phase-I Metabolites Research Articles

Unveiling the spatial distribution of aflatoxin B1 and plant defense metabolites in maize using AP-SMALDI mass spectrometry imaging.

In order to cope with the presence of unfavorable compounds, plants can biotransform xenobiotics, translocate both parent compounds and metabolites, and perform compartmentation and segregation at the cellular or tissue level. Such a scenario also applies to mycotoxins, fungal secondary metabolites with a pre-eminent role in plant infection. In this work, we aimed to describe the effect of the interplay between Zea mays (maize) and aflatoxin B1 (AFB1) at the tissue and organ level. To address this challenge, we used atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) to investigate the biotransformation, localization and subsequent effects of AFB1 on primary and secondary metabolism of healthy maize plants, both insitu and from a metabolomics standpoint. High spatial resolution (5µm) provided fine localization of AFB1, which was located within the root intercellular spaces, and co-localized with its phase-I metabolite aflatoxin M2. We provided a parallel visualization of maize metabolic changes, induced in different organs and tissues by an accumulation of AFB1. According to our untargeted metabolomics investigation, anthocyanin biosynthesis and chlorophyll metabolism in roots are most affected. The biosynthesis of these metabolites appears to be inhibited by AFB1 accumulation. On the other hand, metabolites found in above-ground organs suggest that the presence of AFB1 may also activate the biochemical response in the absence of an actual fungal infection; indeed, several plant secondary metabolites known for their antimicrobial or antioxidant activities were localized in the outer tissues, such as phenylpropanoids, benzoxazinoids, phytohormones and lipids.

Galeon: A Biologically Active Molecule with In Silico Metabolite Prediction, In Vitro Metabolic Profiling in Rat Liver Microsomes, and In Silico Binding Mechanisms with CYP450 Isoforms.

Galeon, a natural cyclic-diarylheptanoid (CDH), which was first isolated from Myrica gale L., is known to have potent cytotoxicity against A549 cell lines, anti-tubercular activity against Mycobacterium tuberculosis H37Rv, chemo-preventive potential, and moderate topoisomerase inhibitory activity. Here, in silico metabolism and toxicity prediction of galeon by CYP450, in vitro metabolic profiling study in rat liver microsomes (RLMs), and molecular interactions of galeon-CYP450 isoforms were performed. An in silico metabolic prediction study showed demethyl and mono-hydroxy galeon were the metabolites with the highest predictability. Among the predicted metabolites, mono-hydroxy galeon was found to have plausible toxicities such as skin sensitization, thyroid toxicity, chromosome damage, and carcinogenicity. An in vitro metabolism study of galeon, incubated in RLMs, revealed eighteen Phase-I metabolites, nine methoxylamine, and three glutathione conjugates. Identification of possible metabolites and confirmation of their structures were carried out using ion-trap tandem mass spectrometry. In silico docking analysis of galeon demonstrated significant interactions with active site residues of almost all CYP450 isoforms.

The Novel Psychoactive Substance Cumyl-CH-MEGACLONE: Human Phase-I Metabolism, Basic Pharmacological Characterization and Comparison to Other Synthetic Cannabinoid Receptor Agonists with a γ-Carboline-1-One Core.

