Identification and structural elucidation of in vitro metabolites of atazanavir by HPLC and tandem mass spectrometry

Atazanavir (marketed as Reyataz®) is an important member of the human immunodeficiency virus protease inhibitor class. LC‐UV‐MSⁿ experiments were designed to identify metabolites of atazanavir after incubations in human hepatocytes. Five major (M1–M5) and seven minor (M7–M12) metabolites were identified. The most abundant metabolite, M1, was formed by a mono‐oxidation on the t‐butyl group at the non‐prime side. The second most abundant metabolite, M2, was also a mono‐oxidation product, which has not yet been definitively identified. Metabolites, M3 and M4, were structural isomers, which were apparently formed by oxidative carbamate hydrolysis. The structure of M5 comprises the non‐prime side of atazanavir which contains a pyridinyl‐benzyl group. Metabolite M6a was formed by the cleavage of the pyridinyl‐benzyl side chain, as evidenced by the formation of the corresponding metabolic product, the pyridinyl‐benzoic acid (M6b). Mono‐oxidation also occurred on the pyridinyl‐benzyl group to produce the low abundance metabolite M8. Oxidation of the terminal methyl groups produced M9 and M10, respectively, which have low chemical stability. Trace‐level metabolites of di‐oxidations, M11 and M12, were also detected, but the complexity of the molecule precluded identification of the second oxidation site. To our knowledge, metabolites M6b and M8 have not been reported. Copyright © 2013 John Wiley & Sons, Ltd.     

Metabolite identification of the antimalarial piperaquine in vivo using liquid chromatography–high‐resolution mass spectrometry in combination with multiple data‐mining tools in tandem

Artemisinin‐based combination therapy is widely used for the treatment of uncomplicated Plasmodium falciparum malaria, and piperaquine (PQ) is one of important partner drugs. The pharmacokinetics of PQ is characterized by a low clearance and a large volume of distribution; however, metabolism of PQ has not been thoroughly investigated. In this work, the metabolite profiling of PQ in human and rat was studied using liquid chromatography tandem high‐resolution LTQ‐Orbitrap mass spectrometry (HRMS). The biological samples were pretreated by solid‐phase extraction. Data processes were carried out using multiple data‐mining techniques in tandem, i.e., isotope pattern filter followed by mass defect filter. A total of six metabolites (M1–M6) were identified for PQ in human (plasma and urine) and rat (plasma, urine and bile). Three reported metabolites were also found in this study, which included N‐oxidation (M1, M2) and carboxylic products (M3). The subsequent N‐oxidation of M3 resulted in a new metabolite M4 detected in urine and bile samples. A new metabolic pathway N‐dealkylation was found for PQ in human and rat, leading to two new metabolites (M5 and M6). This study demonstrated that LC‐HRMSⁿ in combination with multiple data‐mining techniques in tandem can be a valuable analytical strategy for rapid metabolite profiling of drugs. Copyright © 2016 John Wiley & Sons, Ltd.     

Elucidation of the in vitro metabolic profile of stable isotope labeled BAL19403 by accurate mass capillary liquid chromatography/quadrupole time-of-flight mass spectrometry and isotope exchange

The in vitro metabolic pattern of BAL19403, a novel macrolide antibiotic, was investigated by capillary liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/QTOF-MS) in incubations with human microsomes. For the elucidation of the metabolic pathway, BAL19403 labeled with four deuterium atoms (D4) was used, and detection of metabolites performed using mixtures of the unlabeled (H4) BAL19403 and its D4 analogue (1:1) as substrate. All metabolites appeared with similar chromatographic behavior. MS/MS spectra of BAL19403 and its metabolites are dominated by non-informative fragment ions. Therefore, the structure of the metabolites was elucidated mainly by accurate mass measurements with subsequent proposals of elemental compositions. Main biotransformations were N-demethylation, lactone ring hydrolysis, and oxidation. Additionally, N-dealkylation of the aromatic moiety was identified. This dealkylation results not only in formation of an aldehyde, according to the classical pathway, but also in formation of the corresponding alcohol and carboxylic acid. Final elucidation of their structures was possible, since this dealkylation takes place vicinal to the deuterium-labeled part of BAL19403 and interferes with D/H exchange. The degree of D/H exchange, determined by analysis of the metabolite isotopic pattern, was used to elucidate the adjacent functional group.     

