Quantitation of acotiamide in rat plasma by UHPLC‐Q‐TOF‐MS: method development,validation and application to pharmacokinetics

A novel, sensitive and selective ultra‐high‐performance liquid chromatography–electrospray ionization mass spectrometry method was developed and validated for the quantification of acotiamide (ACT), a first‐in‐class drug used in functional dyspepsia, in rat plasma. A simple protein precipitation method with acetonitrile as precipitating solvent was used to extract ACT from rat plasma. ACT and an internal standard (mirabegron, IS) were separated on an Agilent poroshell EC C₁₈ column (50 × 3.0 mm, 2.7 µm) using methanol–10 mM ammonium acetate binary gradient mobile phase at a flow rate of 0.4 mL/min over 4 min run time. Detection was performed using target ions of [M + H]⁺ at m/z 451.2010 for ACT and m/z 397.1693 for IS in selective ion mode. The method was validated in the calibration range of 1.31–1000 ng/mL. All the validation parameters were well within the limits. The method demonstrated good performances in terms of intra‐ and inter‐day precision (3.27–12.60% CV) and accuracy (87.96–104.94%). Thus the present ultra‐high‐pressure liquid chromatograhy–high‐resolution mass spectrometry method for determination of ACT in rat plasma, is highly sensitive and rapid with a short run‐time of 4 min, can be suitable for high sample throughput and for large batches of biological samples in pharmacokinetic studies. This method can be extended to measure plasma concentrations of ACT in humans to understand drug metabolism, drug interaction and adverse effects. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification of the absorptive constituents and their metabolites in vivo of Puerariae Lobatae Radix decoction orally administered in WZS‐miniature pigs by HPLC‐ESI‐Q‐TOFMS

In this study, the technique of high‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight mass spectrometry (HPLC‐ESI‐Q‐TOFMS) was used to analyze and identify the absorptive constituents and their metabolites in drug‐containing urine of Wuzhishan (WZS)‐miniature pigs administered with Puerariae Lobatae Radix (PLR) decoction. With the accurate mass measurements (<5 ppm) and effective MS² fragment ions, 96 compounds, including eight original constituents and 88 metabolites, were identified from the drug‐containing urine. Among these, 64 metabolites were new ones and their structures can be categorized into five types: isoflavones, puerols, O‐desmethylangolensins, equols and isoflavanones. In particular, puerol‐type constituents in PLR were first proved to be absorptive in vivo. Meanwhile, the metabolic pathways of PLR in vivo were investigated. On the basis of relative content of the identified compounds, 13 major metabolites accounting for approximately 50% of the contents, as well as their corresponding 12 prototype compounds, were determined as the major original absorptive constituents and metabolites of PLR in vivo. The HPLC‐ESI‐Q‐TOFMS technique proved to be powerful for characterizing the chemical constituents from the complicated traditional Chinese medicine matrices in this research. Copyright © 2013 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.     

Rapid identification of efavirenz metabolites in rats and humans by ultra high performance liquid chromatography combined with quadrupole time‐of‐flight tandem mass spectrometry

An in vivo study of efavirenz metabolites in rats and human patients with ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry combined with MetabolitePilot^MT software is reported for the first time. Considering the polarity differences between the metabolites, solid‐phase extraction and protein precipitation were both applied as a part of the sample preparation method. The structures of the metabolites and their fragment ions were identified or tentatively characterized based on the accurate mass and MS² data. As a result, a total of 15 metabolites, including 11 from rat samples and 13 from human samples, were identified or tentatively characterized. Two metabolites and several new metabolism pathways are reported for the first time. This study provides a practical approach for identifying complicated metabolites through the rapid and reliable ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry technique, which could be widely used for the investigation of drug metabolites.     

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.     

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.     

Systematic characterization of the metabolites of echinacoside and acteoside from Cistanche tubulosa in rat plasma,bile, urine and feces based on UPLC‐ESI‐Q‐TOF‐MS

Echinacoside (ECH) and acteoside (ACT), as the most and major active components of Cistanche tubulosa, were reported to possess cardioactive, neuroprotective and hepatocyte protective effects, as well as antibacterial, antioxidative effects. Recently, more studies have focused on their pharmacological activities. However, their metabolic profiles in vivo have not been sufficiently investigated. This study proposes an approach for rapidly identifying the complicated and unpredictable metabolites of ECH and ACT in rat plasma, bile, urine and feces, and systematically and comprehensively revealing their major metabolic pathways, based on powerful ultra‐high performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry. Plasma, bile, urine and feces were collected from rats after a single 200 mg/kg oral dose. A total of 49 metabolites were detected in rat biological samples. Through analyzing metabolites in bile samples, it was found that ECH and ACT were subjected to a marked hepatic first‐pass effect in liver. Copyright © 2016 John Wiley & Sons, Ltd.     