Synthetic cannabinoids (SC) remain one of the largest groups of new psychoactive substances on the European drug market. In December 2018, Cumyl-CH-MEGACLONE, a novel SC based on a γ-carboline-1-one core structure, was firstly identified in Hungary and later also other European countries. This work aims to reveal the pharmacological characteristics and phase-I metabolism of Cumyl-CH-MEGACLONE and compare the data to its analogs Cumyl-PEGACLONE and 5F-Cumyl-PEGACLONE. The purified substance was characterized by means of gas chromatography-mass spectrometry (GC-MS), liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QToF-MS), attenuated total reflection infrared spectroscopy (ATR-FTIR) and nuclear magnetic resonance spectroscopy. Phase-I metabolites were identified by LC-QToF-MS analysis combined with a scheduled precursor ion list of authentic urine samples and confirmed by comparison with metabolites built in vitro by pooled human liver microsome assays. Pharmacological data were obtained in a competitive ligand binding assay and a receptor activation assay at the human cannabinoid receptor 1 (hCB1). The structure of 5-cyclohexylmethyl-2-(2-phenylpropan-2-yl)-2,5-dihydro-1H-pyrido[4,3-b]indol-1-one (semisystematic name: Cumyl-CH-MEGACLONE) was identified in a herbal blend as the main active ingredient. Investigation of phase-I biotransformation of Cumyl-CH-MEGACLONE led to three monohydroxylated metabolites (M08, M10 and M13) as reliable urinary markers for proof of consumption. At the hCB1, Cumyl-CH-MEGACLONE shows high binding affinity with Ki=1.01nM (2.5-fold higher than JWH-018), an EC50 of 1.22nM and high efficacy with EMAX=143.4% above constitutive activity of the receptor (1.13-fold higher than JWH-018). Comparison to the analogs 5F-Cumyl-PEGACLONE and Cumyl-PEGACLONE (both are hCB1 full agonists carrying a 5-fluoropentyl or pentyl chain instead of the cyclohexylmethyl moiety) suggests that Cumyl-CH-MEGACLONE is more likely to resemble the pharmacologic profile of the latter one.

A Workflow for the Identification of Mycotoxin Metabolites Using Liquid Chromatography-Ion Mobility-Mass Spectrometry.

The structural identification of phase-I and phase-II metabolites of mycotoxins is a difficult task, mostly due to the lack of standards and because of the large number of isomeric forms. Here, we describe the use of ion mobility-mass spectrometry to analyze cereal extracts and how structural information on newly discovered mycotoxins metabolites could be obtained.

Detection of the recently emerged synthetic cannabinoid 4F-MDMB-BINACA in "legal high" products and human urine specimens.

Synthetic cannabinoids (SCs) remain one of the largest groups of new psychoactive substances (NPS) on the European drug market. Although the number of new derivatives occurring on the market has dropped in the last two years, newly emerging NPS still represent a challenge for laboratories performing forensic drug analysis in biological matrices. The newly emerged SC 4F-MDMB-BINACA has been reported by several law enforcement agencies in Europe and the USA since November 2018. This work aimed at revealing urinary markers to prove uptake of 4F-MDMB-BINACA and differentiate from the use of structurally similar SCs. Phase-I metabolites detected in human urine specimens were confirmed by phase-I metabolites generated in vitro using a pooled human liver microsomes (pHLM) assay. Seized materials and test-purchased "legal high" products were analyzed by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-quadrupole-time-of-flight-mass spectrometry (LC-qToF-MS). Human urine specimens and pHLM assay extracts were measured with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and confirmed by LC-qToF-MS. In January 2019, the Institute of Legal Medicine in Erlangen (Germany) identified 4F-MDMB-BINACA in three herbal blends. During the same time period, the described SC was identified in a research chemical purchased online. Investigation of phase-I metabolism led to the metabolites M10 (ester hydrolysis) and M11 (ester hydrolysis and dehydrogenation) as reliable urinary markers. Widespread distribution on the German drug market was proven by analysis of urine samples from abstinence control programs and by frequent detection of 4F-MDMB-BINACA in "herbal blends" and "'research chemicals" purchased via the Internet.

Simultaneous determination of AM80 (tamibarotene) and WJD-A-1 in rat plasma by ultra high-performance liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study

A compound (WJD-A-1) was previously reported as a candidate prodrug of Am80 (tamibarotene), which was approved in Japan in 2005 as a therapeutic agent for recurrent refractory acute promyelocytic leukaemia. A rapid, selective and sensitive ultra high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed for the first time to simultaneously determine WJD-A-1 and its major phase-I metabolites AM80 in rat plasma. After a simple sample preparation procedure by protein precipitation with methanol and acetonitrile, WJD-A-1, AM80 and the internal standard were chromatographed on an ACQUITY UPLCTM BEH C18 column. The mobile phase consisted of methanol–0.1% formic acid (80:20, v/v) and the flow rate was 0.20 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. Each plasma sample was chromatographed within 2.6 min. The linear calibration curves for WJD-A-1 and the AM80 were obtained in the concentration range of 5.40–5.40 × 103 and 5.08–5.08 × 103 ng/mL, respectively (r ≥ 0.99). The intra- and inter-day precision (relative standard deviation, RSD) values were less than 8% and the accuracy (relative error, RE) was within ±6.8%, determined from quality control (QC) samples for the analytes. The method herein described was fully validated and successfully applied to pharmacokinetic study of WJD-A-1 following an intravenous administration of 300 μg/kg WJD-A-1 to rats.

