Chemical UPLC‐ESI‐MS/MS profiling of aconitum alkaloids and their metabolites in rat plasma and urine after oral administration of Aconitum carmichaelii Debx. Root extract

In this paper, an ultra high performance liquid chromatography tandem mass spectrometric (UPLC‐ESI‐MS/MS) method in positive ion mode was established to systematically identify and to compare the major aconitum alkaloids and their metabolites in rat plasma and urine after oral administration of Fuzi extract. A total twenty‐nine components including twenty‐five C19‐diterpenoid alkaloids and four C20‐diterpenoid alkaloids were identified in Fuzi extract. Thirteen of the parent components and five metabolites were detected in rat plasma and sixteen parent compounds and six metabolites in urine. These parent components found in rat plasma and urine were mainly C19‐diterpenoid alkaloids. All of the metabolites in vivo were demethylated metabolites (phase I metabolites), which suggested that demethylation was the major metabolic pathway of aconitum alkaloids in vivo. A comparison of the parent components in rat plasma and urine revealed that 3‐deoxyacontine was found in plasma but not in urine, while kalacolidine, senbusine and 16‐β‐hydroxycardiopetaline existed in urine but not in plasma, which indicated that most alkaloids components were disposed and excreted in prototype form. This research provides some important information for further metabolic investigations of Fuzi in vivo.     

The biotransformation of oleuropein in rats

A highly sensitive and specific LC‐MS/MS method was developed to investigate the in vivo bio‐transformation of oleuropein in rat. Rat feces and urine samples collected after oral administration were determined by liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the negative‐ion mode. The assay procedure involves a simple liquid–liquid extraction of parent oleuropein and the metabolite from rat feces and urine with ethyl acetate. Chromatographic separation was operated with 0.1% formic acid aqueous and methanol in gradient program at a flow rate of 0.50 mL/min on an RP‐C₁₈ column with a total run time of 31 min. This method was successfully applied to simultaneous determination of oleuropein and its metabolites in rat feces and urine. De‐glucosylation, hydrolysis, oxygenation and methylation were found to comprise the major metabolic pathway of oleuropein in rat gastrointestinal tract and three metabolites were absorbed into the blood circulatory system within 24 h after oral administration. Copyright © 2013 John Wiley & Sons, Ltd.     

An improved highly sensitive method to determine low oxyresveratrol concentrations in rat plasma and its pharmacokinetic application

Existing methods to determine oxyresveratrol, a trans‐polyphenolic stilbene, lack selectivity, require large plasma sample volumes or have time‐consuming sample preparation and chromatographic isolation. Here an improved highly sensitive liquid chromatography–tandem mass spectrometry method was developed to determine low oxyresveratrol concentrations in rat plasma. The plasma samples were prepared by liquid–liquid extraction with acetoacetate. The analyte s were separated on Venusil hydrophilic interaction chromatography (HILIC) column (2.1 × 50 mm, 5.0 µm) guarded by a HILIC column (4 × 3.0 mm, 5.0 µm). The mobile phase consisted of acetonitrile–water (containing 1 mmol/L ammonium formate) at gradient elution mode with a flow rate of 0.3 mL/min. Resveratrol was used as the internal standard. An electrospray ionization source was applied and operated in the negative multiple reaction monitoring (MRM) mode. Oxyresveratrol and resveratrol were detected on MRM by the transitions from the precursor to the product ion (m/z 243.1 → 175.1 and 227.1 → 143.0). The total running time was 5 min and the retention times of oxyresveratrol and resveratrol were 1.97 and 1.82 min. Chromatograms showed no endogenous interfering peaks with blank samples. The linear calibration curve was obtained over the concentration range of 1–500 ng/mL. The injection volume was 10 μL and the limit of quantification was 1 ng/mL. The extraction recovery varied from 78.2 to 84.3% for low, medium and high quality control samples. At the same time, the intra‐ and inter‐day relative standard deviations were <6.78 and <10.02%, respectively, while the corresponding intra‐ and inter‐day accuracy relative error values fell in the range of 3.75–6.67%. The HPLC‐MS/MS method was successfully applied to a pharmacokinetics study, in which the experimental rats received a single dose of oxyresveratrol (10 mg/kg, intragastric administration). The pharmacokinetic results are presented. Copyright © 2013 John Wiley & Sons, Ltd.     

