Metabolite identification of artemether by data-dependent accurate mass spectrometric analysis using an LTQ-Orbitrap hybrid mass spectrometer in combination with the online hydrogen/deuterium exchange technique

Artemether (ARM), the O-methyl ether prodrug of dihydroartemisinin (DHA), is a first-line antimalarial drug used in areas of multi-drug resistance. Artemisinin drugs can be metabolized extensively in vivo and this seems related to their autoinduction pharmacokinetics. In the present study, the metabolite identification of ARM was performed by the generic data-dependent accurate mass spectrometric analysis, using high-resolution (HR) liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) and tandem mass spectrometry (MS/MS) LTQ-Orbitrap hybrid mass spectrometer in conjunction with online hydrogen (H)/deuterium (D) exchange for rapid structural characterization. The LC separation was improved allowing the separation of ARM parent drugs and their metabolites from their diastereomers. A total of 77 phase I metabolites of ARM were identified in rat liver microsomal incubates and rat urine, including dihydroartemisinin and artemisinin. In rat bile, 12 phase II metabolites were found. Accurate mass data were obtained in both full scan and HR-MS/MS mode to support assignments of metabolite structures. Online H/D exchange LC/HR-ESI-MS experiments provided additional evidence in differentiating dihydroxylated deoxy-ARM from mono-hydroxylated ARM. The results showed the main phase I metabolites of artemether are hydroxylated, dehydro, demethylated and deoxy products, and they will undergo subsequent phase II glucuronidation processes. Most metabolites were reported for the first time. This study also demonstrated the effectiveness of high-resolution mass spectrometry in combination with an online H/D exchange LC/HR-MS(n) technique in rapid identification of drug metabolites.     

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.     

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.     

Metabolite identification of triptolide by data-dependent accurate mass spectrometric analysis in combination with online hydrogen/deuterium exchange and multiple data-mining techniques

Triptolide (TP), the primary active component of the herbal medicine Tripterygium wilfordii Hook F, has shown promising antileukemic and anti‐inflammatory activity. The pharmacokinetic profile of TP indicates an extensive metabolic elimination in vivo; however, its metabolic data is rarely available partly because of the difficulty in identifying it due to the absence of appropriate ultraviolet chromophores in the structure and the presence of endogenous interferences in biological samples. In the present study, the biotransformation of TP was investigated by improved data‐dependent accurate mass spectrometric analysis, using an LTQ/Orbitrap hybrid mass spectrometer in conjunction with the online hydrogen (H)/deuterium (D) exchange technique for rapid structural characterization. Accurate full‐scan MS and MS/MS data were processed with multiple post‐acquisition data‐mining techniques, which were complementary and effective in detecting both common and uncommon metabolites from biological matrices. As a result, 38 phase I, 9 phase II and 8 N‐acetylcysteine (NAC) metabolites of TP were found in rat urine. Accurate MS/MS data were used to support assignments of metabolite structures, and online H/D exchange experiments provided additional evidence for exchangeable hydrogen atoms in the structure. The results showed the main phase I metabolic pathways of TP are hydroxylation, hydrolysis and desaturation, and the resulting metabolites subsequently undergo phase II processes. The presence of NAC conjugates indicated the capability of TP to form reactive intermediate species. This study also demonstrated the effectiveness of LC/HR‐MSⁿ in combination with multiple post‐acquisition data‐mining methods and the online H/D exchange technique for the rapid identification of drug metabolites. Copyright © 2011 John Wiley & Sons, Ltd.     

