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

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.

2-Methyl-4'-(methylthio)-2-morpholinopropiophenone: A commercial photoinitiator being used as a new psychoactive substance.

Synthetic cathinones, which are novel psychoactive substances, have caused major social problems worldwide. A substance called 2-methyl-4'-(methylthio)-2-morpholinopropiophenone (MMMP), which is employed as a commercial industrial photoinitiator for triggering polymerization, has a basic cathinone backbone; however, few reports regarding MMMP have been published. In the current study, three potential metabolites of MMMP-namely hydroxy-MMMP (HO-MMMP), HO-MMMP-sulfoxide (HO-MMMP-SO), and HO-MMMP-sulfone (HO-MMMP-SO2)-were successfully synthesized, and MMMP and these three potential metabolites were used as standards to establish an analytic method based on liquid chromatography-tandem mass spectrometry for the quantitative analysis of urine. This analytic method and related parameters-including dynamic range, limit of quantification, selectivity, precision, accuracy, carryover effect, matrix effect, interference, and dilution integrity-were optimized and validated. Forty urine samples from 1,691 individuals who abused drugs were determined to contain MMMP, HO-MMMP, HO-MMMP-SO, or HO-MMMP-SO2; the results of this study indicate that approximately 2.37 % of drug abusers in Taiwan consumed MMMP in 2023. These 40 urine samples were analyzed to investigate the metabolism of MMMP in humans. The results indicate that HO-MMMP-SO is the main metabolite in human urine. This study recommends HO-MMMP-SO with a concentration of 2 ng/mL as a target and cutoff value, respectively, for identifying individuals who have consumed MMMP.

Novel methods for assessing oral direct factor Xa and thrombin inhibitors: use of point-of-care testing and urine samples.

Rivaroxaban and dabigatran are new oral anticoagulants (NOACs) that inhibit directly factor Xa and thrombin, respectively. These NOACs effectively prevent thromboembolic complications using fixed doses without the need for dose adjustment according to laboratory results. About 60% of rivaroxaban is cleared from circulation by glomerular filtration, 30% of which is excreted as active drug. About 80% of dabigatran is excreted into urine as active compound. Accordingly, both NOACs can be determined in urine by means of chromatographic methods. Only a few laboratories are able to perform such methods, and results are not available within short time frames. New methods have to be developed to obtain results within minutes and possibly as point-of-care (POC) techniques. This testing may be useful for special patient populations such as those with acute deterioration of renal function due to any disease, before surgical interventions, during unexpected bleeding or thrombotic episodes while on therapy with NOACs, the oldest and youngest populations, pregnancy, suspicion of overdose and intoxication, and to determine adherence to therapy. Here we describe results of a POC qualitative assay using urine samples from patients on treatment with dabigatran and rivaroxaban.

Stable isotope gas chromatography-tandem mass spectrometry determination of aminoethylcysteine ketimine decarboxylated dimer in biological samples.

Aminoethylcysteine ketimine decarboxylated dimer (AECK-DD; systematic name: 1,2-3,4-5,6-7,8-octahydro-1,8a-diaza-4,6-dithiafluoren-9(8aH)-one) is a previously described metabolite of cysteamine that has been reported to be present in mammalian brain, urine, plasma, and cells in culture and vegetables and to possess potent antioxidative properties. Here, we describe a stable isotope gas chromatography-tandem mass spectrometry (GC-MS/MS) method for specific and sensitive determination of AECK-DD in biological samples. (13)C(2)-labeled AECK-DD was synthesized and used as the internal standard. Derivatization was carried out by N-pentafluorobenzylation with pentafluorobenzyl bromide in acetonitrile. Quantification was performed by selected reaction monitoring of the mass transitions m/z 328 to 268 for AECK-DD and m/z 330 to 270 for [(13)C(2)]AECK-DD in the electron capture negative ion chemical ionization mode. The procedure was systematically validated for human plasma and urine samples. AECK-DD was not detectable in human plasma above approximately 4nM but was present in urine samples of healthy humans at a maximal concentration of 46nM. AECK-DD was detectable in rat brain at very low levels of approximately 8pmol/g wet weight. Higher levels of AECK-DD were detected in mouse brain (∼1nmol/g wet weight). Among nine dietary vegetables evaluated, only shallots were found to contain trace amounts of AECK-DD (∼6.8pmol/g fresh tissue).

