Identification of trimetazidine metabolites in human urine and plasma

1. The objective was to use modern mass spectrometric techniques to update current information on the metabolism of trimetazidine in human subjects found by previous studies.

2. Urine and plasma samples were taken from four healthy human volunteers taking part in a larger kinetic study. Each subject received an oral dose of 80-mg trimetazidine daily for 4 days.

3. Identification and quantitation of trimetazidine and its metabolites in urine and plasma were achieved using modern liquid chromatography-mass spectrometric methods.

4. The major drug-related component observed in urine and plasma was unchanged trimetazidine. In addition to the parent drug, 10 metabolites were detected in urine in concentrations ranging from 0.008 (0.01% dose) to 1.094 μg.ml^-1 (1.4% dose). Metabolic profiles following acute and chronic doses of trimetazidine were qualitatively similar.     

Identification of major metabolites of the catechol-O-methyltransferase-inhibitor nitecapone in human urine.

Metabolites of nitecapone [3-(3,4-dihydroxy-5-nitrobenzylidene)-2,4-pentanedione], a potent new catechol-O-methytransferase-inhibitor, were isolated from human urine both after hydrolysis with beta-glucuronidase and as intact conjugates. Seven phase-I metabolites and corresponding glucuronides were identified using electron ionization and fast atom bombardment mass spectrometry, IR spectroscopy, and proton NMR spectrometry. The most abundant metabolite in urine was the glucuronide of unchanged nitecapone, representing 60-65% of the metabolites found. The main phase-I metabolic reaction was reduction of the side chain double bond and carbonyl groups. One of the major metabolites was formed by cleavage of the side chain by retro aldol condensation. All phase-I metabolites were present mainly as their glucuronic acid conjugates. The 3-nitrocatechol-structure of nitecapone seems to hinder nitro-reduction, catechol-O-methylation, and sulfation reactions.     

Assay of the major (4-hydroxylated) metabolites of diphenylhydantoin in human urine

Summary A modified gas chromatographic procedure for the determination of unconjugated and conjugated 4-hydroxydiphenylhydantoin (4-OH-DPH) in urine has been developed.Unconjugated 4-OH-DPH is determined after selective extraction with toluene — ether (1:1). For the assay of conjugated 4-OH-DPH, the urine is pre-extracted withisoamylalcohol before acid hydrolysis to avoid interference by the dihydrodiol metabolite of DPH. The sensitivity of the method is 0.1 µg per ml. The method has been used to determine the urinary metabolites in two adult volunteers, during steady state plasma concentrations of DPH and in the elimination phase.To the memory of Balzar Alexanderson     

Identification of etazene (etodesnitazene) metabolites in human urine by LC-HRMS

Etazene (or etodesnitazene) is a novel and highly active synthetic opioid belonging to the rapidly evolving and emerging group of “nitazenes.” Etazene metabolites were identified through analysis of a human urine sample. The sample was obtained from a 25-year-old man who attempted suicide by taking a new psychoactive substances (NPS) cocktail purchased online and was analyzed by ultrahigh performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). Etazene metabolites were predicted with BioTransformer 3.0, and the exact masses were added to the inclusion list. Eight possible metabolites were identified in the urine sample. N- and O-deethylation were identified as the predominant metabolism routes, resulting in M1 (O-deethylated etazene; most abundant metabolite based on the peak area), M2 (N-deethylated etazene), and M3 (N,O-dideethylated etazene) metabolites. Less abundant hydroxylated products of these deethylated metabolites and etazene were also found. Additionally, in the analysis without β-glucuronidase treatment, M1- and M3-glucuronide phase II metabolites were found. As N- and O-deethylated products seem to be the predominant urinary metabolites, the detection of these metabolites in urine can be useful to demonstrate etazene exposure.     