Low resolution and high resolution MS for studies on the metabolism and toxicological detection of the new psychoactive substance methoxypiperamide (MeOP)

In 2013, the new psychoactive substance methoxypiperamide (MeOP) was first reported to the European Monitoring Centre for Drug and Drug Addiction. Its structural similarity to already controlled piperazine designer drugs might have contributed to the decision to offer MeOP for online purchase. The aims of this work were to identify the phase I/II metabolites of MeOP in rat urine and the human cytochrome P450 (CYP) isoenzymes responsible for the initial metabolic steps. Finally, the detectability of MeOP in rat urine by gas chromatography–mass spectrometry (GC‐MS) and liquid chromatography coupled with multistage mass spectrometry (LC‐MSⁿ) standard urine screening approaches (SUSAs) was evaluated. After sample preparation by cleavage of conjugates followed by extraction for elucidating phase I metabolites, the analytes were separated and identified by GC‐MS as well as liquid chromatography‐high resolution‐tandem mass spectrometry (LC‐HR‐MS/MS). For detection of phase II metabolites, the analytes were separated and identified after urine precipitation followed by LC‐HR‐MS/MS. The following metabolic steps could be postulated: hydrolysis of the amide, N‐oxide formation, N‐ and/or O‐demethylation, oxidation of the piperazine ring to the corresponding keto‐piperazine, piperazine ring opening followed by oxidation of a methylene group to the corresponding imide, and hydroxylation of the phenyl group. Furthermore, N‐acetylation, glucuronidation and sulfation were observed. Using human CYPs, CYP1A2, CYP2C19, CYP2D6, and/or CYP3A4 were found to catalyze N‐oxide formation and N‐, O‐demethylation and/or oxidation. Mostly MeOP and N‐oxide‐MeOP but to a minor degree also other metabolites could be detected in the GC‐MS and LC‐MSⁿ SUSAs. Copyright © 2015 John Wiley & Sons, Ltd.     

Studies on the metabolism and toxicological detection of glaucine,an isoquinoline alkaloid from Glaucium flavum (Papaveraceae), in rat urine using GC‐MS,LC‐MSn and LC‐high‐resolution MSn

Glaucine ((S)‐5,6,6a,7‐tetrahydro‐1,2,9,10‐tetramethoxy‐6‐methyl‐4H‐dibenzo [de,g]quinoline) is an isoquinoline alkaloid and main component of Glaucium flavum (Papaveraceae). It was described to be consumed as recreational drug alone or in combination with other drugs. Besides this, glaucine is used as therapeutic drug in Bulgaria and other countries as cough suppressant. Currently, there are no data available concerning metabolism and toxicological analysis of glaucine. To study both, glaucine was orally administered to Wistar rats and urine was collected. For metabolism studies, work‐up of urine samples consisted of protein precipitation or enzymatic cleavage followed by solid‐phase extraction. Samples were afterwards measured by liquid chromatography (LC) coupled to low or high‐resolution mass spectrometry (HR‐MS). The phase I and II metabolites were identified by detailed interpretation of the corresponding fragmentations, which were further confirmed by determination of their elemental composition using HR‐MS. From these data, the following metabolic pathways could be proposed: O‐demethylation at position 2, 9 and 10, N‐demethylation, hydroxylation, N‐oxidation and combinations of them as well as glucuronidation and/or sulfation of the phenolic metabolites. For monitoring a glaucine intake in case of abuse or poisoning, the O‐ and N‐demethylated metabolites were the main targets for the gas chromatography‐MS and LC‐MSⁿ screening approaches described by the authors. Both allowed confirming an intake of glaucine in rat urine after a dose of 2 mg/kg body mass corresponding to a common abuser's dose. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

Structural characterization of in vitro rat liver microsomal metabolites of antihistamine desloratadine using LTQ-Orbitrap hybrid mass spectrometer in combination with online hydrogen/deuterium exchange HR-LC/MS

In vitro drug metabolism study is an integral part of drug discovery process. In this report, we have described the application of LTQ-Orbitrap hybrid mass spectrometer in conjunction with online hydrogen (H)/deuterium (D) exchange high resolution (HR)-LC/MS for structural characterization of in vitro rat liver microsomal metabolites of antihistamine desloratadine. Five metabolites M1--M5 have been identified, including three hydroxylated metabolites M1--M3, one N-oxide M4 and one uncommon aromatized N-oxide M5. Accurate mass data have been obtained in both full scan and MSn mode support assignments of metabolite structures with reported mass errors less than 3 ppm. Online H/D exchange HR-LC/MS experiments provide additional evidence in differentiating hydroxylated metabolites from N-oxides. This study demonstrates the effectiveness of this approach in structural characterization of drug metabolites.     