Metabolite profiling of ginsenosides in rat plasma,urine and feces by LC–MS/MS and its application to a pharmacokinetic study after oral administration of Panax ginseng extract

Panax ginseng is widely consumed as a functional food in the form of tea, powder, capsules, among others, and possesses a range of pharmacological activities including adaptogenic, immune‐modulatory, anti‐tumor, anti‐aging and anti‐inflammatory effects. The aim of this study was to identify and quantify the major ginsenosides and their metabolites in rat plasma, urine and feces after administration of P. ginseng extract using LC–MS/MS. We collected rat plasma samples at 0.5, 1, 2, 4, 8, 12, 24 and 48 h, and the amounts of urine and fecal samples accumulated in 24 h. Fourteen major ginsenosides and their metabolites were observed in fecal samples at high levels; however, low levels of 11 ginsenosides were detected in urine samples. The pharmacokinetics of the major ginsenosides and their metabolites was investigated in plasma. The results indicated that the maximum plasma concentration, time to maximum concentration and area under the curve of compound K were significantly greater than those of other ginsenosides. This study thus provides valuable information for drug development and clinical application of P. ginseng.     

Screening and analysis of metabolites in rat urine after oral administration of Apocynum venetum L. 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 in vivo.     

Rapid identification of phase I and II metabolites of artemisinin antimalarials using LTQ‐Orbitrap hybrid mass spectrometer in combination with online hydrogen/deuterium exchange technique

Artemisinin drugs have become the first‐line antimalarials in areas of multi‐drug resistance. However, monotherapy with artemisinin drugs results in comparatively high recrudescence rates. Autoinduction of CYP‐mediated metabolism, resulting in reduced exposure, has been supposed to be the underlying mechanism. To better understand the autoinduction of artemisinin drugs, we evaluated the biotransformation of artemisinin, also known as Qing‐hao‐su (QHS), and its active derivative dihydroartemisinin (DHA) in vitro and in vivo, using LTQ‐Orbitrap hybrid mass spectrometer in conjunction with online hydrogen (H)/deuterium (D) exchange high‐resolution (HR)‐LC/MS (mass spectrometry) for rapid structural characterization. The LC separation was improved allowing the separation of QHS parent drugs and their metabolites from their diastereomers. Thirteen phase I metabolites of QHS have been identified in liver microsomal incubates, rat urine, bile and plasma, including six deoxyhydroxylated metabolites, five hydroxylated metabolites, one dihydroxylated metabolite and deoxyartemisinin. Twelve phase II metabolites of QHS were detected in rat bile, urine and plasma. DHA underwent similar metabolic pathways, and 13 phase I metabolites and 3 phase II metabolites were detected. Accurate mass data were obtained in both full‐scan and MS/MS mode to support assignments of metabolite structures. Online H/D exchange LC‐HR/MS experiments provided additional evidence in differentiating deoxydihydroxylated metabolites from mono‐hydroxylated metabolites. The results showed that the main phase I metabolites of artemisinin drugs are hydroxylated and deoxyl products, and they will undergo subsequent phase II glucuronidation processes. This study also demonstrated the effectiveness of online H/D exchange LC‐HR/MSⁿ technique in rapid identification of drug metabolites. Copyright © 2011 John Wiley & Sons, Ltd.     