Identification and determination of phase I metabolites of propafenone in rat liver perfusate

Propafenone (PF) is a class 1C antiarrhythmic agent. To study the mechanisms of PF interactions with dietary nutrients in isolated, perfused rat livers, metabolites of PF in liver perfusate were identified and an analytical method was developed for these metabolites plus parent drug. Identification of phase I metabolites was performed using HPLC/MS equipped with a Lichrospher RP-18 column and tandem mass spectrometry (MS/MS) with electrospray and atmospheric pressure chemical ionizations. Three major metabolite peaks, whose protonated molecular ions were m/z 358, 358 and 300, were identified as a propafenone derivative hydroxylated in the ω-phenyl ring (ω-OH-PF), 5-hydroxypropafenone (5-OH-PF), and N-despropylpropafenone (N-des-PF). The levels of ω-OH-PF, 5-OH-PF, N-des-PF and PF were determined simultaneously by HPLC with UV detection at 210 nm and a mobile phase of 0.03% triethylamine and 0.05% phosphoric acid in water-acetonitrile-methanol (45:20:35, v/v/v) after extraction with 5 ml diethyl ether at pH 10.0 and evaporation of solvent under nitrogen. The results revealed that ω-OH-PF, which was not found in humans, was the major metabolite of PF in rat liver perfusate, not 5-OH-PF which is the major metabolite in human plasma.     

Effects of the synthesized prostaglandin E2-analogue arbaprostil on gastric mucosal lesions in rats

The effects of arbaprostil (CU-83), a newly synthesized prostaglandin (PG) E2 analogue, on gastric lesions were investigated. Experiment 1: The animals were divided into 3 groups: 1. the control group: untreated, 2. 50% ethanol group: rats were given 1 ml of 50% ethanol intragastrically, and 3. arbaprostil + 50% ethanol group: arbaprostil (10 micrograms/kg) was administered p.o. 30 min before 50% ethanol administration. 1 h after ethanol administration, stomachs were isolated, and gastric mucosal lesions were observed. Experiment 2: Rats were divided into 4 groups; 1. the control group; untreated, 2. 6 h stress group; animals were placed in a stress cage and immersed into a water bath (23 degrees C) for 6 h, 3. arbaprostil (10 micrograms/kg) + 6 h stress group, and 4. arbaprostil (100 micrograms/kg) + 6 h stress group; arbaprostil 10 micrograms/kg or 100 micrograms/kg was administered 30 min before 6 h of water immersion stress, respectively. In each experiment, stomachs were isolated and gastric mucosal lesions were observed. Immediately after the observation of lesions, the fundic mucosal layer was separated from the muscle layer, and mucosal PGs levels were measured by high performance liquid chromatography. Four kinds of PGs, i.e., 6-keto-PG F1 alpha, PGF2 alpha, PGE2, and PGD2 were detected in gastric mucosa. Administration of 50% ethanol and water immersion stress induced gastric lesions and decreased all 4 mucosal PGs levels. Arbaprostil, 10 micrograms/kg, reduced ethanol-induced gastric lesions and maintained mucosal PGs levels, concomitantly, however, the same doses of arbaprostil did not show a protective effect against water immersion stress-induced gastric lesions or decreases in PGs levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the metabolites of di-(2-ethylhexyl) phthalate in urine from the African green monkey

The metabolites of di-(2-ethylhexyl) phthalate (DEHP) found in urine from African Green monkeys after intravenous administration of the 14C-labeled parent compound were isolated and identified. Criteria of identification included cochromatography with rat-derived standards on direct-phase HPLC and a variety of gas-chromatographic columns, as well as correspondence of mass spectra (70-eV electron impact and methane positive chemical ionization) with those of known standards. Approximately 80% of the urinary metabolites were excreted in the form of glucuronide conjugates. This is analogous to what has been reported for the urinary metabolites of DEHP from humans, but in clear contrast to the metabolites found in rat urine. Rat urinary metabolites of DEHP are excreted unconjugated, and consist primarily of derivatives more highly oxidized than the major metabolites produced by monkey or human. It is suggested that the African Green monkey may be a better model for human metabolism of DEHP than is the rat.     