Liquid chromatography/mass spectrometry‐based plasma metabolic profiling study of escitalopram in subjects with major depressive disorder

Liquid chromatography‐mass spectrometry (LC‐MS) method revealed the plasma metabolite profiles in major depressive disorder patients treated with escitalopram (ECTP) (n = 7). Depression severity was assessed according to the 17‐item Hamilton Depression Rating Scale. Metabolic profiles were derived from major depressive disorder subject blood samples collected after ECTP treatment. Blood plasma was separated and processed in order to effectively extract metabolites, which were then analyzed using LC‐MS. We identified 19 metabolites and elucidated their structures using LC‐tandem MS (LC‐MS/MS) combined with elemental compositions derived from accurate mass measurements. We further used online H/D exchange experiments to verify the structural elucidations of each metabolite. Identifying molecular metabolites may provide critical insights into the pharmacological and clinical effects of ECTP treatment and may also provide useful information informing the development of new antidepressant treatments. These detailed plasma metabolite analyses may also be used to identify optimal dose concentrations in psychopharmacotherapeutic treatment through drug monitoring, as well as forming the basis for response predictions in depressed subjects.     

Qualitative–(semi)quantitative data acquisition of artemisinin and its metabolites in rat plasma using an LTQ/Orbitrap mass spectrometer

Artemisinin (QHS) is one of the first‐line antimalarials, and autoinduction of CYP‐mediated metabolism can result in its reduced exposure. To better understand the autoinduction of QHS, we evaluated the pharmacokinetics of QHS and its phase I metabolites in rats using an liquid chromatography‐high resolution mass spectrometry (LC‐HRMS) method. The LC separation was improved, allowing the separation of QHS and its metabolites from their diastereomers, and seven metabolites of QHS with relatively high exposure were identified in rat plasma, including deoxyartemisinin (DQHS), three monoyhydroxylated plus deoxyl metabolites (M1–M3) and three monohydroxylated metabolites (M4–M6). For detection, a high‐resolution LTQ/Orbitrap mass spectrometer with an electrospray ionization (ESI) inlet in the positive ion mode was used. High‐resolution extracted ion chromatograms for each analyte were obtained by processing the full‐scan MS dataset with 10 ppm mass tolerance. The plasma samples were pretreated by protein precipitation with acetonitrile. The standard curve was linear (r² > 0.99) over the QHS and DQHS concentration range of 5.0–200.0 ng/ml in 50 µl of plasma, which offered sufficient sensitivity and accuracy for the determination of QHS and its metabolites. A 3‐day validation approach was used for absolute quantitation of QHS and DQHS. The other six metabolites of QHS were semiquantified based on the calibration curve of QHS. The present method was applied to the pharmacokinetic study of QHS in rats after a single oral administration. The data shown here also suggest that this type of mass analyzer will be capable of a quantitative–qualitative workflow. Copyright © 2012 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.     

Liquid chromatography/mass spectrometric identification of benzimidazole anthelminthics metabolites formed ex vivo by Dicrocoelium dendriticum