Pharmacokinetics of 2,3,5,4′‐tetrahydroxystilbene‐2‐O‐β‐D‐glucoside in rat using ultra‐performance LC‐quadrupole TOF‐MS

2,3,5,4′‐Tetrahydroxystilbene‐2‐O‐β‐D‐glucoside (THSG) from Polygoni multiflori has been demonstrated to possess a variety of pharmacological activities, including antioxidant, anti‐inflammatory and hepatoprotective activities. Ultra‐performance LC‐quadrupole TOF‐MS with MS Elevated Energy data collection technique and rapid resolution LC with diode array detection and ESI multistage MSⁿ methods were developed for the pharmacokinetics, tissue distribution, metabolism, and excretion studies of THSG in rats following a single intravenous or oral dose. The three metabolites were identified by rapid resolution LC‐MSⁿ. The concentrations of the THSG in rat plasma, bile, urine, feces, or tissue samples were determined by ultra‐performance LC‐MS. The results showed that THSG was rapidly distributed and eliminated from rat plasma. After the intravenous administration, THSG was mainly distributing in the liver, heart, and lung. For the rat, the major distribution tissues after oral administration were heart, kidney, liver, and lung. There was no long‐term storage of THSG in rat tissues. Total recoveries of THSG within 24 h were low (0.1% in bile, 0.007% in urine, and 0.063% in feces) and THSG was excreted mainly in the forms of metabolites, which may resulted from biotransformation in the liver.     

New cathinone‐derived designer drugs 3‐bromomethcathinone and 3‐fluoromethcathinone: studies on their metabolism in rat urine and human liver microsomes using GC–MS and LC–high‐resolution MS and their detectability in urine

3‐Bromomethcathinone (3‐BMC) and 3‐Fluoromethcathinone (3‐FMC) are two new designer drugs, which were seized in Israel during 2009 and had also appeared on the illicit drug market in Germany. These two compounds were sold via the Internet as so‐called “bath salts” or “plant feeders.” The aim of the present study was to identify for the first time the 3‐BMC and 3‐FMC Phase I and II metabolites in rat urine and human liver microsomes using GC–MS and LC–high‐resolution MS (HR‐MS) and to test for their detectability by established urine screening approaches using GC–MS or LC–MS. Furthermore, the human cytochrome‐P450 (CYP) isoenzymes responsible for the main metabolic steps were studied to highlight possible risks of consumption due to drug–drug interaction or genetic variations. For the first aim, rat urine samples were extracted after and without enzymatic cleavage of conjugates. The metabolites were separated and identified by GC–MS and by LC–HR‐MS. The main metabolic steps were N‐demethylation, reduction of the keto group to the corresponding alcohol, hydroxylation of the aromatic system and combinations of these steps. The elemental composition of the metabolites identified by GC–MS could be confirmed by LC–HR‐MS. Furthermore, corresponding Phase II metabolites were identified using the LC–HR‐MS approach. For both compounds, detection in rat urine was possible within the authors' systematic toxicological analysis using both GC–MS and LC–MSⁿ after a suspected recreational users dose. Following CYP enzyme kinetic studies, CYP2B6 was the most relevant enzyme for both the N‐demethylation of 3‐BMC and 3‐FMC after in vitro–in vivo extrapolation. Copyright © 2012 John Wiley & Sons, Ltd.     

Metabolite profiling of ginsenoside Rg1 after oral administration in rat

The goal of this study is to investigate the biotransformation of ginsenoside Rg₁ in vivo. A highly sensitive and specific LC‐MS/MS method was developed and used for metabolite identification in rat feces and urine after oral administration of ginsenoside Rg₁. Four metabolites of Rg₁ were detected in rat feces and three metabolites of Rg₁ were detected in rat urine. Deglycosylation and oxygenation were found to be the major metabolic pathways of ginsenoside Rg₁ after oral administration in rat. Except for the reported metabolites Rh₁ and protopanaxatriol, mono‐oxygenated Rg₁ and mono‐oxygenated protopanaxatriol were detected for the first time after oral administration of Rg₁. The in vivo metabolite profiling of ginsenoside Rg₁ in rat was proposed. Viewed collectively, Rg₁ was metabolized to mono‐oxygenated Rg₁, Rh₁, protopanaxatriol and the secondary metabolite mono‐oxygenated protopanaxatriol in rat. Copyright © 2014 John Wiley & Sons, Ltd.     