Identification of the n-heptane metabolites in rat and human urine

Numerous n-heptane metabolites have been identified and quantified by gas chromatography and mass spectrometry in some tissues and in the urine of Sprague Dawley rats exposed for 6 h to 1800 ppm n-heptane. 2-Heptanol and 3-heptanol were the main biotransformation products of the solvent. 2-Heptanone, 3-heptanone, 4-heptanol, 2,5-heptanedione, -valerolactone, 2-ethyl-5-methyl-2, 3-dihydrofuran and 2,6-dimethyl-2,5-dihydropyran were also found as metabolites of n-heptane. In five shoe factory workers and in three rubber factory workers the mean exposure to technical heptane was measured (n-heptane ranged between 5 and 196 mg/m³). In the urine collected at the end of their work shift some n-heptane biotransformation products were found: 2-heptanol, 3-heptanol, 2-heptanone, 4-heptanone and 2,5-heptanedione. 2-Heptanol was the main n-heptane metabolite and its urinary concentrations ranged between 0.1 and 1.9 mg/l. Urinary 2,5-heptanedione was detectable only in some samples and at very low concentration (0.1–0.4 mg/1).These data suggest that n-heptane can be considered as a neurotoxic product, since it gives rise to 2,5-heptanedione, but the small amount of the urinary metabolite is very unlikely to cause clinical damage to the peripheral nervous system.Part of this work was presented at the 48 Congr. Naz. Soc. Ital. Med. Lav. Igiene Ind. Pavia 18–21 September 1985     

Identification of the metabolites of baicalein in human plasma

The metabolites of baicalein in human plasma were investigated after taking baicalein, which is one of the main bioactive flavones in Scutellaria baicalensis Georgi. Five metabolites (M1–M5) together with the parent drug baicalein (P) were detected and identified by the HPLC-diode-array detector (DAD) and LC-MS/MS methods. Among them, 7-methoxybaicalein 6-O-glucuronide (M5) is a new metabolite. Based on the results, the proposed metabolic pathway of baicalein in humans was inferred.     

Identification of the metabolites of baicalein in human plasma

The metabolites of baicalein in human plasma were investigated after taking baicalein, which is one of the main bioactive flavones in Scutellaria baicalensis Georgi. Five metabolites (M1-M5) together with the parent drug baicalein (P) were detected and identified by the HPLC-diode-array detector (DAD) and LC-MS/MS methods. Among them, 7-methoxybaicalein 6-O-glucuronide (M5) is a new metabolite. Based on the results, the proposed metabolic pathway of baicalein in humans was inferred.     

Variable responses to prostaglandin E(2) in human non-pregnant myometrium

Cumulative concentration-effect curves for prostaglandin E(2), sulprostone and butaprost were constructed in matched strips of human non-pregnant myometrium from 14 different donors. All samples were obtained from the mid-lateral wall of the uterus. Prostaglandin E(2) produced four types of concentration-effect curves: monophasic inhibitory (n = 7), monophasic excitatory (n = 2), biphasic consisting of an excitatory phase followed by an inhibitory phase (n = 4), and biphasic consisting of an inhibitory phase followed by an excitatory phase (n = 1). Sulprostone produced excitation of spontaneous contractile activity in all tissues (mean pEC(50) = 9.1+/-0.2, range 8.1-10.1, n = 14). Butaprost produced relaxation of cloprostenol-stimulated contractile activity in all tissues (mean pEC(50) = 5.7 +/- 0.1, range 5.0-6.9, n = 14). The mean pEC(50) value for sulprostone was significantly higher in tissues where prostaglandin E(2) caused some excitation (pEC(50) = 9.4 +/- 0.2, n = 7) compared to those where prostaglandin E(2) caused only inhibition (pEC(50) = 8.8 +/- 0.2, n = 7). Mean pEC(50) values for butaprost were not significantly different between these groups. These data suggest that (a) variability in EP receptor-mediated responses exists within a single anatomical site; (b) both excitatory and inhibitory EP receptor-mediated pathways are always operative in human non-pregnant myometrium, regardless of the type of tissue response to prostaglandin E(2); and (c) regulation of EP receptor-mediated responses occurs predominantly in the excitatory (EP(3) or EP(1) receptor) pathway rather than the inhibitory (EP(2) receptor) pathway.     