Screening for adiponectin receptor agonists and their metabolites in urine and dried blood spots

AdipoRon and 112254 represent two known synthetic adiponectin receptor (adipoR) agonists. Although AdipoRon is the more potent compound, both have physiological properties that are similar to adiponectin - an adipokine with antidiabetic and antiatherogenic effects. Several transcriptional regulators are activated by adipoR-agonists leading to increased mitochondrial DNA content in vitro, an effect that can be abused by athletes for performance enhancing purposes. In the context of preventive anti-doping research, detection of AdipoRon and 112254 in routine doping control specimens would be valuable. Here, we describe our process for incorporating AdipoRon and 112254 into routine doping control methods involving urine and dried blood spot (DBS) analysis. Method validation including evaluation of specificity, limit of detection, identification capability, carryover, matrix interference, recovery, interday and intraday precision and linearity to standards provided by WADA. For identification in human urine, a liquid chromatography-tandem mass spectrometry-based testing approach was implemented for both adipoR agonists and two respective phase-I metabolites. Recovery of 85–104%, satisfactory limits of detection (i.e., 0.5–1ng/mL), and imprecision values over three days at three concentration levels of <19% demonstrated the assay’s fitness-for-purpose. For identification from DBS a liquid chromatography-high-resolution/high-accuracy tandem mass spectrometry with online solid-phase extraction was implemented for AdipoRon and 112254. Here also, acceptable recoveries (i.e., 22–33%), limits of detection of 5–10ng/mL, and imprecision values over three days at three concentration levels of <23%, were demonstrated. Hence, two methods for doping control screening from urine and DBS were established and shown to be fit-for-purpose for routine use.

Identification of in vitro and in vivo human metabolites of the new psychoactive substance nitracaine by liquid chromatography coupled to quadrupole time-of-flight mass spectrometry

The purpose of this work was to investigate the in vitro metabolism of nitracaine, a new psychoactive substance, using human liver microsome incubations, to evaluate the cytochrome P450 (CYP) enzyme isoforms responsible for the phase-I metabolism and to compare the information from the in vitro experiments with data resulting from an authentic user's urine sample. Accurate mass spectra of metabolites were obtained using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) and were used in the structural identification of metabolites. Two major and three minor phase-I metabolites were identified from the in vitro experiments. The observed phase-I metabolites were formed through N-deethylation, N,N-deethylation, N-hydroxylation, and de-esterification, with CYP2B6 and CYP2C19 being the main enzymes catalyzing their formation. One glucuronidated product was identified in the phase-II metabolism experiments. All of these metabolites are reported for the first time in this study except the N-deethylation product. All the in vitro metabolites except the minor N,N-deethylation product were also present in the human urine sample, thus demonstrating the reliability of the in vitro experiments in the prediction of the in vivo metabolism of nitracaine. In addition to the metabolites, three transformation products (p-nitrobenzoic acid, p-aminobenzoic acid, and 3-(diethylamino)-2,2-dimethylpropan-1-ol) were identified, as well as several glucuronides and glutamine derived of them.

Identification of selected in vitro generated phase-I metabolites of the steroidal selective androgen receptor modulator MK-0773 for doping control purposes.