Studies on the metabolism of the Δ9‐tetrahydrocannabinol precursor Δ9‐tetrahydrocannabinolic acid A (Δ9‐THCA‐A) in rat using LC‐MS/MS,LC‐QTOF MS and GC‐MS techniques

In Cannabis sativa, Δ9‐Tetrahydrocannabinolic acid‐A (Δ9‐THCA‐A) is the non‐psychoactive precursor of Δ9‐tetrahydrocannabinol (Δ9‐THC). In fresh plant material, about 90% of the total Δ9‐THC is available as Δ9‐THCA‐A. When heated (smoked or baked), Δ9‐THCA‐A is only partially converted to Δ9‐THC and therefore, Δ9‐THCA‐A can be detected in serum and urine of cannabis consumers. The aim of the presented study was to identify the metabolites of Δ9‐THCA‐A and to examine particularly whether oral intake of Δ9‐THCA‐A leads to in vivo formation of Δ9‐THC in a rat model. After oral application of pure Δ9‐THCA‐A to rats (15 mg/kg body mass), urine samples were collected and metabolites were isolated and identified by liquid chromatography‐mass spectrometry (LC‐MS), liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) and high resolution LC‐MS using time of flight‐mass spectrometry (TOF‐MS) for accurate mass measurement. For detection of Δ9‐THC and its metabolites, urine extracts were analyzed by gas chromatography‐mass spectrometry (GC‐MS). The identified metabolites show that Δ9‐THCA‐A undergoes a hydroxylation in position 11 to 11‐hydroxy‐Δ9‐tetrahydrocannabinolic acid‐A (11‐OH‐Δ9‐THCA‐A), which is further oxidized via the intermediate aldehyde 11‐oxo‐Δ9‐THCA‐A to 11‐nor‐9‐carboxy‐Δ9‐tetrahydrocannabinolic acid‐A (Δ9‐THCA‐A‐COOH). Glucuronides of the parent compound and both main metabolites were identified in the rat urine as well. Furthermore, Δ9‐THCA‐A undergoes hydroxylation in position 8 to 8‐alpha‐ and 8‐beta‐hydroxy‐Δ9‐tetrahydrocannabinolic acid‐A, respectively, (8α‐Hydroxy‐Δ9‐THCA‐A and 8β‐Hydroxy‐Δ9‐THCA‐A, respectively) followed by dehydration. Both monohydroxylated metabolites were further oxidized to their bishydroxylated forms. Several glucuronidation conjugates of these metabolites were identified. In vivo conversion of Δ9‐THCA‐A to Δ9‐THC was not observed. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification of metabolites of isopropyl 3‐(3,4‐dihydroxyphenyl)‐2‐hydroxypropanoate in rats by high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Isopropyl 3‐(3,4‐dihydroxyphenyl)‐2‐hydroxypropanoate (IDHP) is an investigational new drug having the capacity for treating ailments in the cardiovascular and cerebrovascular system. In this work, a rapid and sensitive method using high‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (HPLC‐ESI‐Q‐TOF‐MS) was developed to reveal the metabolic profile of IDHP in rats after oral administration. The method involved pretreatment of the samples by formic acid–methanol solution (v/v, 5:95), chromatographic separation by an Agilent Eclipse XDB‐C₁₈ column (150 × 4.6 mm i.dx., 5 μm) and online identification of the metabolites by Q‐TOF‐MS equipped with electrospray ionizer. A total of 16 metabolites from IDHP, including four phase I metabolites and 12 phase II metabolites, were detected and tentatively identified from rat plasma, urine and feces. Among these metabolites, Danshensu (DSS), a hydrolysis product of IDHP, could be further transformed to 11 metabolites. These results indicated that DSS was the main metabolite of IDHP in rats and the major metabolic pathways of IDHP in vivo were hydrolysis, O‐methylation, sulfation, glucuronidation and reduction. The results also demonstrated that renal route was the main pathway of IDHP clearance in rat. The present study provided valuable information for better understanding the efficacy and safety of IDHP. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification and structural characterization of in vivo metabolites of ketorolac using liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS)