With further use of chemical agents in the control of parasitic infections, an increased number of drug resistance occurrences to antiparasitic drugs has been reported. Induction of enzymes responsible for detoxification of given drugs can contribute to drug resistance development in a parasitic organism. The identification of formed metabolites allows the characterization of the enzymes participating in biotransformation and possibly in drug resistance development. The objective of our work was to find and identify phase I and phase II metabolites of the anthelminthic drugs albendazole, flubendazole and mebendazole formed in ex vivo incubations by the parasitic helminth Dicrocoelium dendriticum, a parasite of ruminants and other grazing animals, using liquid chromatography/mass spectrometric (LC/MS) techniques. In the ex vivo study, approximately 50 living D. dendriticum adults were incubated in 5 mL RPMI‐1640 medium in the presence of 10.0 µmol L^?1 benzimidazole drug (5% CO₂, 38°C) for 24 h. The bodies of the parasite were then removed from the medium. After homogenization of parasites, both parasite homogenates and medium from the incubation were separately extracted using solid‐phase extraction. The extracts were analyzed using LC/MS with electrospray ionization. The results showed that D. dendriticum enzymatic systems are capable of phase I oxidation and reduction as well as phase II conjugation reactions. Detected phase I metabolites comprised albendazole sulfoxide, reduced flubendazole and reduced mebendazole. As for phase II metabolites, methyl derivatives of both reduced flubendazole and reduced mebendazole were observed. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

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.     

Metabolic profile and pharmacokinetics of polyphyllin I,an anticancer candidate,in rats by UPLC‐QTOF‐MS/MS and LC‐TQ‐MS/MS

Polyphyllin I (PPI), a natural steroidal saponin originating from rihzome of Paris polyphylla , is a potential anticancer candidate. Previous pharmacokinetics study showed that the oral bioavailability of PPI was very low, which suggested that certain amount of PPI might be metabolized in vivo . However, to date, information regarding the final metabolic fates of PPI is very limited. In this study, metabolites of PPI and their pharmacokinetics in rats were investigated using UPLC‐QTOF‐MS/MS and LC‐TQ‐MS/MS. A total of seven putative metabolites, including six phase I and one phase II metabolites, were detected and identified with three exact structures by comparison with authentic standards for the first time. Oxidation, deglycosylation and glucuronidation were found to be the major metabolic processes of the compound in rats. The pharmacokinetics of prosapogenin A, trillin and diosgenin, three deglycosylation metabolites of PPI with definite anticancer effects, were further studied, which suggested that the metabolites underwent a prolonged absorption and slower elimination after intragastric administration of PPI at the dose of 500 mg/kg. This study provides valuable and new information on the metabolic fate of PPI, which will be helpful in further understanding its mechanism of action.     

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.     

Characterization and tentative identification of new flunitrazepam metabolites in authentic human urine specimens using liquid chromatography‐Q exactive‐HF hybrid quadrupole‐Orbitrap‐mass spectrometry (LC‐QE‐HF‐MS)

Flunitrazepam (FNZ) is a potent hypnotic, sedative, and amnestic drug used to treat severe insomnia. In our recent study, FNZ metabolic profiles were investigated carefully. Six authentic human urine samples were purified using solid phase extraction (SPE) without enzymatic hydrolysis, and urine extracts were then analyzed by liquid chromatography‐Q exactive‐HF hybrid quadrupole‐Orbitrap‐mass spectrometry (LC‐QE‐HF‐MS), using the full scan positive ion mode and targeted MS/MS (ddms2) technique to make accurate mass measurements. There were 25 metabolites, including 13 phase I and 12 phase II metabolites, which were detected and tentatively identified by LC‐QE‐HF‐MS. In addition, nine previously unreported phase II glucuronide conjugates and four phase I metabolites are reported here for the first time. Eight metabolic pathways, including N‐reduction and O‐reduction, N‐glucuronidation, O‐glucuronidation, mono‐hydroxylation and di‐hydroxylation, demethylation, acetylation, and combinations, were implicated in this work, and 2‐O‐reduction together with dihydroxylation were two novel metabolic pathways for FNZ that were identified tentatively. Although 7‐amino FNZ is widely considered to be the primary metabolite, a previously unreported metabolites (M12) can also serve as a potential biomarker for FNZ misuse.     