In vitro metabolism of β‐lapachone (ARQ 501) in mammalian hepatocytes and cultured human cells

ARQ 501 (3,4‐dihydro‐2,2‐dimethyl‐2H‐naphthol[1,2‐b]pyran‐5,6‐dione, β‐lapachone) is an anticancer agent, currently in multiple phase II clinical trials as monotherapy and in combination with other cytotoxic drugs. This study focuses on in vitro metabolism in cryopreserved hepatocytes from mice, rats, dogs and humans using [¹⁴C]‐labeled ARQ 501. Metabolite profiles were characterized using liquid chromatography/mass spectrometry combined with an accurate radioactivity counter. Ion trap mass spectrometry was employed for further structural elucidation. A total of twelve metabolites were detected in the mammalian hepatocytes studied; all of which but one were generated from phase II conjugation reactions. Ten of the observed metabolites were produced by conjugations occurring at the reduced ortho‐quinone carbonyl groups of ARQ 501. The metabolite profiles revealed that glucuronidation was the major biotransformation pathway in mouse and human hepatocytes. Monosulfation was the major pathway in dog, while, in rat, it appears glucuronidation and sulfation pathways contributed equally. Three major metabolites were found in rats: monoglucuronide M1, monosulfate M6, and glucuronide‐sulfate M9. Two types of diconjugation metabolites were formed by attachment of the second glycone to an adjacent hydroxyl or to an existing glycone. Of the diconjugation metabolites, glucosylsulfate M10, diglucuronide M5, and glucuronide‐glucoside M11 represent rarely observed phase II metabolites in mammals. The only unconjugated metabolite was generated through hydrolysis and was observed in rat, dog and human hepatocytes. ARQ 501 appeared less stable in human hepatocytes than in those of other species. To further elucidate the metabolism of ARQ 501 in extrahepatic sites, its metabolism in human kidney, lung and intestine cells was also studied, and only monoglucuronide M1 was observed in all the cell types examined. Copyright © 2008 John Wiley & Sons, Ltd.     

Comparison of sulfo‐conjugated and gluco‐conjugated urinary metabolites for detection of methenolone misuse in doping control by LC‐HRMS,GC‐MS and GC‐HRMS

Methenolone (17β‐hydroxy‐1‐methyl‐5α‐androst‐1‐en‐3‐one) misuse in doping control is commonly detected by monitoring the parent molecule and its metabolite (1‐methylene‐5α‐androstan‐3α‐ol‐17‐one) excreted conjugated with glucuronic acid using gas chromatography‐mass spectrometry (GC‐MS) and liquid chromatography mass spectrometry (LC‐MS) for the parent molecule, after hydrolysis with β‐glucuronidase. The aim of the present study was the evaluation of the sulfate fraction of methenolone metabolism by LC‐high resolution (HR)MS and the estimation of the long‐term detectability of its sulfate metabolites analyzed by liquid chromatography tandem mass spectrometry (LC‐HRMSMS) compared with the current practice for the detection of methenolone misuse used by the anti‐doping laboratories. Methenolone was administered to two healthy male volunteers, and urine samples were collected up to 12 and 26 days, respectively. Ethyl acetate extraction at weak alkaline pH was performed and then the sulfate conjugates were analyzed by LC‐HRMS using electrospray ionization in negative mode searching for [M‐H]^? ions corresponding to potential sulfate structures (comprising structure alterations such as hydroxylations, oxidations, reductions and combinations of them). Eight sulfate metabolites were finally detected, but four of them were considered important as the most abundant and long term detectable. LC clean up followed by solvolysis and GC/MS analysis of trimethylsilylated (TMS) derivatives reveal that the sulfate analogs of methenolone as well as of 1‐methylene‐5α‐androstan‐3α‐ol‐17‐one, 3z‐hydroxy‐1β‐methyl‐5α‐androstan‐17‐one and 16β‐hydroxy‐1‐methyl‐5α‐androst‐1‐ene‐3,17‐dione were the major metabolites in the sulfate fraction. The results of the present study also document for the first time the methenolone sulfate as well as the 3z‐hydroxy‐1β‐methyl‐5α‐androstan‐17‐one sulfate as metabolites of methenolone in human urine. The time window for the detectability of methenolone sulfate metabolites by LC‐HRMS is comparable with that of their hydrolyzed glucuronide analogs analyzed by GC‐MS. The results of the study demonstrate the importance of sulfation as a phase II metabolic pathway for methenolone metabolism, proposing four metabolites as significant components of the sulfate fraction. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterization of the metabolite of AdipoRon in rat and human liver microsomes by ultra‐high‐performance liquid chromatography combined with Q‐Exactive Orbitrap tandem mass spectrometry