Research into developing anabolic agents for various therapeutic purposes has been pursued for decades. As the clinical utility of anabolic-androgenic steroids has been found to be limited because of their lack of tissue selectivity and associated off-target effects, alternative drug entities have been designed and are commonly referred to as selective androgen receptor modulators (SARMs). While most of these SARMs are of nonsteroidal structure, the drug candidate MK-0773 comprises a 4-aza-steroidal nucleus. Besides the intended therapeutic use, SARMs have been found to be illicitly distributed and misused as doping agents in sport, necessitating frequently updated doping control analytical assays. As steroidal compounds reportedly undergo considerable metabolic transformations, the phase-I metabolism of MK-0773 was simulated using human liver microsomal (HLM) preparations and electrochemical conversion. Subsequently, major metabolic products were identified and characterized employing liquid chromatography-high-resolution/high- accuracy tandem mass spectrometry with electrospray (ESI) and atmospheric pressure chemical ionization (APCI) as well as nuclear magnetic resonance (NMR) spectroscopy. MK-0773 produced numerous phase-I metabolites under the chosen in vitro incubation reactions, mostly resulting from mono- and bisoxygenation of the steroid. HLM yielded at least 10 monooxygenated species, while electrochemistry-based experiments resulted predominantly in three monohydroxylated metabolites. Elemental composition data and product ion mass spectra were generated for these analytes, ESI/APCI measurements corroborated the formation of at least two N-oxygenated metabolites, and NMR data obtained from electrochemistry-derived products supported structures suggested for three monohydroxylated compounds. Hereby, the hydroxylation of the A-ring located N- bound methyl group was found to be of particular intensity. In the absence of controlled elimination studies, the produced information enables the implementation of new target analytes into routine doping controls and expands the focus of anti-doping efforts concerning this new anabolic agent.

Identification and characterization of in vitro and in vivo generated metabolites of the adiponectin receptor agonists AdipoRon and 112254

Peroxisome proliferator-activated receptors (PPARs), peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), sirtuin 1 (SIRT1) and adenosine monophosphate-activated protein kinase (AMPK) are regulators of transcriptional processes and effects of exercise and pseudo-exercise situations. Compounds occasionally referred to as endurance exercise mimetics such as AdipoRon and 112254, both adiponectin receptor agonists, can be used to simulate the physiology of endurance exercise via pathways including these transcriptional regulators. Adiponectin supports fatty acid utilization and triglyceride-content reduction in cells and increases both the mitochondrial biogenesis and the oxidative metabolism in muscle cells. In routine doping control analysis, knowledge about phase-I and -II metabolic products of target analytes is essential. Hence, in vitro- and in vivo-metabolism experiments are frequently employed tools in preventive doping research to determine potential urinary metabolites for sports drug testing purposes, especially concerning new, (yet) unapproved compounds. In the present study, in vitro assays were conducted using human liver microsomal and S9 fractions, and rat in vivo experiments were performed using both AdipoRon and 112254. For AdipoRon, obtained samples were analyzed using liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry with both electrospray ionization or atmospheric-pressure chemical ionization techniques. Overall, more than five phase I-metabolites were found in vitro and in vivo, including particularly monohydroxylated and hydrogenated species. No phase II-metabolites were found in vitro; conversely, signals suggesting the presence of glucuronic acid or other conjugates in samples collected from in vivo experiment were observed, the structures of which were however not conclusively identified. Also for 112254, several phase-I metabolites were found in vitro, e.g. monohydroxylated and demethylated species. Here, no phase II-metabolites were observed neither using in vitro nor in vivo samples. Based on the generated data, the implementation of metabolites and unmodified drug candidates into routine doping control protocols is deemed warranted for comprehensive sports drug testing programs until human elimination study data are available.

Use of genetically manipulated Salmonella typhimurium strains to evaluate the role of human sulfotransferases in the bioactivation of nitro- and aminotoluenes.

