Structure identification and elucidation of mosapride metabolites in human urine,feces and plasma by ultra performance liquid chromatography-tandem mass spectrometry method

Abstract

1.?Mosapride citrate (mosapride) is a potent gastroprokinetic agent. The only previous study on mosapride metabolism in human reported one phase I oxidative metabolite, des-p-fluorobenzyl mosapride, in human plasma and urine using HPLC method. Our aim was to identify mosapride phase I and phase II metabolites in human urine, feces and plasma using UPLC-ESI-MS/MS.

2.?A total of 16 metabolites were detected. To the best of our knowledge, 15 metabolites have not been reported previously in human.

3.?Two new metabolites, morpholine ring-opened mosapride (M15) and mosapride N-oxide (M16), alone with one known major metabolite, des-p-fluorobenzyl mosapride (M3), were identified by comparison with the reference standards prepared by our group. The chemical structures of seven phase I and six phase II metabolites of mosapride were elucidated based on UPLC–MS/MS analyses.

4.?There were two major phase I reactions, dealkylation and morpholine ring cleavage. Phase II reactions included glucuronide, glucose and sulfate conjugation. The comprehensive metabolic pathway of mosapride in human was proposed for the first time.

5.?The metabolites in humans were compared with those in rats reported previously. In addition to M10, the other 15 metabolites in humans were also found in rats. This result suggested that there was little qualitative species difference in the metabolism of mosapride between rats and humans.

6.?In all, 16 mosapride metabolites including 15 new metabolites were reported. These results allow a better understanding of mosapride disposition in human.     

Development and validation of an LC‐MS/MS method after chiral derivatization for the simultaneous stereoselective determination of methylenedioxy‐methamphetamine (MDMA) and its phase I and II metabolites in human blood plasma

3,4‐Methylenedioxymethamphetamine (MDMA, ecstasy) is a racemic drug of abuse and its two enantiomers are known to differ in their dose‐response curves. The S‐enantiomer was shown to be eliminated at a higher rate than the R‐enantiomer. The most likely explanation for this is a stereoselective metabolism also claimed in in vitro studies. Urinary excretion studies showed that the main metabolites in humans are 4‐hydroxy 3‐methoxymethamphetamine (HMMA) 4‐sulfate, HMMA 4‐glucuronide and 3,4‐dihydroxymethamphetamine (DHMA) 3‐sulfate. For stereoselective pharmacokinetic analysis of phase I and phase II metabolites in human blood plasma useful analytical methods are needed. Therefore the aim of the presented study was the development and validation of a stereoselective liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method for the simultaneous quantification of MDMA, 3,4‐methylenedioxyamphetamine, DHMA, DHMA 3‐sulfate, HMMA, HMMA 4‐glucuronide, HMMA 4‐sulfate, and 4‐hydroxy 3‐methoxyamphetamine in blood plasma for evaluation of the stereoselective pharmacokinetics in humans. Blood plasma samples were prepared by simple protein precipitation and afterwards all analytes were derivatized using N‐(2,4‐dinitro‐5‐fluorophenyl) L‐valinamide resulting in the formation of diastereomers which were easily separable on standard reverse phase stationary phases. This simple and fast method was validated according to international guidelines including specificity, recovery, matrix effects, accuracy and precision, stabilities, and limits of quantification. The method proved to be selective, sensitive, accurate and precise for all tested analytes except for DHMA. Copyright © 2014 John Wiley & Sons, Ltd.     

Fatal intoxication by 5F–ADB and diphenidine: Detection,quantification, and investigation of their main metabolic pathways in humans by LC/MS/MS and LC/Q‐TOFMS