AdipoRon is an orally active adiponectin receptor agonist. The aim of this study was to characterize the metabolites of AdipoRon in rat and human liver microsomes using ultra‐high performance liquid chromatography combined with Q‐Exactive Orbitrap tandem mass spectrometry (UPLC‐Q‐Exactive‐Orbitrap‐MS) together with data processing techniques including extracted ion chromatograms and a mass defect filter. AdipoRon (10 μm ) was incubated with liver microsomes in the presence of NADPH and this resulted in a total of 11 metabolites being detected. The identities of these metabolites were characterized by comparing their accurate masses and fragment ions as well as their retention times with those of AdipoRon using MetWorks software. Metabolites M1–M3, M6, and M8–M11 were identified for the first time. Metabolite M4, the major metabolite both in rat and human liver microsomes, was further confirmed using the reference standard. Our results revealed that the metabolic pathways of AdipoRon in liver microsomes were N‐dealkylation (M2), hydroxylation (M, M5–M9), carbonyl reduction (M4) and the formation of amide (M10 and M11). Our results provide valuable information about the in vitro metabolism of AdipoRon, which would be helpful for us to understand the mechanism of the elimination of AdipoRon and, in turn, its effectiveness and toxicity.     

Two‐injection workflow for a liquid chromatography/LTQ‐Orbitrap system to complete in vivo biotransformation characterization: demonstration with buspirone metabolite identification

The relatively high background matrix in in vivo samples typically poses difficulties in drug metabolite identification, and causes repeated analytical runs on unit resolution liquid chromatography/mass spectrometry (LC/MS) systems before the completion of biotransformation characterization. Ballpark parameter settings for the LTQ‐Orbitrap are reported herein that enable complete in vivo metabolite identification within two HPLC/MS injections on the hybrid LTQ‐Orbitrap data collection system. By setting the FT survey full scan at 60K resolution to trigger five dependent LTQ MS² scans, and proper parameters of Repeat Duration, Exclusion Duration and Repeat Count for the first run (exploratory), the Orbitrap achieved the optimal parallel data acquisition capability and collected maximum number of product ion scans. Biotransformation knowledge based prediction played the key role in exact mass ion extraction and multiple mass defect filtration when the initial data was processed. Meanwhile, product ion extraction and neutral loss extraction of the initial dependent data provided additional bonus in identifying metabolites. With updated parent mass list and the data‐dependent setting to let only the ions on the parent mass list trigger dependent scans, the second run (confirmatory) ensures that all precursor ions of identified metabolites trigger not only dependent product ion scans, but also at or close to the highest concentration of the eluted metabolite peaks. This workflow has been developed for metabolite identification of in vivo or ADME studies, of which the samples typically contain a high level of complex matrix. However, due to the proprietary nature of the in vivo studies, this workflow is presented herein with in vitro buspirone sample incubated with human liver microsomes (HLM). The major HLM‐mediated biotransformation on buspirone was identified as oxidation or hydroxylation since five mono‐ (+16 Da), seven di‐ (+32 Da) and at least three tri‐oxygenated (+48 Da) metabolites were identified. Besides the metabolites 1‐pyrimidinylpiperazine (1‐PP) and hydroxylated 1‐PP that formed by N‐dealkylation, a new metabolite M308 was identified as the result of a second N‐dealkylation of the pyrimidine unit. Two new metabolites containing the 8‐butyl‐8‐azaspiro[4,5]decane‐7,9‐dione partial structure, M240 and M254, were also identified that were formed apparently due to the first N‐dealkylation of the 1‐PP moiety. Copyright © 2009 John Wiley & Sons, Ltd.     