Excretion of compound M-11 and its metabolites with urine and feces in rats.

The excretion of compound M-11 and its metabolites with the urine and feces was studied in rats after intraperitoneal and oral administration in a dose of 25 mg/kg. Experiments showed that 1% metabolites were detected in excretions over 24 h irrespective of the route of administration, while the initial compound was not found even in trace amounts.

[Pharmacokinetics of afobazole metabolite (M-11) in rats].

Pharmacokinetics of compound M-11 (main metabolite of afobazole) after administration via different routes was studied in rats. After oral and intravenous administration, M-11 exhibited weakly pronounced bioconversion with the formation of a few metabolites that could be detected in plasma samples for about 3 hours. The absolute bioavailability of M-11 after oral administration was 68.3%. It was found that M-11 was completely absorbed from gastrointestinal tract of rats and characterized by "the first pass effect", after which approximately 70% of administered dose entered the circulation. The parent substance was determined neither in urine nor in feces.

Toxicological detection of pholcodine in blood, urine and hair in three cases of fatal intoxication.

Pholcodine is an opioid antitussive reputed for its low toxicity and absence of addictive effect. We report three cases of pholcodine intoxication with fatal outcome. Large concentrations of pholcodine were quantified by gas chromatography coupled to mass spectrometry (GC/MS) in peripheral postmortem blood (respectively 2890 ng/mL, 979 ng/mL and 12,280 ng/mL). Segmental hair analyses by GC/MS and detected pholcodine in three 1.5-2 cm segments (38-161 ng/mg, 8.54-41.6 ng/mg, and 0.26-2.66 ng/mg, respectively). These findings underline that pholcodine can be involved in fatal poisoning and raise the question of misuse or abuse and of taking account of this drug in opioid overdose prevention policies.

Metabolism, pharmacokinetics, and excretion of the 5-hydroxytryptamine1b receptor antagonist elzasonan in humans.

The metabolism, pharmacokinetics, and excretion of a potent and selective 5-hydroxytryptamine(1B) receptor antagonist elzasonan have been studied in six healthy male human subjects after oral administration of a single 10-mg dose of [(14)C]elzasonan. Total recovery of the administered dose was 79% with approximately 58 and 21% of the administered radioactive dose excreted in feces and urine, respectively. The average t(1/2) for elzasonan was 31.5 h. Elzasonan was extensively metabolized, and excreta and plasma were analyzed using mass spectrometry and NMR spectroscopy to elucidate the structures of metabolites. The major component of drug-related material in the excreta was in the feces and was identified as 5-hydroxyelzasonan (M3), which accounted for approximately 34% of the administered dose. The major human circulating metabolite was identified as the novel cyclized indole metabolite (M6) and accounted for ∼65% of the total radioactivity. A mechanism for the formation of M6 is proposed. Furthermore, metabolism-dependent covalent binding of drug-related material was observed upon incubation of [(14)C]elzasonan with liver microsomes, and data suggest that an indole iminium ion is involved. Overall, the major metabolic pathways of elzasonan were due to aromatic hydroxylation(s) of the benzylidene moiety, N-oxidation at the piperazine ring, N-demethylation, indirect glucuronidation, and oxidation, ring closure, and subsequent rearrangement to form M6.

Distribution, Metabolism, and Excretion of Gedatolisib in Healthy Male Volunteers After a Single Intravenous Infusion.

In this open-label study (NCT02142920), we investigated the distribution, pharmacokinetics, and metabolism of the pan-class-I isoform phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor gedatolisib (PF-05212384), following a single intravenous administration in healthy male subjects. A single, 89-mg, intravenous dose of gedatolisib was associated with a favorable safety profile in the 6 healthy subjects evaluated. Peak plasma concentrations for unchanged gedatolisib and total radioactivity were observed at the end of the 30-minute infusion. The only observed drug-related material in plasma was the parent drug, gedatolisib. Terminal half-life for plasma gedatolisib was ∼37 hours. Following the dose, 66%-73% of drug-related material was recovered in the feces. Metabolism of gedatolisib was trace; only 1 oxidative metabolite, M5, was identified in feces (<1% of total dose). Identification of gedatolisib in feces suggests that biliary and/or intestinal secretion of unchanged parent drug significantly contributes to gedatolisib clearance.