Despite the implementation of a new blanket scheduling system in 2013, new psychoactive substance (NPS) abuse remains a serious social concern in Japan. We present a fatal intoxication case involving 5F–ADB (methyl 2‐[1‐(5‐fluoropentyl)‐1H–indazole‐3‐carboxamido]‐3,3‐dimethylbutanoate) and diphenidine. Postmortem blood screening by liquid chromatography/quadrupole time‐of‐flight mass spectrometry (LC/Q‐TOFMS) in the information‐dependent acquisition mode only detected diphenidine. Further urinary screening using an in‐house database containing NPS and metabolites detected not only diphenidine but also possible 5F–ADB metabolites; subsequent targeted screening by LC/tandem mass spectrometry (LC/MS/MS) allowed for the detection of a very low level of unchanged 5F–ADB in postmortem heart blood. Quantification by standard addition resulted in the postmortem blood concentrations being 0.19 ± 0.04 ng/mL for 5F–ADB and 12 ± 2.6 ng/mL for diphenidine. Investigation of the urinary metabolites revealed pathways involving ester hydrolysis (M1) and oxidative defluorination (M2), and further oxidation to the carboxylic acid (M3) for 5F–ADB. Mono‐ and di‐hydroxylated diphenidine metabolites were also found. The present case demonstrates the importance of urinary metabolite screening for drugs with low blood concentration. Synthetic cannabinoids (SCs) fluorinated at the terminal N‐alkyl position are known to show higher cannabinoid receptor affinity relative to their non‐fluorinated analogues; 5F–ADB is no exception with high CB₁ receptor activity and much greater potency than Δ⁹‐THC and other earlier SCs, thus we suspect its acute toxicity to be high compared to other structurally related SC analogues. The low blood concentration of 5F–ADB may be attributed to enzymatic and/or non‐enzymatic degradation, and further investigation into these possibilities is underway.     

Pharmacokinetics and metabolism of pemafibrate,a novel selective peroxisome proliferator‐activated receptor‐alpha modulator,in rats and monkeys

The metabolic profiles and pharmacokinetics of pemafibrate, a novel selective peroxisome proliferator activated receptor‐alpha modulator currently launched as an antidyslipidemic drug, were investigated in vitro using hepatocytes from rats, monkeys and humans and in vivo in rats and monkeys. Hepatocytes from rats, monkeys and humans all biotransformed pemafibrate to its demethylated form (M1). The bioavailabilities of pemafibrate in Sprague–Dawley rats and cynomolgus monkeys were 15% and 87%, respectively, after a single oral administration of pemafibrate (1 mg/kg). In rat plasma, unmetabolized pemafibrate was the major form, accounting for 29% of the area under the curve (AUC) of total radioactivity. In monkey plasma, in contrast, the major circulating metabolites were M2/3 (dearylated/dicarboxylic acid forms, 15%), M4 (N‐dealkylated form, 21%) and M5 (benzylic oxidative form, 9%), but pemafibrate was the notable minor form (3%). These results, in combination with the reported findings in humans, suggest that the metabolite profile of pemafibrate in plasma was different for rats and monkeys, and that monkeys could be a suitable animal model for further pharmacokinetic studies of pemafibrate in humans.     

Metabolism of the tryptamine‐derived new psychoactive substances 5‐MeO‐2‐Me‐DALT, 5‐MeO‐2‐Me‐ALCHT,and 5‐MeO‐2‐Me‐DIPT and their detectability in urine studied by GC–MS,LC–MSn,and LC‐HR‐MS/MS

Many N,N‐dialkylated tryptamines show psychoactive properties and were encountered as new psychoactive substances. The aims of the presented work were to study the phase I and II metabolism and the detectability in standard urine screening approaches (SUSA) of 5‐methoxy‐2‐methyl‐N,N‐diallyltryptamine (5‐MeO‐2‐Me‐DALT), 5‐methoxy‐2‐methyl‐N‐allyl‐N‐cyclohexyltryptamine (5‐MeO‐2‐Me‐ALCHT), and 5‐methoxy‐2‐methyl‐N,N‐diisopropyltryptamine (5‐MeO‐2‐Me‐DIPT) using gas chromatography–mass spectrometry (GC–MS), liquid chromatography coupled with multistage accurate mass spectrometry (LC–MSⁿ), and liquid chromatography‐high‐resolution tandem mass spectrometry (LC‐HR‐MS/MS). For metabolism studies, urine was collected over a 24 h period after administration of the compounds to male Wistar rats at 20 mg/kg body weight (BW). Phase I and II metabolites were identified after urine precipitation with acetonitrile by LC‐HR‐MS/MS. 5‐MeO‐2‐Me‐DALT (24 phase I and 12 phase II metabolites), 5‐MeO‐2‐Me‐ALCHT (24 phase I and 14 phase II metabolites), and 5‐MeO‐2‐Me‐DIPT (20 phase I and 11 phase II metabolites) were mainly metabolized by O‐demethylation, hydroxylation, N‐dealkylation, and combinations of them as well as by glucuronidation and sulfation of phase I metabolites. Incubations with mixtures of pooled human liver microsomes and cytosols (pHLM and pHLC) confirmed that the main metabolic reactions in humans and rats might be identical. Furthermore, initial CYP activity screenings revealed that CYP1A2, CYP2C19, CYP2D6, and CYP3A4 were involved in hydroxylation, CYP2C19 and CYP2D6 in O‐demethylation, and CYP2C19, CYP2D6, and CYP3A4 in N‐dealkylation. For SUSAs, GC–MS, LC‐MSⁿ, and LC‐HR‐MS/MS were applied to rat urine samples after 1 or 0.1 mg/kg BW doses, respectively. In contrast to the GC–MS SUSA, both LC–MS SUSAs were able to detect an intake of 5‐MeO‐2‐Me‐ALCHT and 5‐MeO‐2‐Me‐DIPT via their metabolites following 1 mg/kg BW administrations and 5‐MeO‐2‐Me‐DALT following 0.1 mg/kg BW dosage. Copyright © 2017 John Wiley & Sons, Ltd.     