Validation and application of a liquid chromatography-tandem mass spectrometric method for the determination of GDC-0834 and its metabolite in human plasma using semi-automated 96-well protein precipitation

A liquid chromatographic–tandem mass spectrometric (LC‐MS/MS) method was developed and validated for the determination of GDC‐0834 and its amide hydrolysis metabolite (M1) in human plasma to support clinical development. The method consisted of semi‐automated 96‐well protein precipitation extraction for sample preparation and LC‐MS/MS analysis in positive ion mode using TurboIonSpray® for analysis. D6‐GDC‐0834 and D6‐M1 metabolite were used as internal standards. A linear regression (weighted 1/concentration²) was used to fit calibration curves over the concentration range of 1 – 500 ng/mL for both GDC‐0834 and M1 metabolite. The accuracy (percentage bias) at the lower limit of quantitation (LLOQ) was 5.20 and 0.100% for GDC‐0834 and M1 metabolite, respectively. The precision (CV) for samples at the LLOQ was 3.13–8.84 and 5.20–8.93% for GDC‐0834 and M1 metabolite, respectively. For quality control samples at 3, 200 and 400 ng/mL, the between‐run CV was ≤7.38% for GDC‐0834 and ≤8.20% for M1 metabolite. Between run percentage bias ranged from ?2.76 to 6.98% for GDC‐0834 and from ?6.73 to 2.21% for M1 metabolite. GDC‐0834 and M1 metabolite were stable in human plasma for 31 days at ?20 and ?70°C. This method was successfully applied to support a GDC‐0834 human pharmacokinetic‐based study. Copyright © 2012 John Wiley & Sons, Ltd.     

The use of 18O‐exchange and base‐catalyzed N‐dealkylation with liquid chromatography/tandem mass spectrometry to identify carbinolamide metabolites

Oxidation of N‐alkyl‐substituted amides is a common transformation observed in metabolism studies of drugs and other chemicals. Metabolism at the alpha carbon atom can produce stable carbinolamide compounds, which may be abundant enough to require complete confidence in structural assignments. In a drug discovery setting, rapid structural elucidation of test compounds is critical to inform the compound selection process. Traditional approaches to the analysis of carbinolamides have relied upon the time‐consuming synthesis of authentic standards or purification of large enough quantities for characterization by nuclear magnetic resonance (NMR). We describe a simple technique used in conjunction with liquid chromatography/tandem mass spectrometry (LC/MS/MS) which demonstrates the chemical identity of a carbinolamide by its distinctive ability to reversibly exchange [¹⁸O]water through an imine intermediate. A key advantage of the technique is that the chromatographic retention times of metabolites are preserved, allowing direct comparisons of mass chromatograms from non‐treated and [¹⁸O]water‐treated samples. Metabolites susceptible to the treatment are clearly indicated by the addition of 2 mass units to their original mass. An additional test which can be used in conjunction with ¹⁸O‐exchange is base‐catalyzed N‐dealkylation of N‐(α‐hydroxy)alkyl compounds. The use of the technique is described for carbinolamide metabolites of dirlotapide, loperamide, and a proprietary compound. Copyright © 2010 John Wiley & Sons, Ltd.     

Challenges in the indirect quantitation of acyl‐glucuronide metabolites of a cardiovascular drug from complex biological mixtures in the absence of reference standards

This paper describes the quantitation of acyl‐glucuronide metabolites (M26 and M5) of a cardiovascular‐drug (torcetrapib) from monkey urine, in the absence of their reference standards. LC/MS/MS assays for M1 and M4 (aglycones of M26 and M5, respectively) were characterized from normal and base‐treated urine, as their respective reference standards were available. The in vivo study samples containing M26 and M5 were treated with 1 n sodium hydroxide to hydrolyze them to their respective aglycones. The study samples were assayed for M1 and M4 before and after alkaline hydrolysis and the difference in the concentrations provided an estimate of the urinary levels of M26 and M5. Prior to the main sample analysis, conditions for alkaline hydrolysis of the glucuronides were optimized by incubating pooled study samples. During incubations, a prolonged increase in M4 levels over time was observed, which is inconsistent with the base‐hydrolysis of an acyl‐glucuronide (expected to hydrolyze rapidly). Possible interference of the metabolite M9 (an ether‐glucuronide metabolite isobaric to M4) was investigated to explain this observation using chromatographic and wet‐chemistry approaches. The strategies adopted herein established that the LC/MS/MS assay and our approach were reliable. The metabolite exposure was then correlated to toxicological observations to gain initial insights into the physiological role of these metabolites. Copyright © 2009 John Wiley & Sons, Ltd.     