Various nitro- and aminotoluenes demonstrated carcinogenic activity in rodent studies, but were inactive or weakly active in conventional in vitro mutagenicity assays. Standard in vitro tests do not take into account activation by certain classes of enzymes. This is true in particular for sulfotransferases (SULTs). These enzymes may convert aromatic hydroxylamines and benzylic alcohols, two major classes of phase-I metabolites of nitro- and aminotoluenes, to reactive esters. Here it is shown that expression of certain human SULTs in Salmonella typhimurium TA1538 or TA100 strongly enhanced the mutagenicity of various nitrotoluenes and nitro- and amino-substituted benzyl alcohols. Human SULT1A1, SULT1A2, and SULT1C2 showed the strongest activation. The observation that some nitrotoluenes as well as some aminobenzyl alcohols were activated by SULTs in the absence of cytochromes P450 implies that mutagenic sulfuric esters were formed at both the exocyclic nitrogen and the benzylic carbon, respectively. Nitroreductase deficiency (using strain YG7131 instead of TA1538 for SULT1A1 expression) did not affect the SULT-dependent mutagenicity of 1-hydroxymethylpyrene (containing no nitro group), moderately enhanced that of 2-amino-4-nitrobenzyl alcohol, and drastically attenuated the effects of nitrobenzyl alcohols without other substituents. The last finding suggests that either activation occurred at the hydroxylamino group formed by nitroreductase or the nitro group (having a strong -M effect) had to be reduced to an electron-donating substituent to enhance the reactivity of the benzylic sulfuric esters. The results pointed to an important role of SULTs in the genotoxicity of nitrotoluenes and alkylated anilines. Activation occurs at nitrogen functions as well as benzylic positions.

Emerging drugs affecting skeletal muscle function and mitochondrial biogenesis - Potential implications for sports drug testing programs.

A plethora of compounds potentially leading to drug candidates that affect skeletal muscle function and, more specifically, mitochondrial biogenesis, has been under (pre)clinical investigation for rare as well as more common diseases. Some of these compounds could be the object of misuse by athletes aiming at artificial and/or illicit and drug-facilitated performance enhancement, necessitating preventive and proactive anti-doping measures. Early warnings and the continuous retrieval and dissemination of information are crucial for sports drug testing laboratories as well as anti-doping authorities, as they assist in preparation of efficient doping control analytical strategies for potential future threats arising from new therapeutic developments. Scientific literature represents the main source of information, which yielded the herein discussed substances and therapeutic targets, which might become relevant for doping controls in the future. Where available, mass spectrometric data are presented, supporting the development of analytical strategies and characterization of compounds possibly identified in human sports drug testing samples. Focusing on skeletal muscle and mitochondrial biogenesis, numerous substances exhibiting agonistic or antagonistic actions on different cellular 'control centers' resulting in increased skeletal muscle mass, enhanced performance (as determined with laboratory animal models), and/or elevated amounts of mitochondria have been described. Substances of interest include agonists for REV-ERBα (e.g. SR9009, SR9011, SR10067, GSK4112), sirtuin 1 (e.g. SRT1720, SRT2104), adenosine monophosphate-activated protein kinase (AMPK, e.g. AICAR), peroxisome proliferator-activated receptor (PPAR)δ (e.g. GW1516, GW0742, L165041), and inhibitory/antagonistic agents targeting the methionine-folate cycle (MOTS-c), the general control non-derepressible 5 (GCN5) acetyl transferase (e.g. CPTH2, MB-3), myostatin (e.g. MYO-029), the myostatin receptor (bimagrumab), and myostatin receptor ligands (e.g. sotatercept, ACE-031). In addition, potentially relevant drug targets were identified, e.g. with the sarcoplasmic transmembrane peptide myoregulin and the nuclear receptor corepressor 1 (NCOR-1). The antagonism of these has shown to result in substantially enhanced physical performance in animals, necessitating the monitoring of strategies such as RNA interference regarding these substances. Most drug candidates are of lower molecular mass and comprise non-natural compositions, facts which suggest approaches for their qualitative identification in doping control samples by mass spectrometry. Electrospray ionization/collision-induced dissociation mass spectra of representatives of the aforementioned substances and selected in vitro derived phase-I metabolites support this assumption, and test methods for a subset of these have been recently established. Expanding the knowledge on analytical data will further facilitate the identification of such analytes and related compounds in confiscated material as well as sports drug testing specimens.

Determination of metabolic profile of novel triethylamine containing thiophene S006-830 in rat, rabbit, dog and human liver microsomes.