Acute antidepressant response and plasma levels of bupropion and metabolites in a pediatric-aged sample: an exploratory study

Studies examining associations between antidepressant response and plasma levels of bupropion and its metabolites have yielded contradictory findings. There have been no such studies in youth. This study explored such associations in 8 boys and 8 girls, age 11 to 17 years, all prescribed bupropion sustained release (SR) for major depression (n = 6) or depressive disorder not otherwise specified (n = 10) as part of a pharmacokinetic (PK) study. All were started on morning doses of bupropion SR of 100 mg/day, and most eventually had doses increased to 200 mg/day because of inadequate clinical response. After taking prescribed dose of bupropion SR at least 14 days (median = 21 days), subjects had steady-state serial plasma levels of bupropion and its metabolites measured during a 24-hour period after morning doses. A total of 9 subjects underwent these PK assessments on doses of 100 mg/day, and 6 underwent these on doses of 200 mg/day, with 4 studied on both doses. In this 24-hour assessment, the treating psychiatrist rated subjects' antidepressant response using the Clinical Global Impression's Improvement scale (CGI-I), blind to plasma levels, but informed by child and parent rating scales of depressive symptoms and clinical interviews. Relative to 7 nonresponders, 9 responders (CGI-I < or = 2) had significantly higher mean areas under concentration curves for bupropion (P = 0.03), threohydrobupropion (P = 0.02), and erythrohydrobupropion (P = 0.02), and especially hyroxybupropion (P = 0.006). Plasma levels 7.5 hours after morning doses reaching the following cut points discriminated responders from nonresponders: bupropion > or = 37 ng/mL (P = 0.001), hydroxybupropion > or = 575 ng/mL (P = 0.003), threohydrobupropion > or = 240 ng/mL (P = 0.009), or erythrohydrobupropion > or = 45 ng/mL (P = 0.009). These preliminary findings suggest that plasma levels of bupropion and metabolites, particularly hydroxybupropion, may predict acute antidepressant response in depressed youths taking bupropion SR.     

Detection of stanozolol O‐ and N‐sulfate metabolites and their evaluation as additional markers in doping control

Stanozolol (STAN) is one of the most frequently detected anabolic androgenic steroids in sports drug testing. STAN misuse is commonly detected by monitoring metabolites excreted conjugated with glucuronic acid after enzymatic hydrolysis or using direct detection by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). It is well known that some of the previously described metabolites are the result of the formation of sulfate conjugates in C17, which are converted to their 17‐epimers in urine. Therefore, sulfation is an important phase II metabolic pathway of STAN that has not been comprehensively studied. The aim of this work was to evaluate the sulfate fraction of STAN metabolism by LC‐MS/MS to establish potential long‐term metabolites valuable for doping control purposes. STAN was administered to six healthy male volunteers involving oral or intramuscular administration and urine samples were collected up to 31 days after administration. Sulfation of the phase I metabolites commercially available as standards was performed in order to obtain MS data useful to develop analytical strategies (neutral loss scan, precursor ion scan and selected reaction monitoring acquisitions modes) to detect potential sulfate metabolites. Eleven sulfate metabolites (M‐I to M‐XI) were detected and characterized by LC‐MS/MS. This paper provides valuable data on the ionization and fragmentation of O‐ sulfates and N‐ sulfates. For STAN, results showed that sulfates do not improve the retrospectivity of the detection compared to the previously described long‐term metabolite (epistanozolol‐N ‐glucuronide). However, sulfate metabolites could be additional markers for the detection of STAN misuse. Copyright © 2016 John Wiley & Sons, Ltd.     

Single-dose pharmacokinetics of bupropion in adolescents: effects of smoking status and gender.