Stability of bupropion and its major metabolites in human plasma

Bupropion (BUP) is a new, monocyclic, second-generation antidepressant with efficacy comparable with that of the tricyclics but possessing a more favorable side-effect profile. Therapeutic drug monitoring services are expected to be widely available following the marketing of BUP, yet relatively little has been published on its clinical pharmacokinetics. The purpose of this study was to assess the drug's plasma stability and to determine the best storage conditions for conducting pharmacokinetic studies of BUP and its major metabolites. Human plasma was spiked with known concentrations of BUP and its major basic metabolites, threo-amino alcohol (TB), erythro-amino alcohol (EB), and hydroxy metabolite (HB), and incubated under varying conditions of temperature and pH. BUP showed a log linear degradation that was both temperature and pH dependent. BUP half-life in pH 7.4 plasma stored at 22 and 37 degrees C was 54.2 and 11.4 h, respectively. Incubation at 37 degrees C, pH 2.5 to 10, for 48 h had minimal effect on metabolite concentrations. These results indicate that drug degradation must be considered in studies involving the incubation of plasma for protein binding determinations and the collection and storage of plasma samples for drug concentration analysis during therapeutic drug monitoring.     

Identification of the in vivo liver metabolites of (--)-delta 7-tetrahydrocannabinol produced by the mouse

The major in vivo metabolites of (--)-delta 7-tetrahydrocannabinol were extracted from the livers of mice after a single intraperitoneal dose and were examined by combined gas-liquid chromatography-mass spectrometry. Twenty-six metabolites were identified; most of these appeared to be produced via epoxidation of the double bond, although the epoxides themselves were not detected. Major metabolites involved hydroxylation of the double bond, to give the isomeric 1 alpha- and 1 beta, 7-dihydroxyhexahydrocannabinols. Of these, the 1 alpha, 7-isomer was the major product and appeared to be produced by hydrolysis of 1 alpha, 7-epoxyhexahydrocannabinol. It was further oxidized to 1 alpha-hydroxyhexahydrocannabinol-7-oic acid. Rearrangement of the epoxide to the 7-aldehyde and subsequent reduction to either the 7-alcohol or oxidation to the 7-acid was another major metabolic route. Derivatives of all these metabolites, hydroxylated in the 2"-, 3"- and 4"-positions of the sidechain were also identified. Allylic hydroxylation, predominantly at position 6, was also observed, but this was a relatively minor route. Reduction of the double bond yielded the isomeric axial and equatorial hexahydrocannabinols in low yield.     

罂粟碱的体内与体外代谢物研究

利用HPLC-MSⁿ检测罂粟碱及其在大鼠体内(大鼠粪便)和体外(肝微粒体, 肠道菌)代谢物。体内与体外的代谢物经C₁₈小柱进行富集和纯化后, 直接采用优化的HPLC-ESI/ITMSⁿ方法对样品进样分析。以罂粟碱标准品为对象优化高效液相色谱/电喷雾离子阱质谱(HPLC-ESI/ITMSⁿ )实验条件, 分析总结其电喷雾质谱的一级电离规律和多级质谱裂解规律, 作为罂粟碱大鼠体内与体外代谢物结构分析的依据。代谢物的结构推导主要依据代谢物的色谱保留时间及其电喷雾离子阱质谱HPLC-ESI/ITMSⁿ电离规律。在大鼠粪便中有原药及其8种代谢产物。体外代谢物检测到原药的脱甲基和羟基化。     

Isolation and identification of major metabolites of tixocortol pivalate in human urine