UPLC‐HR‐MS/MS‐based determination study on the metabolism of four synthetic cannabinoids,ADB‐FUBICA,AB‐FUBICA,AB‐BICA and ADB‐BICA,by human liver microsomes

Since 2012, several cannabimimetic indazole and indole derivatives with valine amino acid amide residue have emerged in the illicit drug market, and have gradually replaced the old generations of synthetic cannabinoids (SCs) with naphthyl or adamantine groups. Among them, ADB‐FUBICA [N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐(4‐fluorobenzyl)‐1H–indole‐3‐carboxamide], AB‐FUBICA [N‐(1‐amino‐3‐methyl‐1‐oxobutan‐2‐yl)‐1‐(4‐fluorobenzyl)‐1H–indole‐3‐carboxamide], AB‐BICA [N‐(1‐amino‐3‐methyl‐1‐oxobutan‐2‐yl)‐1‐benzyl‐1H‐indole‐3‐carboxamide] and ADB‐BICA [N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐benzyl‐1H‐indole‐3‐carboxamide] were detected in China recently, but unfortunately no information about their in vitro human metabolism is available. Therefore, biomonitoring studies to screen their consumption lack any information about the potential biomarkers (e.g. metabolites) to target. To bridge this gap, we investigated their phase I metabolism by incubating with human liver microsomes, and the metabolites were identified by ultra‐performance liquid chromatography–high resolution–tandem mass spectrometry. Metabolites generated by N‐dealkylation and hydroxylation on the 1‐amino‐alkyl moiety were found to be predominant for all these four substances, and others which underwent hydroxylation, amide hydrolysis and dehydrogenation were also observed in our investigation. Based on our research, we recommend that the N‐dealkylation and hydroxylation metabolites are suitable and appropriate analytical markers for monitoring their intake.     

New analytical method for the study of the metabolism of gentiopicroside in rats after oral administration by LC–TOF‐MS following picolinoyl derivatization

The metabolism of gentiopicroside (GPS) in vivo was studied for the first time by LC–MS following picolinoyl derivatization. Incubation of erythrocentaurin, one of the main in vitro metabolites of GPS by intestinal bacteria, with liver microsome indicated that GPS might be metabolized to a final metabolite 3,4‐dihydro‐5‐(hydroxymethyl)isochroman‐1‐one (HMIO) in vivo. After hydrolysis with sulfatase, HMIO was successfully detected in rat plasma after oral administration of GPS by LC–MS following picolinoyl derivatization. 4‐Methoxyphenyl methanol was used as an internal standard to quantify HMIO in rat plasma. A metabolic pathway of GPS in rats is proposed. The monoterpene compound GPS was found to be metabolized to dihydroisocoumarin, which may be responsible for the pharmacological effect of GPS.     

In silico methods for predicting metabolism and mass fragmentation applied to quetiapine in liquid chromatography/time‐of‐flight mass spectrometry urine drug screening

Current in silico tools were evaluated for their ability to predict metabolism and mass spectral fragmentation in the context of analytical toxicology practice. A metabolite prediction program (Lhasa Meteor), a metabolite detection program (Bruker MetaboliteDetect), and a fragmentation prediction program (ACD/MS Fragmenter) were used to assign phase I metabolites of the antipsychotic drug quetiapine in the liquid chromatography/time‐of‐flight mass spectrometry (LC/TOFMS) accurate mass data from ten autopsy urine samples. In the literature, the main metabolic routes of quetiapine have been reported to be sulfoxidation, oxidation to the corresponding carboxylic acid, N‐ and O‐dealkylation and hydroxylation. Of the 14 metabolites predicted by Meteor, eight were detected by LC/TOFMS in the urine samples with use of MetaboliteDetect software and manual inspection. An additional five hydroxy derivatives were detected, but not predicted by Meteor. The fragment structures provided by ACD/MS Fragmenter software confirmed the identification of the metabolites. Mean mass accuracy and isotopic pattern match (SigmaFit) values for the fragments were 2.40 ppm (0.62 mDa) and 0.010, respectively. ACD/MS Fragmenter, in particular, allowed metabolites with identical molecular formulae to be differentiated without a need to access the respective reference standards or reference spectra. This was well exemplified with the hydroxy/sulfoxy metabolites of quetiapine and their N‐ and O‐dealkylated forms. The procedure resulted in assigning 13 quetiapine metabolites in urine. The present approach is instrumental in developing an extensive database containing exact monoisotopic masses and verified retention times of drugs and their urinary metabolites for LC/TOFMS drug screening. Copyright © 2009 John Wiley & Sons, Ltd.     