Sustained-release (SR) bupropion (Zyban) is approved as a smoking cessation aid for adults. Since smoking often begins in adolescence, we determined the single-dose pharmacokinetics of bupropion SR in 75 adolescent subjects ranging from 13 to 18 years old. Subjects self-reported their smoking status. Urinary cotinine concentration was used to verify smoking status. Thirty-seven subjects (18 males, 19 females) were classified as cigarette smokers and 38 were nonsmokers (19 males, 19 females). Fasted subjects received one tablet (150 mg) of bupropion SR, and plasma samples were collected before (0) and 1/2, 1, 2, 3, 4, 6, 8, 24, 48, and 72 hours after dosing. Plasma samples were analyzed for bupropion and its three major metabolites (hydroxybupropion and the aminoalcohol isomers, erythrohydrobupropion plus threohydrobupropion, expressed as a composite) by solid-phase extraction, followed by LC/MS/MS. Factorial analysis of variance (ANOVA) was used to evaluate the effects of smoking and gender on pharmacokinetic parameters. Smokers and nonsmokers differed significantly (p < 0.05) in age and urinary cotinine (p < 0.01) concentration but did not differ significantly in mean weight, height, body surface area, or body mass index. The pharmacokinetic (PK) parameters for bupropion and hydroxybupropion did not differ between smokers and nonsmokers, but differences were found between male and female subjects. Mean values for area under the plasma concentration versus time curve (AUC0-->infinity), volume of distribution (Vd beta) normalized to body weight, maximum plasma concentration (Cmax), and elimination half-life (t1/2 beta) for bupropion were significantly (p < 0.05) greater in females than males, while clearance of bupropion normalized to body weight (CL/f) did not differ between males and females. Females also exhibited significantly (p < 0.05) larger values for hydroxybupropion mean AUC0-->infinity and Cmax than males. The mean ratio of hydroxybupropion to bupropion AUC for adolescents was approximately 4 to 5, which is lower than that previously reported for adults. In conclusion, smoking status does not affect the single-dose pharmacokinetics of bupropion SR in adolescents. However, females differ from males in several potentially important PK parameters for bupropion and its major metabolite, hydroxybupropion.     

Pharmacokinetics of cytisine,an α4β2 nicotinic receptor partial agonist,in healthy smokers following a single dose

Cytisine, an α₄β₂ nicotinic receptor partial agonist, is a plant alkaloid that is commercially extracted for use as a smoking cessation medication. Despite its long history of use, there is very little understanding of the pharmacokinetics of cytisine. To date, no previous studies have reported cytisine concentrations in humans following its use as a smoking cessation agent. A high performance liquid chromatography‐ultraviolet (HPLC‐UV) method was developed and validated for analysis of Tabex® and nicotine‐free oral strips, two commercial products containing cytisine. A sensitive liquid chromatography‐mass spectrometry (LC‐MS) method was developed and validated for the quantification of cytisine in human plasma and for the detection of cytisine in urine. Single‐dose pharmacokinetics of cytisine was studied in healthy smokers. Subjects received a single 3 mg oral dose administration of cytisine. Cytisine was detected in all plasma samples collected after administration, including 15 min post‐dose and at 24 h. Cytisine was renally excreted and detected as an unchanged drug. No metabolites were detected in plasma or urine collected in the study. No adverse reactions were reported. Copyright © 2014 John Wiley & Sons, Ltd.     

Quantitation of escitalopram and its metabolites by liquid chromatography‐tandem mass spectrometry in psychiatric patients: New metabolic ratio establishment

Therapeutic drug monitoring (TDM) is used to determine the concentration of drug in plasma/serum to adjust the dose of the therapeutic drug. Selective and sensitive analytical methods are used to determine drug and metabolite levels for the successful application of TDM. The aim of the study was to develop and validate using LC‐MS/MS to analyse quantitative assay of escitalopram (S‐CT) and metabolites in human plasma samples. In order to provide a convenient and safe treatment dose, it was aimed to determine the levels of S‐CT and its metabolites in the patients’ plasma. A new method with short sample preparation and analysis time was developed and validated using LC‐MS/MS to analyse quantitative assay of S‐CT and its metabolites in plasma. Also, plasma samples of 30 patients using 20 mg S‐CT between the ages of 18 and 65 years were analysed by the validated method. The mean values of S‐CT, demethyl escitalopram and didemethyl escitalopram in plasma of patients were 27.59, 85.52 and 44.30 ng/mL, respectively. At the end of the analysis, the metabolic ratio of S‐CT and metabolites was calculated. It is considered that the method for the quantitative analysis of S‐CT and its metabolites in human plasma samples may contribute to the literature on account of its sensitive and easy application. Additionally, the use of our data by physicians will contribute to the effective drug treatment for their patients who take S‐CT.     