Use of on-line hydrogen/deuterium exchange to facilitate metabolite identification

Biotransformation studies performed on an investigational compound (I, represented by R1-CH(NH(2))-CO-N(R2)-CH(2)-S-R3) led to the identification of five metabolites (M1-M5). Based on LC/MS (liquid chromatography/mass spectrometry) analysis which included the use of H(2)O and D(2)O in the mobile phases, they were identified as the sulfoxide (M1), sulfone (M2), carbamoyl glucuronide (M3), N-glucuronide (M4), and N-glucoside (M5) metabolites, respectively. The structure of M3, a less commonly seen carbamoyl glucuronide metabolite, was established using on-line H/D (hydrogen/deuterium) exchange experiments conducted by LC/MS. H/D exchange experiments were also used to distinguish the S-oxidation structures of M1 and M2 from hydroxylation. Herein, the application of deuterium oxide as the LC/MS mobile phase for structural elucidation of drug metabolites in biological matrices is demonstrated.     

High‐resolution mass spectrometry and hydrogen/deuterium exchange study of mitorubrin azaphilones and nitrogenized analogues

Azaphilones represent numerous groups of wild fungal secondary metabolites that exhibit exceptional tendency to bind to nitrogen atoms in various molecules, especially those containing the amine group. Nitrogenized analogues of mitorubrin azaphilones, natural secondary metabolites of Hypoxylon fragiforme fungus, have been detected in the fungal methanol extract in very low concentrations. Positive electrospray ionization interfaced with high‐resolution mass spectrometry was applied for confirmation of the elemental composition of protonated species. Collision‐induced dissociation (CID) experiments have been performed, and fragmentation mechanisms have been proposed. Additional information regarding both secondary metabolite analogue families has been reached by application of gas‐phase proton/deuterium (H/D) exchanges performed in the collision cell of a triple quadrupole mass spectrometer. An incomplete H/D exchange with one proton less than expected was observed for both protonated mitorubrin azaphilones and their nitrogenized analogues. By means of the density functional theory, an appropriate explanation of this behavior was provided, and it revealed some information concerning gas‐phase H/D exchange mechanism and protonation sites. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification and characterization of in vitro and in vivo fidarestat metabolites: Toxicity and efficacy evaluation of metabolites

The progression of diabetic complications can be prevented by inhibition of aldose reductase and fidarestat considered to be highly potent. To date, metabolites of the fidarestat, toxicity, and efficacy are unknown. Therefore, the present study on characterization of hitherto unknown in vitro and in vivo metabolites of fidarestat using liquid chromatography–electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) is undertaken. In vitro and in vivo metabolites of fidarestat have been identified and characterized by using LC/ESI/MS/MS and accurate mass measurements. To identify in vivo metabolites, plasma, urine, and feces samples were collected after oral administration of fidarestat to Sprague–Dawley rats, whereas for in vitro metabolites, fidarestat was incubated in human S9 fraction, human liver microsomes, and rat liver microsomes. Furthermore, in silico toxicity and efficacy of the identified metabolites were evaluated. Eighteen metabolites have been identified. The main in vitro phase I metabolites of fidarestat are oxidative deamination, oxidative deamination and hydroxylation, reductive defluroniation, and trihydroxylation. Phase II metabolites are methylation, acetylation, glycosylation, cysteamination, and glucuronidation. Docking studies suggest that oxidative deaminated metabolite has better docking energy and conformation that keeps consensus with fidarestat whereas the rest of the metabolites do not give satisfactory results. Aldose reductase activity has been determined for oxidative deaminated metabolite (F‐1), and it shows an IC50 value of 0.44 μM. The major metabolite, oxidative deaminated, did not show any cytotoxicity in H9C2, HEK, HEPG2, and Panc1 cell lines. However, in silico toxicity, the predication result showed toxicity in skin irritation and ocular irritancy SEV/MOD versus MLD/NON (v5.1) model for fidarestat and its all metabolites. In drug discovery and development research, it is distinctly the case that the potential for pharmacologically active metabolites must be considered. Thus, the active metabolites of fidarestat may have an advantage as drug candidates as many drugs were initially observed as metabolites.