CDRI S006-830 is a potent triethylamine containing thiophene antitubercular compound of the Central Drug Research Institute, India. The present study aimed to conduct comprehensive metabolic investigations of CDRI S006-830 to corroborate its preclinical investigations. Preliminary metabolic investigations were performed to assess the metabolic stability, enzyme kinetics, reaction phenotyping, and metabolite identification of CDRI S006-830 in rat, rabbit, dog, and human liver microsomes using liquid chromatography with mass spectrometry. The observed in vitro t1/2 and Clint values were 9.9 ± 1.29, 4.5 ± 0.52, 4.5 ± 0.86, 17 ± 5.21 min and 69.60 ± 8.37, 152.0 ± 17.26, 152.34 ± 27.63, 33.62 ± 21.04 μL/min/mg in rat, rabbit, dog and human liver microsomes respectively. These observations suggested that CDRI S006-830 rapidly metabolized in the presence of NADPH in liver microsomes of rat, rabbit and dog while moderately metabolized in human liver microsomes. It was observed that CDRI S006-830 exhibited monophasic Michaelis-Menten kinetics. The metabolism of CDRI S006-830 was primarily mediated by CYP3A4 and was deduced by CYP reaction phenotyping with known potent inhibitors. CYP3A4 involvement was also confirmed by cDNA-expressed recombinant human isozyme activity with different CYPs. Four major phase-I metabolites of S006-830, (M-1 to M-4) were detected in rat, rabbit, dog (except M4) and human liver microsomes.

Mitochondrial toxicity of diclofenac and its metabolites via inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria: Possible role in drug induced liver injury (DILI)

Diclofenac is a widely prescribed NSAID, which by itself and its reactive metabolites (Phase-I and Phase-II) may be involved in serious idiosyncratic hepatotoxicity. Mitochondrial injury is one of the mechanisms of drug induced liver injury (DILI). In the present work, an investigation of the inhibitory effects of diclofenac (Dic) and its phase I [4-hydroxy diclofenac (4′-OH-Dic) and 5-hydroxy diclofenac (5-OH-dic)] and Phase-II [diclofenac acyl glucuronide (DicGluA) and diclofenac glutathione thioester (DicSG)] metabolites, on ATP synthesis in rat liver mitochondria was carried out. A mechanism based inhibition of ATP synthesis is exerted by diclofenac and its metabolites. Phase-I metabolite (4′-OH-Dic) and Phase-II metabolites (DicGluA and DicSG) showed potent inhibition (2–5 fold) of ATP synthesis, where as 5-OH-Dic, one of the Phase-I metabolite, was a less potent inhibitor as compared to Dic. The calculated kinetic constants of mechanism based inhibition of ATP synthesis by Dic showed maximal rate of inactivation (Kinact) of 2.64±0.15min−1 and half maximal rate of inactivation (KI) of 7.69±2.48μM with Kinact/KI ratio of 0.343min−1μM−1. Co-incubation of mitochondria with Dic and reduced GSH exhibited a protective effect on Dic mediated inhibition of ATP synthesis. Our data from this study strongly indicate that Dic as well as its metabolites could be involved in the hepato-toxic action through inhibition of ATP synthesis.

Quantification of trantinterol, its two metabolites and their primary conjugated metabolites in human plasma by ultra-high-performance liquid chromatography- tandem mass spectrometry and its application to a pharmacokinetic study

A highly rapid, selective and sensitive ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed to simultaneously determine trantinterol, its major phase-I metabolites and their primary conjugated metabolites in human plasma. Waters Oasis HLB C18 solid phase extraction cartridges were used in the sample preparation. The separation was carried out on an ACQUITY UPLC™ BEH C18 column with methanol/0.2% formic acid (30:70, v/v) as the mobile phase at a flow rate of 0.25mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in selective reaction monitoring (SRM) mode with the use of an electrospray ionization (ESI) source. The linear calibration curves for trantinterol, tert-butyl hydroxylated trantinterol (tert-OH-trantinterol) and 1-carbonyl trantinterol (trantinterol-COOH) were obtained in the concentration ranges of 0.200–250, 0.108–4.00 and 0.0840–5.02ng/mL, respectively (r2≥0.99). The intra- and inter-day precision (relative standard deviation, RSD) values were less than 13%, and the accuracy (relative error, RE) was within ±9.9%, as determined from quality control (QC) samples for the analytes. The concentrations of conjugated forms of trantinterol and tert-OH- trantinterol in plasma were determined using selective enzyme hydrolysis. The method described herein was fully validated and successfully applied for the pharmacokinetic study of trantinterol in healthy volunteers after oral administration.