The metabolism of tixocortol pivalate (PIVALONE), an anti-inflammatory steroid without systemic glucocorticoid effects, has been investigated in man. The analysis was conducted using urine samples collected from two volunteers who had received a 2-g oral dose of 14C-tixocortol pivalate as an oral suspension. Metabolites were purified and isolated from urine by normal phase HPLC, and structural identification was achieved by desorption chemical ionization/NH3 and electron impact/direct line introduction mass spectrometry. Unchanged tixocortol pivalate was not detected in urine; all metabolites were sulfo- and glucurono-conjugates. Metabolites were identified in the neutral steroid fraction obtained after hydrolysis of conjugates. Metabolic transformations in common with cortisol were reduction of the 3-keto, delta 4 system, reduction of the C-20 carbonyl group, oxidation of the C-11 alcohol, and cleavage of the side chain at C-17. Specific metabolic pathways involving the C-21 thiol ester function were transformations into methylthio, methylsulfinyl and methylsulfonyl derivatives, and a reductive cleavage of the C-21-S bond leading to 21-methyl structures. Since none of these metabolites had binding affinity for glucocorticoid receptors in vitro, fast and extensive transformation of tixocortol pivalate into inactive metabolites provides an explanation for the large dissociation between the topical and systemic activities of this drug.     

Identification of the major covalent adduct formed in vitro and in vivo between acetaminophen and mouse liver proteins

Improved analytical methodology has been developed for the structural characterization of covalently bound drug-protein adducts and has been applied to an investigation of the conjugates formed in vivo and in vitro between [14C]acetaminophen and mouse liver proteins. The major adduct released by acid hydrolysis of hepatic protein samples, which accounted for approximately 70% of the bound radioactivity in vivo and in vitro, was identified as 3-cystein-S-yl-4-hydroxyaniline, a derivative whose structure reflects the predominance of acetaminophen thioether adducts in drug-modified proteins. It is concluded that the reactive, electrophilic metabolite of acetaminophen, which most likely is N-acetyl-p-benzoquinoneimine, binds with a high degree of selectivity to cysteinyl thiol groups on protein, formally in a Michael-type addition reaction. Cysteine residues thus represent primary target sites for arylation by the reactive metabolite of acetaminophen, and proteins rich in free thiols may be especially vulnerable to damage by this toxic intermediate.     

Novel metabolites of buprenorphine detected in human liver microsomes and human urine.

The in vitro metabolism of buprenorphine was investigated to explore new metabolic pathways and identify the cytochromes P450 (P450s) responsible for the formation of these metabolites. The resulting metabolites were identified by liquid chromatography-electrospray ionization-tandem mass spectrometry. In addition to norbuprenorphine, two hydroxylated buprenorphine (M1 and M2) and three hydroxylated norbuprenorphine (M3, M4, and M5) metabolites were produced by human liver microsomes (HLMs), with hydroxylation occurring at the tert-butyl group (M1 and M3) and at unspecified site(s) on the ring moieties (M2, M4, and M5). Time course and other data suggest that buprenorphine is N-dealkylated to form norbuprenorphine, followed by hydroxylation to form M3; buprenorphine is hydroxylated to form M1 and M2, followed by N-dealkylation to form M3 and M4 or M5. The involvement of selected P450s was investigated using cDNA-expressed P450s coupled with scaling models, chemical inhibition, monoclonal antibody (MAb) analysis, and correlation studies. The major enzymes involved in buprenorphine elimination and norbuprenorphine and M1 formation were P450s 3A4, 3A5, 3A7, and 2C8, whereas 3A4, 3A5, and 3A7 produced M3 and M5. Based on MAb analysis and chemical inhibition, the contribution of 2C8 was higher in HLMs with higher 2C8 activity, whereas 3A4/5 played a more important role in HLMs with higher 3A4/5 activity. Examination of human urine from subjects taking buprenorphine showed the presence of M1 and M3; most of M1 was conjugated, whereas 60 to 70% of M3 was unconjugated.