A Fatality Involving Furanylfentanyl and MMMP, with Presumptive Identification of Three MMMP Metabolites in Urine.

The prevalence of new psychoactive substances (NPS) on the illicit drug market continues to grow, with new analogs being routinely synthesized. Routes of administration for these compounds are also diversifying, and recent research has shown an increase in the incorporation of NPS into vaping liquids. Among the most commonly encountered NPS are the cathinone and fentanyl analogs. Fentanyl analogs in particular have been implicated in a significant number of deaths, usually in combination with other prescription and illicit drugs. We report the case of a 44-year-old male with a history of polysubstance abuse found deceased at his home address. Items located within the vicinity of the deceased were found to contain furanylfentanyl and 2-methyl-4'-(methylthio)-2-morpholinopropiophenone (MMMP also known as MTMP, MMTMP, Irgacure 907 and Caccure 907). Both of these compounds were detected in the post-mortem peripheral blood of the deceased: furanylfentanyl at 1.6 ng/mL and MMMP at 6.7 ng/mL. MMMP is an unrestricted, commercially available photo-initiator used in the printing and polymer industry, which structurally can be classed as a highly modified cathinone. Although MMMP has been found previously in drug seizures, this is the first fatality in which MMMP has been detected. A number of other prescription and illicit drugs were also detected in the blood. MMMP was not detected in the post-mortem urine; however three metabolites, beta-hydroxy-MMMP, beta-hydroxy-MMMP-sulfoxide and beta-hydroxy-MMMP-sulfone, were presumptively identified. The significance of MMMP to the cause of death is uncertain as its pharmacological and toxicological profile is unclear.

[Afobazole metabolism in rats].

Metabolism of afobazole in rats has been studied using mass-spectrometry and HPLC, which revealed 17 products of afobazole biotransformation along with the parent compound. The structures of six afobazole metabolites were established and confirmed by comparison of HPLC retention times with the synthetic reference compounds and HPLC/mass spectrometry. Other metabolites were characterized by the masses of molecular ions. A significant fraction of the drug dose is biotransformed with the formation of hydroxylated benzimidazole moiety and oxidated morpholine.

[Pharmacokinetics of afobazole in rats].

Afobazole pharmacokinetics was studied after the administration via different ways in rats. After oral administration, afobazole is subject to intensive biotransformation with the formation of several metabolites (M-6, M-7, and M-11). The drug and its metabolites were detected in the blood plasma for 3 h and characterized by a high elimination rate after both oral and intravenous administration. Afobazole and its main metabolite (M-11) had a medium rate of permeability into brain (the target organ): the tissue availability was 0.584 for afobazole and 0.793 for M-11. The absolute bioavailability of afobazole upon oral administration was 43.6% for. Afobazole was completely absorbed from the gastrointestinal tract of rats and characterized by the first-pass effect, after which more than 40% of administered dose entered the circulation. The parent compound was determined in extremely low amounts in urine and feces: its content in 24-h urine on the average did not exceed 0.07% of the administered dose; in 24-h feces, it did not exceed 0.05 % after intravenous administration and 0.01% after oral administration).

Penetration of rat skin by N-nitrosodiethanolamine and N-nitrosomorpholine.

N-Nitrosomorpholine (NMOR) and N-nitrosodiethanolamine (NDELA) were painted on the clipped upper dorsal skin of male F344 rats. NDELA was applied undiluted, dissolved in water, and dissolved in cutting oil; NMOR was applied dissolved in water and in ethyl acetate. Aqueous solutions of the nitrosamines were used for gavage. Rats were housed individually. Blood and urine samples were analyzed for nitrosamines by chromatography combined with a Thermal Energy Analyzer. Maximum penetration of NMOR was approximately equal to 34% 2 hours after application of 5 mg to the skin or by gavage; less than 1% appeared in the urine in 24 hours. Skin painting with NDELA in water (20 mg/100 microliters) and in cutting oil (25 mg/25 microliters) yielded small concentrations of NDELA (always < 25 micrograms/ml blood). When 50 mg of undiluted NDELA was painted on the skin, 130 to 220 micrograms/ml of blood was recovered after 1 hour. Administering 50 mg NDELA in water by gavage yielded similar blood concentrations. Maximum skin penetration observed with NDELA was 78% 1 hour after application of 50 mg. From 20 to 30% of the NDELA applied undiluted and by gavage appeared in the urine in 24 hours. Although animals and humans differ, skin exposure to NMOR or NDELA represents a risk due to absorption.