Interaction of bupropion and zinc with neuronal nicotinic acetylcholine receptors

Zinc is known to exert antidepressant-like actions and to make the effects of some antidepressants more efficient in animal models of depression. Both zinc and bupropion interact with nicotinic acetylcholine receptors (nAChRs) which are related with depression. Here we examined the effects of bupropion, in the absence and presence of zinc, on the ion current elicited by acetylcholine (ACh-current) in Xenopus oocytes expressing neuronal α4β4 nAChRs. We found that bupropion-inhibited ACh-currents depending on ACh and bupropion concentrations. Thus, the IC₅₀ of bupropion was lower with a higher ACh concentration: 3.51 and 2.27 μM for the current elicited with 0.5 and 2 μM ACh, respectively. The inhibitory effect of bupropion was more potent in the presence of zinc, e.g. the IC₅₀ was 0.81 μM in the presence of 100 μM zinc and 2 μM ACh. Furthermore, the zinc-potentiated ACh-current decreased with increasing bupropion concentration. Thus, zinc potentiation was 5.05 and 1.25 fold of the ACh-current inhibited by 10 nM and 5 μM bupropion, respectively. The ACh-current inhibited by 3 μM bupropion was voltage-independent, decreasing to 0.48 of the ACh-current at all voltages. Zinc potentiation of the bupropion-inhibited ACh-current was slight and voltage-independent. In addition, the zinc-potentiated ACh-current was slightly voltage-dependent: 1.8 fold of the ACh-current at −120 mV and 2.3 at −40 mV. Bupropion inhibition of the zinc-potentiated ACh-current was strong and voltage-independent, decreasing to 0.15 of the zinc-potentiated ACh-current at all voltages. Accordingly, zinc may interact within the ion channel, whereas bupropion, and bupropion in the presence of zinc (which causes greater inhibition) interact in an external region of the receptor–channel complex. These results suggest that bupropion interacts with α4β4 nAChRs in a non-competitive manner, that zinc increases the sensitivity of nAChRs to bupropion, and that bupropion decreases the sensitivity of nAChRs to zinc.     

Wuzhi capsule regulates chloroacetaldehyde pharmacokinetics behaviour and alleviates high‐dose cyclophosphamide‐induced nephrotoxicity and neurotoxicity in rats

High‐dose cyclophosphamide (HD‐CTX) treatment often leads to severe nephrotoxicity and neurotoxicity, which are mainly caused by one of its metabolites, chloroacetaldehyde (CAA). However, there are no effective antidotes to prevent these side effects. The objective of this study was to evaluate the effect of Wuzhi Capsule (WZC) on the pharmacokinetics of CTX and its metabolites in rats, and the attenuation of CAA induced kidney and brain injuries, which was produced at equimolar with 2‐dechloroethylcyclophosphamide. Rats were treated with single‐ or multiple‐dose of WZC when giving HD‐CTX, and the plasma concentration of CTX and its metabolites were quantitated by UHPLC‐MS/MS Single‐dose, not multiple‐dose of WZC co‐administration (300 mg/kg) significantly reduced C_max and AUC_0→24 h of DC‐CTX by 33.10% and 35.51%, respectively. Biochemical assay suggested oxidative stress was involved in kidney and brain injuries by HD‐CTX, which were attenuated by single‐dose WZC (300 mg/kg) pre‐treatment, with increased glutathione, glutathione peroxidase and superoxide dismutase contents/or activities in both tissues and plasma (P < 0.05). Meanwhile, WZC pre‐treatment could also significantly decrease the plasma levels of creatinine, blood urea nitrogen and malondialdehyde (P < 0.05). Additionally, WZC treatment improved the morphology and pathology condition of the kidneys and brains in rats. In conclusion, single‐dose WZC co‐administration decreased CAA production and exerted protective effect on CTX‐induced oxidative stress in kidney and brain, whereas repetitive WZC co‐administration with CTX was probably not recommended.     