Determination of metabolic profile of novel triethylamine containing thiophene S006-830 in rat, rabbit, dog and human liver microsomes.

CDRI S006-830 is a potent triethylamine containing thiophene antitubercular compound of the Central Drug Research Institute, India. The present study aimed to conduct comprehensive metabolic investigations of CDRI S006-830 to corroborate its preclinical investigations. Preliminary metabolic investigations were performed to assess the metabolic stability, enzyme kinetics, reaction phenotyping, and metabolite identification of CDRI S006-830 in rat, rabbit, dog, and human liver microsomes using liquid chromatography with mass spectrometry. The observed in vitro t1/2 and Clint values were 9.9 ± 1.29, 4.5 ± 0.52, 4.5 ± 0.86, 17 ± 5.21 min and 69.60 ± 8.37, 152.0 ± 17.26, 152.34 ± 27.63, 33.62 ± 21.04 μL/min/mg in rat, rabbit, dog and human liver microsomes respectively. These observations suggested that CDRI S006-830 rapidly metabolized in the presence of NADPH in liver microsomes of rat, rabbit and dog while moderately metabolized in human liver microsomes. It was observed that CDRI S006-830 exhibited monophasic Michaelis-Menten kinetics. The metabolism of CDRI S006-830 was primarily mediated by CYP3A4 and was deduced by CYP reaction phenotyping with known potent inhibitors. CYP3A4 involvement was also confirmed by cDNA-expressed recombinant human isozyme activity with different CYPs. Four major phase-I metabolites of S006-830, (M-1 to M-4) were detected in rat, rabbit, dog (except M4) and human liver microsomes.

Characterization of a non-approved selective androgen receptor modulator drug candidate sold via the Internet and identification of in vitro generated phase-I metabolites for human sports drug testing.

Potentially performance-enhancing agents, particularly anabolic agents, are advertised and distributed by Internet-based suppliers to a substantial extent. Among these anabolic agents, a substance referred to as LGD-4033 has been made available, comprising the core structure of a class of selective androgen receptor modulators (SARMs). In order to provide comprehensive analytical data for doping controls, the substance was obtained and characterized by nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography/electrospray ionization high resolution/high accuracy tandem mass spectrometry (LC/ESI-HRMS). Following the identification of 4-(2-(2,2,2-trifluoro-1-hydroxyethyl)pyrrolidin-1-yl)-2-(trifluoromethyl)benzonitrile, the substance was subjected to in vitro metabolism studies employing human liver microsomes and Cunninghamella elegans (C. elegans) preparations as well as electrochemical metabolism simulations. By means of LC/ESI-HRMS, five main phase-I metabolites were identified as products of liver microsomal preparations including three monohydroxylated and two bishydroxylated species. The two most abundant metabolites (one mono- and one bishydroxylated product) were structurally confirmed by LC/ESI-HRMS and NMR. Comparing the metabolic conversion of 4-(2-(2,2,2-trifluoro-1-hydroxyethyl)pyrrolidin-1-yl)-2-(trifluoromethyl)benzonitrile observed in human liver microsomes with C. elegans and electrochemically derived metabolites, one monohydroxylated product was found to be predominantly formed in all three methodologies. The implementation of the intact SARM-like compound and its presumed urinary phase-I metabolites into routine doping controls is suggested to expand and complement existing sports drug testing methods.

Urinary excretion and metabolism of the α-pyrrolidinophenone designer drug 1-phenyl-2-(pyrrolidin-1-yl)octan-1-one (PV9) in humans