In vivo metabolites of ketorolac (KTC) have been identified and characterized by using liquid chromatography positive ion electrospray ionization high resolution tandem mass spectrometry (LC/ESI‐HR‐MS/MS) in combination with online hydrogen/deuterium exchange (HDX) experiments. To identify in vivo metabolites, blood urine and feces samples were collected after oral administration of KTC to Sprague–Dawley rats. The samples were prepared using an optimized sample preparation approach involving protein precipitation and freeze liquid separation followed by solid‐phase extraction and then subjected to LC/HR‐MS/MS analysis. A total of 12 metabolites have been identified in urine samples including hydroxy and glucuronide metabolites, which are also observed in plasma samples. In feces, only O‐sulfate metabolite and unchanged KTC are observed. The structures of metabolites were elucidated using LC‐MS/MS and MSⁿ experiments combined with accurate mass measurements. Online HDX experiments have been used to support the structural characterization of drug metabolites. The main phase I metabolites of KTC are hydroxylated and decarbonylated metabolites, which undergo subsequent phase II glucuronidation pathways. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of circulatory and excretory metabolites of meisoindigo in rat plasma,urine and feces by high‐performance liquid chromatography coupled with positive electrospray ionization tandem mass spectrometry

Meisoindigo has been a routine therapeutic agent in the clinical treatment of chronic myelogenous leukemia in China since the 1980s. However, information relevant to in vivo metabolism of meisoindigo is absent so far. In this study, in vivo circulatory metabolites of meisoindigo in rat plasma, as well as excretory metabolites in rat urine and feces, were identified by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Integration of multiple reaction monitoring with conventional metabolic profiling methodology was adopted to enable a more sensitive detection of in vivo metabolites. By comparing with the MS/MS spectra and retention times of the in vitro reduced metabolites, the major metabolites in rat plasma were proposed to form from 3,3′ double bond reduction, whereas the minor metabolites were formed from reduction followed by N‐demethylation, and reduction followed by phenyl mono‐oxidation. The major metabolites in the rat urine were proposed to form from reduction followed by phenyl mono‐oxidation, and its glucuronide conjugation and sulfate conjugation, whereas the minor metabolites were formed from 3,3′ double bond reduction, N‐demethylation, reduction followed by N‐demethylation, phenyl di‐oxidation, phenyl mono‐oxidation and its glucuronide conjugation and sulfate conjugation. The major metabolites in the rat feces were proposed to form from reduction followed by phenyl mono‐oxidation, whereas the minor metabolites were formed from reduction followed by N‐demethylation, and reduction followed by phenyl di‐oxidation. The phase I metabolic pathways showed a significant in vitro–in vivo correlation in rat. Copyright © 2010 John Wiley & Sons, Ltd.     

Development and validation of a HPLC method for quantification of rivastigmine in rat urine and identification of a novel metabolite in urine by LC‐MS/MS

A sensitive, specific and accurate HPLC method for the quantification of rivastigmine (RSM) in rat urine was developed and validated. The method involves the simple liquid–liquid extraction of RSM and pyridostigmine as an internal standard (IS) from rat urine with tertiary methyl butyl ether. The chromatographic separation of RSM and IS was achieved with 20 mm ammonium acetate buffer (pH 6.5) and acetonitrile (65:35, v/v) delivered at flow‐rate of 1 mL/min on a Kromasil KR‐100. The method was in linear range from 50 to 5000 ng/mL. The validation was done as per FDA guidelines and the results met the acceptance criteria. The method was successfully applied for the quantification of RSM in rat urine. Besides method validation, we have identified two metabolites of RSM in urine. Both the metabolites were characterized by HPLC‐PDA and LC‐MS/MS and it was found that one metabolite is novel. Copyright © 2010 John Wiley & Sons, Ltd.     