New potential biomarkers for mesterolone misuse in human urine by liquid chromatography quadrupole time‐of‐flight mass spectrometry

In this paper, mesterolone metabolic profiles were investigated carefully. Mesterolone was administered to one healthy male volunteer. Urinary extracts were analyzed by liquid chromatography quadruple time‐of‐flight mass spectrometry (LC‐QTOFMS) for the first time. Liquid–liquid extraction was applied to processing urine samples, and dilute‐shoot analyses of intact metabolites were also presented. In LC‐QTOFMS analysis, chromatographic peaks for potential metabolites were hunt down by using the theoretical [M–H]^? as target ions in full scan experiment, and their actual deprotonated ions were analyzed in targeted MS/MS mode. Ten metabolites including seven new sulfate and three glucuronide conjugates were found for mesterolone. Because of no useful fragment ion for structural elucidation, gas chromatography–mass spectrometry instrumentation was employed to obtain structural details of the trimethylsilylated phase I metabolite released after solvolysis. Thus, their potential structures were proposed particularly by a combined MS approach. All the metabolites were also evaluated in terms of how long they could be detected, and S1 (1α‐methyl‐5α‐androst‐3‐one‐17β‐sulfate) together with S2 (1α‐methyl‐5α‐androst‐17‐one‐3β‐sulfate) was detected up to 9 days after oral administration, which could be the new potential biomarkers for mesterolone misuse. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

Evaluation of tramadol and its main metabolites in horse plasma by high‐performance liquid chromatography/fluorescence and liquid chromatography/electrospray ionization tandem mass spectrometry techniques

Tramadol is a centrally acting analgesic drug that has been used clinically for the last two decades to treat pain in humans. The clinical response of tramadol is strictly correlated to its metabolism, because of the different analgesic activity of its metabolites. O‐Desmethyltramadol (M1), its major active metabolite, is 200 times more potent at the µ‐receptor than the parent drug. In recent years tramadol has been widely introduced in veterinary medicine but its use has been questioned in some species. The aim of the present study was to develop a new sensible method to detect the whole metabolic profile of the drug in horses, through plasma analyses by high‐performance liquid chromatography (HPLC) coupled with fluorimetric (FL) and photodiode array electrospray ionization mass spectrometric (PDA‐ESI‐MS) detection, after its sustained release by oral administration (5 mg/kg). In HPLC/FL experiments the comparison of the horse plasma chromatogram profile with that of a standard mixture suggested the identification of the major peaks as tramadol and its metabolites M1 and N,O‐desmethyltramadol (M5). LC/PDA‐ESI‐MS/MS analysis confirmed the results obtained by HPLC/FL and also provided the identification of two more metabolites, N‐desmethyltramadol (M2), and N,N‐didesmethyltramadol (M3). Another metabolite, M6, was also detected and identified. The present findings demonstrate the usefulness and the advantage of LC/ESI‐MS/MS techniques in a search for tramadol metabolites in horse plasma samples. Copyright © 2008 John Wiley & Sons, Ltd.     