Identification of metabolites of Helicid in vivo using ultra‐high performance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry

Helicid is an active natural aromatic phenolic glycoside ingredient originating from a well‐known traditional Chinese herbal medicine and has the significant effects of sedative hypnosis, anti‐inflammatory analgesia and antidepressant. In this study, we analyzed the potential metabolites of Helicid in rats by multiple mass defect filter and dynamic background subtraction in ultra‐high‐performance liquid chromatography–quadrupole time‐of‐flight mass spectrometry (UHPLC‐Q‐TOF‐MS). Moreover, we used a novel data processing method, ‘key product ions’, to rapidly detect and identify metabolites as an assistant tool. MetabolitePilot™ 2.0 software and PeakView™ 2.2 software were used for analyzing metabolites. Twenty metabolites of Helicid (including 15 phase I metabolites and five phase II metabolites) were detected by comparison with the blank samples. The biotransformation route of Helicid was identified as demethylation, oxidation, dehydroxylation, hydrogenation, decarbonylation, glucuronide conjugation and methylation. This is the first study simultaneously detecting and identifying Helicid metabolism in rats employing UHPLC‐Q‐TOF‐MS technology. This experiment not only proposed a method for rapidly detecting and identifying metabolites, but also provided useful information for further study of the pharmacology and mechanism of Helicid in vivo. Furthermore, it provided an effective method for the analysis of other aromatic phenolic glycosides metabolic components in vivo.     

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

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

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.     

Profiling primaquine metabolites in primary human hepatocytes using UHPLC‐QTOF‐MS with 13C stable isotope labeling

Therapeutic efficiency and hemolytic toxicity of primaquine (PQ), the only drug available for radical cure of relapsing vivax malaria are believed to be mediated by its metabolites. However, identification of these metabolites has remained a major challenge apparently due to low quantities and their reactive nature. Drug candidates labeled with stable isotopes afford convenient tools for tracking drug‐derived metabolites in complex matrices by liquid chromatography‐tandem mass spectrometry (LC‐MS‐MS) and filtering for masses with twin peaks attributable to the label. This study was undertaken to identify metabolites of PQ from an in vitro incubation of a 1:1 w/w mixture of ¹³C₆‐PQ/PQ with primary human hepatocytes. Acquity ultra‐performance LC (UHPLC) was integrated with QTOF‐MS to combine the efficiency of separation with high sensitivity, selectivity of detection and accurate mass determination. UHPLC retention time, twin mass peaks with difference of 6 (originating from ¹³C₆‐PQ/PQ), and MS‐MS fragmentation pattern were used for phenotyping. Besides carboxy‐PQ (cPQ), formed by oxidative deamination of PQ to an aldehyde and subsequent oxidation, several other metabolites were identified: including PQ alcohol, predictably generated by oxidative deamination of PQ to an aldehyde and subsequent reduction, its acetate and the alcohol's glucuronide conjugate. Trace amounts of quinone‐imine metabolites of PQ and cPQ were also detected which may be generated by hydroxylation of the PQ/cPQ quinoline ring at the 5‐position and subsequent oxidation. These findings shed additional light on the human hepatic metabolism of PQ, and the method can be applied for identification of reactive PQ metabolites generated in vivo in preclinical and clinical studies. 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.     

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.     

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

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

Structural elucidation of new urinary tamoxifen metabolites by liquid chromatography quadrupole time‐of‐flight mass spectrometry

In this study, tamoxifen metabolic profiles were investigated carefully. Tamoxifen was administered to two healthy male volunteers and one female patient suffering from breast cancer. Urinary extracts were analyzed by liquid chromatography quadruple time‐of‐flight mass spectrometry using full scan and targeted MS/MS techniques with accurate mass measurement. Chromatographic peaks for potential metabolites were selected by using the theoretical [M + H]⁺ as precursor ion in full‐scan experiment and m/z 72, 58 or 44 as characteristic product ions for N,N‐dimethyl, N‐desmethyl and N,N‐didesmethyl metabolites in targeted MS/MS experiment, respectively. Tamoxifen and 37 metabolites were detected in extraction study samples. Chemical structures of seven unreported metabolites were elucidated particularly on the basis of fragmentation patterns observed for these metabolites. Several metabolic pathways containing mono‐ and di‐hydroxylation, methoxylation, N‐desmethylation, N,N‐didesmethylation, oxidation and combinations were suggested. All the metabolites were detected in the urine samples up to 1 week. Copyright © 2014 John Wiley & Sons, Ltd.     

