthrin; 4-hydroxymethyl-2,3,5,6-tetrafluorobenzoic acid (HOCH2–FB–Ac), hydroxylated MCA and its lactone for profluthrin; 4-methoxymethyl-2,3,5,6-tetrafluorobenzoic acid, HOCH2–FB–Ac, hydroxylated MCA, and its lactone for metofluthrin. The pyrethroids administered underwent metabolic reactions such as ester hydrolysis and oxidation, and most of the metabolites were excreted as their conjugates into urine. These findings should be useful as basic data for evaluating the health effects of exposure to pyrethroids for the general population.

Identification of 5,7,3′,4′-Tetramethoxyflavone Metabolites in Rat Urine by the Isotope-Labeling Method and Ultrahigh-Performance Liquid Chromatography–Electrospray Ionization–Mass Spectrometry

5,7,3',4'-Tetramethoxyflavone (TMF), one of the major polymethoxyflavones (PMFs) isolated from Kaempferia parviflor , has been reported possessing various bioactivities, including antifungal, antimalarial, antimycobacterial, and anti-inflammatory activities. Although several studies on the TMF have been reported, the information about the metabolism of TMF and the structures of TMF metabolites is still not yet clear. In this study, an isotope-labeling method was developed for the identification of TMF metabolites. Three isotope-labeled TMFs (5,7,3',4'-tetramethoxy[3'-D(3)]flavone, 5,7,3',4'-tetramethoxy[4'-D(3)]flavone, and 5,7,3',4'-tetramethoxy[5,4'-D(6)]flavone) were synthesized and administered to rats. The urine samples were collected, and the main metabolites were monitored by ultrahigh-performance liquid chromatography-electrospray ionization-mass spectrometry. Five TMF metabolites were unambiguously identified as 3'-hydroxy-5,7,4'-trimethoxyflavone, 7-hydroxy-5,3',4'-trimethoxyflavone sulfate, 7-hydroxy-5,3',4'-trimethoxyflavone, 4'-hydroxy-5,7,3'-trimethoxyflavone, and 5-hydroxy-7,3',4'-trimethoxyflavone.

Identification of in vivo and in vitro metabolites of triptolide by liquid chromatography–tandem mass spectrometry

Triptolide, a major active constituent of Tripterygium wilfordii Hook F, has multiple pharmacological activities. In this work, a rapid, sensitive and specific liquid chromatography coupled to an ion trap mass spectrometer (MS) with electrospray ionization (ESI) interface has been developed for identification of triptolide and some of its metabolites in rat urine after oral administration of a single dose (0.6 mg/kg) of triptolide to healthy rats, as well as some metabolites in vitro after incubation with rat liver microsome (RLM) and rat intestinal flora, respectively. All samples were separated on a reversed-phase C18 column using a mobile phase of acetonitrile/water (70:30, v/v) and detected by an on-line MS(n) detector. Identification and structural elucidation of the selected metabolites were performed by comparing their full scan MS(n) spectra with those of the parent drug. In this paper we identified ten metabolites in rat urine, four metabolites in RLM incubation solution and one metabolite in rat intestinal flora incubation solution, after drug administration. The metabolic reactions of triptolide that we observed in vivo were hydrolysis reaction, hydroxylation reaction, and the conjugate reaction with sulfate, glucuronide and GSH, respectively. The in vitro metabolic reactions of triptolide observed were hydrolysis and hydroxylation reactions.

Metabolism of (2S)-Pterosin A: Identification of the Phase I and Phase II Metabolites in Rat Urine

The metabolic profile of the potent hypoglycemic agent, (2S)-pterosin A (1), in rat urine via intragastrical oral administration was investigated. In total, 19 metabolites (M1-M19) were identified. Among these, 16 metabolites were characterized by high-performance liquid chromatography solid-phase extraction-tube transfer-NMR, and seven metabolites were further isolated from the treated urine to enable further structural determination. Twelve of these are new compounds. The phase I metabolites of 1 were formed via various oxidations at positions C-3, C-10, C-12, C-13, or C-1 followed by decarboxylation of C-10 or C-14, and lactonization at C-12/C-14 or C-14/C-12. The phase II metabolites were glucuronide conjugates from the parent compound or phase I metabolites. The major metabolites were found to be (2S)-14-O-glucuronylpterosin A (M9), (2S)-2-hydroxymethylpterosin E (M14), and (±)-pterosin B (M19). Quantitative HPLC analysis of metabolites, based on similar UV absorption and use of the regression equation of 1, indicated that ∼71% 1 was excreted as metabolites in rat urine.