Identification of 1,4-thiomorpholine-3-carboxylic acid (TMA) in normal human urine.

The sulfur-containing cyclic imino acid 1,4-thiomorpholine-3-carboxylic acid (TMA) has been identified in normal human urine. After the enrichment procedure with ion-exchange chromatography, the urine extracts were reacted with diazomethane. Gas-liquid chromatography revealed the presence of two peaks with the same retention times exhibited by authentic TMA after the same derivatization. The two compounds have been identified by mass-spectrometry as the monomethylated and dimethylated derivatives of TMA. This result represents the first indication of the occurrence of TMA in a mammalian sample.

Pharmacokinetics of single-dose reboxetine in volunteers with renal insufficiency.

Reboxetine is a new selective norepinephrine reuptake inhibitor (selective NRI) for the short- and long-term treatment of depression that is effective and well tolerated at a dose of 8 to 10 mg/day. This study assessed the pharmacokinetics of reboxetine in volunteers with renal impairment. A single 4 mg dose of reboxetine was administered to a total of 18 volunteers with mild (n = 6), moderate (n = 6), or severe (n = 6) renal impairment (creatinine clearance: 56-64, 26-51, and 9-19 ml/min, respectively), and reboxetine concentrations were measured in plasma by HPLC. Mean AUC infinity increased by 43% (mild vs. severe; p < 0.01) as renal function declined, while renal clearance and total urinary excretion of unchanged reboxetine decreased by 67% and 62%, respectively (mild vs. severe; p < 0.01 for both parameters). tmax and t1/2 were not significantly different between groups. In comparison with historical data from young healthy volunteers, AUC infinity and t1/2 are at least doubled in volunteers with renal impairment, while CLr is halved. This pharmacokinetic study has shown that increasing renal dysfunction leads to increasing systemic exposure to reboxetine, particularly in severe renal insufficiency, although reboxetine was well tolerated by all volunteers. Thus, a reduction of the starting dose of reboxetine to 2 mg twice daily would be prudent in patients with renal dysfunction.

Review of the pharmacokinetics and metabolism of reboxetine, a selective noradrenaline reuptake inhibitor.

The pharmacokinetics and metabolism of reboxetine, a selective noradrenaline reuptake inhibitor, in humans and animal models are reviewed here. Reboxetine has potent antidepressant activity, low affinity for alpha-adrenergic and muscarinic receptors and low toxicity in animals. It is a mixture of (R,R) and (S,S) enantiomer, the latter being more potent but no qualitative differences in pharmacodynamic properties are observed between the two. Humans rapidly absorb reboxetine (tmax about 2 h) with a terminal half-life of elimination (t1/2) of 13 h, allowing twice-daily administration. Animal models also rapidly absorb reboxetine (tmax 0.5-2 h) but t1/2 was 1-2 h. Food does not affect bioavailability. There were no major inter-species differences in the metabolic profile of reboxetine. Elimination is principally renal in humans and monkeys. Reboxetine has linear pharmacokinetics in young, healthy males for single doses of 1-5 mg and in elderly, female depressed patients (up to 4 mg b.i.d.). Multiple dosing, gender or liver insufficiency had no significant effects on the pharmacokinetics. Elderly (particularly frail elderly) patients and patients with severe renal impairment may need dose reduction. Reboxetine shows no clinically relevant interaction with lorazepam and has no inhibitory effects on the major enzymes involved in drug metabolism. It may be possible to use reboxetine in combination with monoamine oxidase inhibitors as it has no inhibitory effect on this enzyme; in addition, it may protect patients against tyramine-induced reactions. In conclusion, reboxetine seems to be an antidepressant with negligible interference with the pharmacokinetics of other drugs thus fewer drug-drug interactions are expected.

Determination of N-methylmorpholine in air samples from a polyurethane foam factory. Comparison between two methods using gas chromatography and isotachophoresis for analysis.