1-Phenyl-2-(pyrrolidin-1-yl)octan-1-one (PV9) and 16 metabolites, including diastereomers and conjugates, were identified or tentatively detected in human urine by gas chromatography–mass spectrometry and liquid chromatography–high-resolution tandem mass spectrometry. These urinary metabolites indicated that the metabolic pathways of PV9 include: (1) the reduction of ketone groups to their corresponding alcohols; (2) oxidation of the pyrrolidine ring to the corresponding pyrrolidone; (3) aliphatic oxidation of the terminal carbon atom to the corresponding carboxylate form, possibly through an alcohol intermediate (not detected); and (4) hydroxylation at the penultimate carbon atom to the corresponding alcohols followed by further oxidation to ketones, and combinations of these steps. In addition, results from the quantitative analyses of five phase-I metabolites using newly synthesized authentic standards suggested that the main metabolic pathway includes the aliphatic oxidation of terminal and/or penultimate carbons. Human metabolism of PV9 differed significantly from those of α-pyrrolidinovalerophenone and α-pyrrolidinobutiophenone, suggesting that the main metabolic pathways of α-pyrrolidinophenones significantly change depending on the alkyl chain length of the parent molecule.

In vitro metabolism studies on the selective androgen receptor modulator (SARM) LG121071 and its implementation into human doping controls using liquid chromatography-mass spectrometry.

LG121071 is a member of the tetrahydroquinolinone-based class of selective androgen receptor modulator (SARM) drug candidates. These nonsteroidal compounds are supposed to act as full anabolic agents with reduced androgenic properties. As SARMs provide an alternative to anabolic androgenic steroids, they represent an emerging class of potential doping substances abused by athletes for illicit performance enhancement. According to the World Anti-Doping Agency's regulations, SARMs are banned substances and part of the Prohibited List since 2008. In consideration of the increasing number of adverse analytical findings in doping controls caused by SARMs abuse, potential drug candidates such as LG121071 have been proactively investigated to enable a timely integration into routine testing procedures even though clinical trials are not yet complete. In the present approach, the collision-induced dissociation (CID) of LG121071 was characterized by means of electrospray ionization-high resolution/high accuracy mass spectrometry, MS(n), and isotope labeling experiments. Interestingly, the even-electron precursor ion [M + H](+) at m/z297 was found to produce a radical cation at m/z268 under CID conditions, violating the even-electron rule that commonly applies. For doping control purposes, metabolites were generated invitro and a detection method for urine samples based on liquid chromatography-tandem mass spectrometry was established. The overall metabolic conversion of LG121071 was modest, yielding primarily mono-, bis- and trishydroxylated species. Notable, however, was the identification of a glucuronic acid conjugate of the intact drug, attributed to an N-glucuronide structure. The sample preparation procedure included the enzymatic hydrolysis of glucuronides prior to liquid-liquid extraction, allowing intact LG121071 to be measured, as well as the corresponding phase-I metabolites. The method was characterized concerning inter alia lower limit of detection (0.5 ng mL(-1) in urine), recovery (40%), and intra-/interday precision (2.3% to 11.7%) to assess its fitness for purpose. Prospectively, the assay can serve as detection method for LG121071 in drug testing and/or doping controls.

In vitro metabolism of 2-ethylhexyldiphenyl phosphate (EHDPHP) by human liver microsomes

2-ethylhexyl diphenyl phosphate (EHDPHP) is used as flame retardant and plasticizer additive in a variety of consumer products. Since EHDPHP is toxic to aquatic organisms and has been detected in environmental samples, concerns about human exposure and toxicity are emerging. With the aim of identifying human-specific metabolites, the biotransformation of EHDPHP was investigated using human liver microsomes. Using an in silico program (Meteor) for the prediction of metabolites, untargeted screening tools (agilent Mass Hunter) and a suitable analysis platform based on ultra-high performance liquid chromatography (UPLC) and quadrupole time-of-flight high resolution mass spectrometer (QTOF-MS), for the first time a wide variety of phases-I and II metabolites of EHDPHP were identified. Mono- and di-hydroxylated metabolites, keto metabolites, mixed keto and hydroxylated metabolites and diphenyl phosphate were the major phase-I metabolites of EHDPHP. Glucuronidated metabolites of phase-I metabolites of EHDPHP were also formed by human liver microsomes. Using these results, we propose a general metabolism pathway for EHDPHP in humans and a number of candidate biomarkers for assessing the human exposure to this ubiquitous phosphate flame retardant and plasticizer in future biomonitoring studies. Furthermore, we provide a template analytical approach based on the combination of untargeted and targeted screening and UPLC–QTOF-MS analysis suitable for use in future metabolism studies.