Solid‐phase extraction–liquid chromatography–tandem mass spectrometry method for the qualitative analysis of 61 synthetic cannabinoid metabolites in urine

This article comprises the development and validation of a protocol for the qualitative analysis of 61 phase I synthetic cannabinoid metabolites in urine originating from 29 synthetic cannabinoids, combining solid‐phase extraction (SPE) utilizing a reversed phase silica‐based sorbent (phenyl) with liquid chromatography–tandem mass spectrometry (LC?MS/MS). Validation was performed according to the guidelines of the German Society of Toxicological and Forensic Chemistry. Sufficient chromatographic separation was achieved within a total runtime of 12.3 minutes. Validation included specificity and selectivity, limit of detection (LOD), recovery and matrix effects, as well as auto‐sampler stability of processed urine samples. LOD ranged between 0.025 ng/mL and 0.5 ng/mL in urine. Recovery ranged between 43% and 97%, with only two analytes exhibiting recoveries below 50%. However, for those two analytes, the LODs were 0.05 ng/mL in urine. In addition, matrix effects between 81% and 185% were determined, whereby matrix effects over 125% were observed for 10 non‐first‐generation synthetic cannabinoid metabolites. The developed method enables the rapid and sensitive detection of synthetic cannabinoid metabolites in urine, complementing the spectrum of existing analytical tools in forensic case work. Finally, application to 61 urine samples from both routine and autopsy case work yielded one urine sample that tested positive for ADB‐PINACA N‐pentanoic acid.     

Metabolism and pharmacokinetics in rats of ganoderiol F,a highly cytotoxic and antitumor triterpene from <Emphasis Type="Italic">Ganoderma lucidum</Emphasis>

The metabolism of ganoderiol F (GF), a cytotoxic and antitumor triterpene from Ganoderma lucidum, by intestinal bacteria and its pharmacokinetics in rats were investigated by using liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). GF was converted to ganodermatriol by anaerobic incubation with bacterial mixtures from rats and humans. This metabolite was detected in rat feces, but not in plasma and urine, after oral administration of GF. The fate of GF after oral (p.o.) and intravenous (i.v.) administration to rats was examined in pharmacokinetics studies. Plasma samples pretreated by solid-phase extraction were quantified by HPLC/MS/MS over a GF concentration range of 1.25–100 ng/ml (S/N = 5). The intra- and interday precision (CV%) was below 8% and accuracy was within the range of 95.9–103.6% for all samples. The range of recovery ratios was 89.2–98.2%. After the administration of GF at 0.5 mg/kg i.v., the plasma concentrations of GF quickly declined and the elimination half-life values (t _1/2α and t _1/2β) were about 2.4 and 34.8 min. On the other hand, the elimination half-life values (t _1/2α) after p.o. administration of GF at doses of 20 and 50 mg/kg were 14.4 and 143.3 min for the former, and 18.6 and 114.6 min for the latter. The AUC_0–t value was 11.17 (ng/ml) h at a GF dose of 0.5 mg/kg i.v., but 49.4 and 111.6 (ng/ml) h at GF doses of 20 and 50 mg/kg p.o., respectively, indicating that the AUC_0–t value is proportional to the administered oral doses. The estimated absolute bioavailability of GF in rats was F = 0.105.     

Mechanism of an unusual,but clinically significant,digoxin–bupropion drug interaction

An unusual, but clinically significant, digoxin (DIG)–bupropion (BUP) drug interaction (DDI), in which BUP increased DIG renal clearance by 80% is reported. To investigate the mechanism(s) of this unusual DDI, first the effect of BUP, its circulating metabolites or their combination on [³H]‐DIG transport by cells expressing human P‐gp or human OATP4C1 was determined. Second, the study asked whether this DDI could be replicated in the rat so that it could be used to conduct mechanistic studies. Then, the effect of BUP and its rat metabolites on [³H]‐DIG transport were tested by cells expressing rat Oatp4c1. Bupropion and its metabolites had no effect on human P‐gp mediated transepithelial transport of [³H]‐DIG. Bupropion and hydroxybupropion (HBUP) significantly stimulated H‐OATP4C1 mediated transport of [³H]‐DIG. In addition, BUP cocktail (BUP plus its metabolites) significantly increased the H‐OATP4C1 mediated transport of [³H]‐DIG, and partially reversed the inhibition by 100 µm DIG. However, erythro‐hydrobupropion (EBUP) and threo‐hydrobupropion (TBUP) did not affect the [³H]‐DIG uptake by H‐OATP4C1 cells. Bupropion administration significantly increased digoxin renal clearance in rats. Surprisingly, bupropion significantly inhibited r‐Oatp4c1 mediated transport of [³H]‐DIG at clinically relevant unbound plasma concentrations of BUP or those observed in the rat study, while HBUP or TBUP did not. These data support our hypothesis that at clinically relevant plasma concentrations, bupropion and its metabolites activate H‐OATP4C1 mediated DIG tubular secretion, and could possibly explain the increase in digoxin renal clearance produced by bupropion. While bupropion increased digoxin renal clearance in the rat, it appeared to do so by inhibiting r‐Oatp4c1‐mediated digoxin renal reabsorption. Copyright © 2014 John Wiley & Sons, Ltd.     