Identification of phase I and II metabolites of the new designer drug α‐pyrrolidinohexiophenone (α‐PHP) in human urine by liquid chromatography quadrupole time‐of‐flight mass spectrometry (LC‐QTOF‐MS)

Pyrrolidinophenones represent one emerging class of newly encountered drugs of abuse, also known as ‘new psychoactive substances’, with stimulating psychoactive effects. In this work, we report on the detection of the new designer drug α‐pyrrolidinohexiophenone (α‐PHP) and its phase I and II metabolites in a human urine sample of a drug abuser. Determination and structural elucidation of these metabolites have been achieved by liquid chromatography electrospray ionisation quadrupole time‐of‐flight mass spectrometry (LC‐ESI‐QTOF‐MS). By tentative identification, the exact and approximate structures of 19 phase I metabolites and nine phase II glucuronides were elucidated. Major metabolic pathways revealed the reduction of the ß‐keto moieties to their corresponding alcohols, didesalkylation of the pyrrolidine ring, hydroxylation and oxidation of the aliphatic side chain leading to n‐hydroxy, aldehyde and carboxylate metabolites, and oxidation of the pyrrolidine ring to its lactam followed by ring cleavage and additional hydroxylation, reduction and oxidation steps and combinations thereof. The most abundant phase II metabolites were glucuronidated ß‐keto‐reduced alcohols. Besides the great number of metabolites detected in this sample, α‐PHP is still one of the most abundant ions together with its ß‐keto‐reduced alcoholic dihydro metabolite. Monitoring of these metabolites in clinical and forensic toxicology may unambiguously prove the abuse of the new designer drug α‐PHP. Copyright © 2015 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.     

Metabolic profiles of neratinib in rat by using ultra‐high‐performance liquid chromatography coupled with diode array detector and Q‐Exactive Orbitrap tandem mass spectrometry

Neratinib is a tyrosine kinase inhibitor that has been approved by the US Food and Drug Administration for the treatment of breast cancer. However, its metabolism remains unknown. This study was carried out to investigate the in vitro and in vivo metabolism of neratinib using an UHPLC‐DAD‐Q Exactive Orbitrap‐MS instrument with dd‐MS² on‐line data acquisition mode. The post‐acquisition data was processed using MetWorks software. Under the current conditions, a total of 12 metabolites were detected and structurally identified based on their accurate masses, fragment ions and chromatographic retention times. Among these metabolites, M3, M10 and M12 were unambiguously identified using chemically synthesized reference standards. M6 and M7 (GSH conjugates) were the major metabolites. The metabolic pathways of neratinib were proposed accordingly. Our findings suggested that neratinib was mainly metabolized via O‐dealkylation (M3), oxygenation (M8), N‐demethylation (M10), N‐oxygenation (M12), GSH conjugation (M1, M2, M4, M5, M6 and M7) and N‐acetylcysteine conjugation (M9 and M11). The α,β‐unsaturated ketone was the major metabolic site and GSH conjugation was the predominant metabolic pathway. In conclusion, this study provided valuable metabolic data and would benefit the assessment of the contributions to the overall activity or toxicity from the key metabolites.     

Metabolite identification of the antimalarial naphthoquine using liquid chromatography–tandem high‐resolution mass spectrometry in combination with multiple data‐mining tools

Naphthoquine (NQ) is one of important partner drugs of artemisinin‐based combination therapy (ACT), which is recommended for the treatment of uncomplicated Plasmodium falciparum. NQ shows a high cure rate after a single oral administration. It is absorbed quickly (time to peak concentration 2–4 h) and has a long elimination half‐life (255 h). However, the metabolism of NQ has not been clarified. In this work, the metabolite profiling of NQ was studied in six liver microsomal incubates (human, cynomolgus monkey, beagle dog, mini pig, rat and CD1 mouse), seven recombinant CYP enzymes (1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) and rat (plasma, urine, bile and feces) using liquid chromatography tandem high‐resolution LTQ‐Orbitrap mass spectrometry (HRMSⁿ) in conjunction with online hydrogen/deuterium exchange. The biological samples were pretreated by protein precipitation and solid‐phase extraction. For data processing, multiple data‐mining tools were applied in tandem, i.e. background subtraction and followed by mass defect filter. NQ metabolites were characterized by accurate MS/MS fragmentation characteristics, the hydrogen/deuterium exchange data and cLogP simulation. As a result, five phase I metabolites (M1–M5) of NQ were characterized for the first time. Two metabolic pathways were involved: hydroxylation and N‐oxidation. This study demonstrates that LC‐HRMSⁿ in combination with multiple data‐mining tools in tandem can be a valuable analytical strategy for rapid metabolite profiling of drugs.