The N-methylmorpholine levels in workroom air in a polyurethane foam factory were determined by two methods in which midget impinger flasks were used for sampling. The analyses were performed by gas chromatography and isotachophoresis. The values obtained by the gas chromatographic method were 19% higher than those of the isotachophoretic method. Determinations of the amine in samples generated in laboratory experiments showed no statistical difference between the two methods. The mean air concentrations of N-methylmorpholine in different work areas of the factory ranged from 7 to 22 mg/m3. Urine was collected from seven workers and analyzed for N-methylmorpholine by gas chromatography. The amine concentrations and the excretion rates increased considerably during the workday.

Determination of a substance P antagonist in human plasma and urine using high-performance liquid chromatography with ultraviolet absorbance and tandem mass spectrometric detection.

A high-performance liquid chromatographic (HPLC) assay using ultraviolet (UV) detection was developed and compared with a HPLC method with tandem mass spectrometric (HPLC/MS-MS) detection for the determination of a substance P receptor antagonist 2(S)-((3,5-bis(trifluoromethyl)benzyl)-oxy)-3(S)-phenyl-4-((3-oxo-1,2,4- triazol-5-yl) methyl)morpholine (Fig. 1, Ia, L-742 694) in human plasma and urine. The drug was isolated from the biological matrix through liquid-liquid extraction. In the HPLC/UV method, the samples were initially injected onto a cyano Hypersil column, and the chromatographic region containing the peaks of interest was heart-cut onto an analytical C-18 Hypersil column via a column switching device. The analyte was quantified by monitoring absorbance at 205 nm. The limit of quantification for I extracted from 1 ml of plasma or urine was 2.5 ng ml-1, and the assays were validated in the concentration range 2.5-500 ng ml-1. The HPLC/MS-MS method were validated in the concentration range 0.2-500 ng ml-1. Both assays provided data with precision, measured as coefficient of variation, better than 10% at all points within the standard curve range and with adequate accuracy.

Pholcodine interference in the immunoassay for opiates in urine.

The excretion in urine after single oral therapeutic doses of morphine derivatives was analysed with radioimmunoassay (RIA) and homogeneous enzyme immunoassay (EMIT) for opiates. In contrast to the rapid excretion of ethylmorphine and codeine, pholcodine showed positive results for opiates 2-6 weeks after intake when the urines were analysed with the RIA-method. When analysed with the EMIT-method, positive results were obtained for pholcodine for approximately 10 days. As pholcodine is a common component in cough mixtures, its prolonged excretion could represent a hazard in interpreting the results from drug analyses of urines.

Interpretation of GC-MS opiate results in the presence of pholcodine.

Although the cross reactivity of pholcodine with opiate immunoassays has been well documented there is little published information the potential for pholcodine interference with chromatographic analyses. Wilson and Smith [Ann. Clin. Biochem. 36 (1999) 592] recently described the 'misidentification' of morphine in quality control specimens that had been spiked with pholcodine. This report describes a sensitive, rapid gas chromatography-mass spectrometry (GC-MS) method for the detection and quantitation of pholcodine and morphine, together with 6-monoacetylmorphine (6-MAM), codeine and dihydrocodeine in urine. This method was used to analyse urine specimens collected from volunteers given single and multiple doses of pholcodine to establish the significance this drug on the analytical results obtained when performing drug screening according to the proposed UK and EU legally defensible workplace drug testing guidelines. The maximum urinary free morphine concentration achieved following a single 10mg oral dose of pholcodine was 1.39 mg/l at 2-4h post dose. Following multiple 10mg oral doses of pholcodine the maximum urinary free morphine concentration was determined as 0.4 mg/l at 170 h after the final dose was administered. This apparent anomaly in the morphine concentrations obtained following single and multiple pholcodine doses can be explained in part by differences in the concentration of the specimens, and may be overcome by applying a correction factor for specimen dilution using their creatinine concentration. The data from this study suggests that even following one single 10mg dose of pholcodine, free morphine concentrations greater than both the proposed UK workplace drug testing guidelines threshold of 0.3mg/l total morphine and the proposed European Union threshold of 0.2mg/l total morphine can be achieved. This highlights the need for caution when interpreting confirmatory opiate data, especially in medicolegal and clinical cases, and in cases where the use of pholcodine is suspected.