Identification and quantification of predominant metabolites of synthetic cannabinoid MAB‐CHMINACA in an authentic human urine specimen

An autopsy case in which the cause of death was judged as drug poisoning by two synthetic cannabinoids, including MAB‐CHMINACA, was investigated. Although unchanged MAB‐CHMINACA could be detected from solid tissues, blood and stomach contents in the case, the compound could not be detected from a urine specimen. We obtained six kinds of reference standards of MAB‐CHMINACA metabolites from a commercial source. The MAB‐CHMINACA metabolites from the urine specimen of the abuser were extracted using a QuEChERS method including dispersive solid‐phase extraction, and analyzed by liquid chromatography–tandem mass spectrometry with or without hydrolysis with β‐glucuronidase. Among the six MAB‐CHMINACA metabolites tested, two predominant metabolites could be identified and quantified in the urine specimen of the deceased. After hydrolysis with β‐glucuronidase, an increase of the two metabolites was not observed. The metabolites detected were a 4‐monohydroxycyclohexylmethyl metabolite M1 (N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐((4‐hydroxycyclohexyl)methyl)‐1H–indazole‐3‐carboxamide) and a dihydroxyl (4‐hydroxycyclohexylmethyl and tert‐butylhydroxyl) metabolite M11 (N‐(1‐amino‐4‐hydroxy‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐((4‐hydroxycyclohexyl)methyl)‐1H–indazole‐3‐carboxamide). Their concentrations were 2.17 ± 0.15 and 10.2 ± 0.3 ng/mL (n = 3, each) for M1 and M11, respectively. Although there is one previous in vitro study showing the estimation of metabolism of MAB‐CHMINACA using human hepatocytes, this is the first report dealing with in vivo identification and quantification of MAB‐CHMINACA metabolites in an authentic human urine specimen.     

Decarbonylation: A metabolic pathway of cannabidiol in humans

Cannabidiol (CBD) is a non‐psychoactive cannabinoid, which is of growing medical interest. Previous studies on the metabolism of CBD showed mainly the formation of hydroxylated or oxidized derivatives, the formation of carboxylic acids or modifications of the aliphatic side chain. Using incubation of CBD with hepatic microsomes of mice, the formation of carbon monoxide was reported. We investigated the phase I metabolism of CBD and cannabidivarin (CBDV) using in vitro experiments with human liver microsomes in order to discover so far not considered metabolites. Identification of metabolites was done by liquid chromatography coupled with quadrupole time of flight mass spectrometry (LC?QToF?MS). Within these experiments, we came across decarbonylation of CBD and CBDV. Further investigations were focused on observed decarbonylated CBD (DCBD). To confirm this metabolite in humans in vivo, plasma samples containing large amounts of cannabinoids as well as serum and urine samples, collected after a voluntary intake of a CBD‐containing food supplement, were analyzed by LC coupled to triple quadrupole mass spectrometry (LC?QQQ?MS). DCBD was detected in in vitro incubation mixtures, serum samples, and urine samples (after alkaline or enzymatic hydrolysis) collected after the voluntary intake, as well as in plasma samples of cannabis users. DCBD appears to be an important supplementary human metabolite that might be helpful for the analytical confirmation of a CBD uptake and might improve the interpretation of the consumption of CBD‐containing products. Results of this study indicate a prolonged detectability of DCBD (in serum) in comparison to CBD after oral CBD ingestion.     