Liquid chromatography/quadrupole time‐of‐flight mass spectrometry in combination with online hydrogen/deuterium exchange technique for structural elucidation of phase I metabolites of iso‐phenylcyclopentylamine in rat bile

MS/MS experiment and accurate mass measurement are powerful tools in metabolite identification. However, sometimes these data do not provide enough information to assign an unambiguous structure to a metabolite. In combination with MS techniques, hydrogen/deuterium (H/D) exchange can provide additional information for structural elucidation by determination of the number of exchangeable hydrogen atoms in a structure. In this study, the principal phase I metabolites of iso‐phenylcyclopentylamine in rat bile were identified by high‐performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight mass spectrometry (ESI‐Q‐TOF‐MS). Since N‐oxidation may occur because of the existence of the primary amino group in the structure, it was difficult to differentiate the hydroxylated metabolites from N‐oxides by ESI‐Q‐TOF‐MS alone. Therefore, online H/D exchange technique was applied to solve this problem. Finally, 25 phase I metabolites were detected and structurally described, in which 11 were confirmed to be N‐oxides. This study demonstrated the effectiveness of high‐resolution mass spectrometry in combination with an online H/D exchange technique in rapid identification of drug metabolites, especially in discriminating hydroxylated metabolites from N‐oxides. Copyright © 2014 John Wiley & Sons, Ltd.     

In vivo metabolic investigation of silodosin using UHPLC–QTOF–MS/MS and in silico toxicological screening of its metabolites

Silodosin (SLD) is a novel α1‐adrenoceptor antagonist which has shown promising clinical efficacy and safety in patients with benign prostatic hyperplasia (BPH). However, lack of information about metabolism of SLD prompted us to investigate metabolic fate of SLD in rats. To identify in vivo metabolites of SLD, urine, feces and plasma were collected from Sprague–Dawley rats after its oral administration. The samples were prepared using an optimized sample preparation approach involving protein precipitation followed by solid‐phase extraction and then subjected to LC/HR‐MS/MS analysis. A total of 13 phase I and six phase II metabolites of SLD have been identified in rat urine which includes hydroxylated, N‐dealkylated, dehydrogenated, oxidative, glucosylated, glucuronide and N‐sulphated metabolites, which are also observed in feces. In plasma, only dehydrogenated, N‐dealkylated and unchanged SLD are observed. The structure elucidation of metabolites was done by fragmentation in MS/MS in combination with HRMS data. The potential toxicity profile of SLD and its metabolites were predicted using TOPKAT software and most of the metabolites were proposed to show a certain degree of skin sensitization and occular irritancy. Copyright © 2016 John Wiley & Sons, Ltd.     

Application of a LC–MS/MS method developed for the determination of p‐phenylenediamine,N‐acetyl‐p‐phenylenediamine and N,N‐diacetyl‐p‐phenylenediamine in human urine specimens

Cases of poisoning by p‐phenylenediamine (PPD) are detected sporadically. Recently an article on the development and validation of an LC–MS/MS method for the detection of PPD and its metabolites, N‐acetyl‐p‐phenylenediamine (MAPPD) and N,N‐diacetyl‐p‐phenylenediamine (DAPPD) in blood was published. In the current study this method for detection of these compounds was validated and applied to urine samples. The analytes were extracted from urine samples with methylene chloride and ammonium hydroxide as alkaline medium. Detection was performed by LC–MS/MS using electrospray positive ionization under multiple reaction‐monitoring mode. Calibration curves were linear in the range 5–2000 ng/mL for all analytes. Intra‐ and inter‐assay imprecisions were within 1.58–9.52 and 5.43–9.45%, respectively, for PPD, MAPPD and DAPPD. Inter‐assay accuracies were within ?7.43 and 7.36 for all compounds. The lower limit of quantification was 5 ng/mL for all analytes. The method, which complies with the validation criteria, was successfully applied to the analysis of PPD, MAPPD and DAPPD in human urine samples collected from clinical and postmortem cases.     