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

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

Identification of metabolites of gardenin A in rats by combination of high‐performance liquid chromatography with linear ion trap–Orbitrap mass spectrometer based on multiple data processing techniques

Gardenin A is one of the less abundant hydroxylated polymethoxyflavonoids (OH‐PMFs) in nature, and has many potential significant health benefits. In the present study, an efficient strategy was established using high‐performance liquid chromatography coupled with linear ion trap–Orbitrap mass spectrometer to profile the in vivo metabolic fate of gardenin A in rat plasma and various tissues. First, an online LC‐MSⁿ data acquisition method was developed to trace all the probable metabolites. Second, a combination of offline data processing methods including extracted ion chromatography and multiple mass defect filters was employed to screen the common and uncommon metabolites from the background noise and endogenous components. Finally, structures of the metabolites were elucidated based on an accurate mass measurement, the diagnostic product ions of PMFs, and relevant drug biotransformation knowledge. Based on the proposed strategy, a total of 26 metabolites were observed and characterized. The results indicate that some biotransformations, such as methylation, demethoxylation, demethylation, glucuronide conjugation, sulfate conjugation and their composite reactions, have been discovered for OH‐PMFs. Moreover, some diagnostic biotransformation pathways are summarized. Overall, this study gives us a first insight into the in vivo metabolism of gardenin A. The study also provides a practical strategy for rapidly screening and identifying metabolites, which can be widely applied for the other biotransformations. Copyright © 2014 John Wiley & Sons, Ltd.     

Biotransformation and metabolic profile of caudatin‐2,6‐dideoxy‐3‐O‐methy‐β‐d‐cymaropyranoside with human intestinal microflora by liquid chromatography quadrupole time‐of‐flight mass spectrometry

In our previous studies, caudatin‐2,6‐dideoxy‐3‐O‐methy‐β‐d‐ cymaropyranoside (CDMC) was for the first time isolated from Cynanchum auriculatum Royle ex Wightand and was reported to possess a wide range of biological activities. However, the routes and metabolites of CDMC produced by intestinal bacteria are not well understood. In this study, ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF‐MS) technique combined with Metabolynx^TMsoftware was applied to analyze metabolites of CDMC by human intestinal bacteria. The incubated samples collected for 48 h in an anaerobic incubator and extracted with ethyl acetate were analyzed by UPLC‐Q‐TOF‐MS within 12 min. Eight metabolites were identified based on MS and MS/MS data. The results indicated that hydrolysis, hydrogenation, demethylation and hydroxylation were the major metabolic pathways of CDMC in vitro. Seven strains of bacteria including Bacillus sp. 46, Enterococcus sp. 30 and sp. 45, Escherichia sp. 49A, sp. 64, sp. 68 and sp. 75 were further identified using 16S rRNA gene sequencing owing to their relatively strong metabolic capacity toward CDMC. The present study provides important information about metabolic routes of CDMC and the roles of different intestinal bacteria in the metabolism of CDMC. Moreover, those metabolites might influence the biological effect of CDMC in vivo, which affects the clinical effects of this medicinal plant. Copyright © 2015 John Wiley & Sons, Ltd.     

Preparing the key metabolite of Z‐ligustilide in vivo by a specific electrochemical reaction

The key in vivo metabolites of a drug play an important role in its efficacy and toxicity. However, due to the low content and instability of these metabolites, they are hard to obtain through in vivo methods. Electrochemical reactions can be an efficient alternative to biotransformation in vivo for the preparation of metabolites. Accordingly, in this study, the metabolism of Z‐ligustilide was investigated in vitro by electrochemistry coupled online to mass spectrometry. This work showed that five oxidation products of the electrochemical reaction were detected and that two of the oxidation products (senkyunolide I and senkyunolide H) were identified from liver microsomal incubation as well. Furthermore, after intragastric administration of Z‐ligustilide in rats, senkyunolide I and senkyunolide H were detected in the rat plasma and liver, while 6,7‐epoxyligustilide, a key intermediate metabolite of Z‐ligustilide, was difficult to detect in vivo. By contrast, 6,7‐epoxyligustilide was obtained from the electrochemical reaction. In addition, for the first time, 6 mg of 6,7‐epoxyligustilide was prepared from 120 mg of Z‐ligustilide. Therefore, electrochemical reactions represent an efficient laboratory method for preparing key drug metabolites.     

New clostebol metabolites in human urine by liquid chromatography time‐of‐flight tandem mass spectrometry and their application for doping control

In this study, clostebol metabolic profiles were investigated carefully. Clostebol was administered to one healthy male volunteer. Urinary extracts were analyzed by liquid chromatography quadrupole time‐of‐flight mass spectrometry (MS) using full scan and targeted MS/MS techniques with accurate mass measurement for the first time. Liquid–liquid extraction and direct injection were applied to processing urine samples. Chromatographic peaks for potential metabolites were found by using the theoretical [M–H]^? as target ion in full scan experiment, and their actual deprotonated ions were analyzed in targeted MS/MS mode. Fourteen metabolites were found for clostebol, and nine unreported metabolites (two free ones and seven sulfate conjugates) were identified by MS, and their potential structures were proposed based on fragmentation and metabolism pathways. Four glucuronide conjugates were also first reported. All the metabolites were evaluated in terms of how long they could be detected and S1 (4ξ‐chloro‐5ξ‐androst‐3ξ‐ol‐17‐one‐3ξ‐sulfate) was considered to be the long‐term metabolite for clostebol misuse detected up to 25 days by liquid–liquid extraction and 14 days by direct injection analysis after oral administration. Five conjugated metabolites (M2, M5, S2, S6 and S7) could also be the alternative biomarkers for clostebol misuse. Copyright © 2015 John Wiley & Sons, Ltd.