Development and validation of an LC–MS/MS method for the determination of quetiapine and four related metabolites in human plasma

Pharmacokinetic measurement of the psychotropic compound quetiapine and four related metabolites in human plasma was conducted using a sensitive and specific liquid–chromatography tandem mass spectrometry (LC–MS/MS) assay that has been developed and validated for this purpose. The assay employs a single liquid–liquid extraction of quetiapine and its N-desalkyl (norquetiapine, M211,803, M1), 7-hydroxy (M214,227, M2), 7-hydroxy N-desalkyl (M236,303, M3), and sulfoxide (M213,841, M4) metabolites from human plasma, and utilizes dual-column separation, using Luna C₁₈ columns (50 mm × 2.0 mm, 5 μm) and positive ionization tandem MS detection in the multiple reaction monitoring (MRM) mode of the analytes and their respective stable labeled internal standards. The method provides a linear response from a quantitation range of <0.70 ng/ml to at least 500 ng/ml for each analyte using 40 μl of plasma. The applicable range was extended by dilution up to 100-fold with blank matrix. The accuracy and precision for quetiapine were less than 6.0% and 6.4% for quetiapine, respectively. The accuracy (and precision) was less than 9.4% (5.9%) for norquetiapine; 6.4% (6.2%) for M2; and 10.0% (6.4%) for M3; and 8.6% (9.5%) for M4. This methodology enabled the determination of the pharmacokinetics of quetiapine and its metabolites in human plasma, and an example of its application is presented.     

Species differences in metabolism of a new antiepileptic drug candidate,DSP‐0565 [2‐(2′‐fluoro[1,1′‐biphenyl]‐2‐yl)acetamide]

The metabolism and pharmacokinetics of DSP‐0565 [2‐(2′‐fluoro[1,1′‐biphenyl]‐2‐yl)acetamide], an antiepileptic drug candidate, was investigated in rats, dogs, and humans. In human hepatocytes, [¹⁴C]DSP‐0565 was primarily metabolized via amide bond hydrolysis to (2′‐fluoro[1,1′‐biphenyl]‐2‐yl)acetic acid (M8), while in rat and dog hepatocytes, it was primarily metabolized via both hydrolysis to M8 and hydroxylation at the benzene ring or the benzyl site to oxidized metabolites. After single oral administration of [¹⁴C]DSP‐0565 to rats and dogs, the major radioactivity fraction was recovered in the urine (71–72% of dose) with a much smaller fraction recovered in feces (23–25% of dose). As primary metabolites in their excreta, M8, oxidized metabolites, and glucuronide of DSP‐0565 were detected. The contribution of metabolic pathways was estimated from metabolite profiles in their excreta: the major metabolic pathway was oxidation (57–62%) and the next highest was the hydrolysis pathway (23–33%). These results suggest that there are marked species differences in the metabolic pathways of DSP‐0565 between humans and animals. Finally, DSP‐0565 human oral clearance (CL/F) was predicted using in vitro–in vivo extrapolation (IVIVE) with/without animal scaling factors (SF, in vivo intrinsic clearance/in vitro intrinsic clearance). The SF improved the underestimation of IVIVE (fold error = 0.22), but the prediction was overestimated (fold error = 2.4–3.3). In contrast, the use of SF for hydrolysis pathway was the most accurate for the prediction (fold error = 1.0–1.4). Our findings suggest that understanding of species differences in metabolic pathways between humans and animals is important for predicting human metabolic clearance when using animal SF.     

Identification of domperidone metabolites in plasma and urine of gastroparesis patients with LC–ESI-MS/MS

Abstract

1. Domperidone is a prokinetic agent used to treat gastroparesis. Previous studies reported oxidative metabolites of domperidone, detected by radiometric high-performance liquid chromatography or single quadrupole mass spectrometric techniques. Our aim was to identify domperidone Phase I and Phase II metabolites using liquid chromatography combined with electrospray ionization-enabled tandem mass spectrometry.

2. Domperidone metabolites were identified in the plasma and urine of 11 gastroparesis patients currently being treated with domperidone. In addition, oxidative and conjugative metabolites of domperidone were characterized in human liver subcellular fractions.

3. Seven metabolites were detected in vivo. Domperidone was metabolized to two mono-hydroxylated metabolites (M1 and M2), a de-alkylated metabolite (M5) and a di-hydroxylated metabolite (M7). The mono-hydroxylated metabolites were further glucuronidated to M8, M9 and sulfated to M11. To the best of our knowledge, M7, M8, M9 and M11 have not been reported previously. Five additional metabolites were identified in vitro in human subcellular fractions which comprise two additional mono-hydroxylated metabolites (M3 and M4), an alcohol metabolite (M6) possibly formed from an aldehyde intermediate, and other conjugative metabolites (M10 and M12). M6, M10 and M12 have not been characterized previously.

4. In total, 12 domperidone metabolites including 7 new metabolites were identified in the present study. These results allow a better understanding of domperidone disposition in humans.