Characterization of metabolites of sweroside in rat urine using ultra‐high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry and NMR spectroscopy

Sweroside, a major active iridoid in Swertia pseudochinensis Hara, is recognized as an effective agent in the treatment of liver injury. Based on previous reports, the relatively short half‐life (64 min) and poor bioavailability (approximately 0.31%) in rats suggested that not only sweroside itself but also its metabolites could be responsible for the observed hepato‐protective effect. However, few studies have been carried out on the metabolism of sweroside. Therefore, the present study aimed at identifying the metabolites of sweroside in rat urine after a single oral dose (100 mg/kg). With ultra‐high‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (UHPLC/Q‐TOF‐MS), the metabolic profile revealed 11 metabolites in rat urine, including phase I, phase II and aglycone‐related products. The chemical structures of metabolites were proposed based on accurate mass measurements of protonated or deprotonated molecules and their fragmentation patterns. Our findings showed that the aglycone of sweroside (M05) and its glucuronide conjugate (M06) were principal circulating metabolites in rats. While several other metabolic transformations, occurring via reduction, N‐heterocyclization and N‐acetylation after deglycosylation, were also observed. Two metabolites (M05 and M06) were isolated from the rat urine for structural elucidation and identifcation of reaction sites. Both M05 and M06 were characterized by ¹H, ¹³C and two‐dimensional nuclear magnetic resonance (NMR) spectroscopy. UHPLC/Q‐TOF‐MS analysis has provided an important analytical platform to gather metabolic profile of sweroside. Copyright © 2014 John Wiley & Sons, Ltd.     

Identification of the major urinary metabolites in man of seven synthetic cannabinoids of the aminoalkylindole type present as adulterants in ‘herbal mixtures’ using LC‐MS/MS techniques

Herbal mixtures, such as ‘Spice’, containing cannabimimetic compounds are easily available on the Internet and have become increasingly popular among people having to undergo urine drug testing, as these compounds are not detected by current immunochemical tests. For analysis of urine samples, knowledge of the main metabolites is necessary as the unchanged compounds are usually not found in urine after consumption. In this paper, the identification of the major metabolites of the currently most common seven synthetic cannabinoids is presented. Urine samples from patients of psychiatric facilities known to have consumed synthetic cannabinoids were screened by LC‐MS/MS and HR‐MS/MS techniques, and the major metabolites for each of the following synthetic cannabinoids were identified by their enhanced product ion spectra and accurate mass measurement: JWH‐018, JWH‐073, JWH‐081, JWH‐122, JWH‐210, JWH‐250 and RCS‐4. The major metabolic pathway is monohydroxylation either at the N‐alkyl side chain, the naphthyl moiety or the indole moiety. In addition, metabolites with carboxylated alkyl chains were identified for some of the compounds. These results facilitate the design of urine screening methods for detecting consumption of synthetic cannabinoids. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification and characterization of fluvastatin metabolites in rats by UHPLC/Q‐TOF/MS/MS and in silico toxicological screening of the metabolites

The present study reports the in vivo and in vitro identification and characterization of metabolites of fluvastatin, the 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A reductase inhibitor, using liquid chromatography–mass spectrometry (LC–MS). In vitro studies were conducted by incubating the drug with human liver microsomes and rat liver microsomes. In vivo studies were carried out by administration of the drug in the form of suspension to the Sprague–Dawley rats followed by collection of urine, faeces and blood at different time points up to 24 h. Further, samples were prepared by optimized sample preparation method, which includes freeze liquid extraction, protein precipitation and solid phase extraction. The extracted and concentrated samples were analysed using ultrahigh‐performance liquid chromatography–quadruple time‐of‐flight tandem mass spectrometry. A total of 15 metabolites were observed in urine, which includes hydroxyl, sulphated, desisopropyl, dehydrogenated, dehydroxylated and glucuronide metabolites. A few of the metabolites were also present in faeces and plasma samples. In in vitro studies, a few metabolites were observed that were also present in in vivo samples. All the metabolites were characterized using ultrahigh‐performance liquid chromatography–quadruple time‐of‐flight tandem mass spectrometry in combination with accurate mass measurement. Finally, in silico toxicity studies indicated that some of the metabolites show or possess carcinogenicity and skin sensitization. Several metabolites that were identified in rats are proposed to have toxicological significance on the basis of in silico evaluation. However, these metabolites are of no human relevance. Copyright © 2017 John Wiley & Sons, Ltd.