Ultrasound-assisted emulsification microextraction for rapid determination of unmetabolized synthetic polycyclic and nitro-aromatic musks in human urine.

An effective method to rapidly determine the presence of seven unmetabolized synthetic musks in human urine samples is developed. The target musks are five synthetic polycyclic musks (i.e., celestolide (ADBI), phantolide (AHMI), traseolide (ATII), galaxolide (HHCB), tonalide (AHTN)), and two nitro-aromatic musks (i.e., musk xylene (MX) and musk ketone (MK)). The method involved an ultrasound-assisted emulsification microextraction (USAEME) coupled with gas chromatography-mass spectrometry (GC-MS). The factors that affect USAEME efficiency were optimized in detail, and the optimized procedure involved the rapid injection of 50 µL of carbon tetrachloride into 1.0 mL of urine sample (contained 0.1-g of sodium chloride) in a conical bottom glass tube. After 1.0 min ultrasonication and 3 min centrifugation (at 7000 rpm), the sedimented extract 10 µL was directly injected into the GC-MS system. The limits of quantitation (LOQs) varied from 0.1 to 0.5 ng/mL. The precisions for both repeatability and reproducibility were <8%. The trueness varied from 79 to 96% with the RSD ranging from 2 to 8%. The total concentrations of the seven unmetabolized target musks in collected human urine samples were in the range from 0.93 to 3.74 ng/mL. HHCB and AHTN were detected in all the collected samples, and the daily excretion doses were evaluated.

Identification and characterization of fluvastatin metabolites in rats by UHPLC/Q-TOF/MS/MS and in silico toxicological screening of the metabolites.

The present study reports the in vivo and in vitro identification and characterization of metabolites of fluvastatin, the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitor, using liquid chromatography-mass spectrometry (LC-MS). In vitro studies were conducted by incubating the drug with human liver microsomes and rat liver microsomes. In vivo studies were carried out by administration of the drug in the form of suspension to the Sprague-Dawley rats followed by collection of urine, faeces and blood at different time points up to 24 h. Further, samples were prepared by optimized sample preparation method, which includes freeze liquid extraction, protein precipitation and solid phase extraction. The extracted and concentrated samples were analysed using ultrahigh-performance liquid chromatography-quadruple time-of-flight tandem mass spectrometry. A total of 15 metabolites were observed in urine, which includes hydroxyl, sulphated, desisopropyl, dehydrogenated, dehydroxylated and glucuronide metabolites. A few of the metabolites were also present in faeces and plasma samples. In in vitro studies, a few metabolites were observed that were also present in in vivo samples. All the metabolites were characterized using ultrahigh-performance liquid chromatography-quadruple time-of-flight tandem mass spectrometry in combination with accurate mass measurement. Finally, in silico toxicity studies indicated that some of the metabolites show or possess carcinogenicity and skin sensitization. Several metabolites that were identified in rats are proposed to have toxicological significance on the basis of in silico evaluation. However, these metabolites are of no human relevance. Copyright © 2017 John Wiley & Sons, Ltd.

Traditional Chinese medicine and sports drug testing: identification of natural steroid administration in doping control urine samples resulting from musk (pod) extracts.

The administration of musk extract, that is, ingredients obtained by extraction of the liquid secreted from the preputial gland or resulting grains of the male musk deer (eg, Moschus moschiferus), has been recommended in Traditional Chinese Medicine (TCM) applications and was listed in the Japanese pharmacopoeia for various indications requiring cardiovascular stimulation, anti-inflammatory medication or androgenic hormone therapy. Numerous steroidal components including cholesterol, 5α-androstane-3,17-dione, 5ß-androstane-3,17-dione, androsterone, etiocholanolone, epiandrosterone, 3ß-hydroxy-androst-5-en-17-one, androst-4-ene-3,17-dione and the corresponding urea adduct 3α-ureido-androst-4-en-17-one were characterised as natural ingredients of musk over several decades, implicating an issue concerning doping controls if used for the treatment of elite athletes. In the present study, the impact of musk extract administration on sports drug testing results of five females competing in an international sporting event is reported. In the course of routine doping controls, adverse analytical findings concerning the athletes' steroid profile, corroborated by isotope-ratio mass spectrometry (IRMS) data, were obtained. The athletes' medical advisors admitted the prescription of TCM-based musk pod preparations and provided musk pod samples for comparison purposes to clarify the antidoping rule violation. Steroid profiles, IRMS results, literature data and a musk sample obtained from a living musk deer of a local zoo conclusively demonstrated the use of musk pod extracts in all cases which, however, represented a doping offence as prohibited anabolic-androgenic steroids were administered.

Liquid chromatography-mass spectrometry methods for urinary biomarker detection in metabonomic studies with application to nutritional studies.

The effects of sample preparation and chromatographic method differences on the classification and recovery of metabolic biomarkers from UPLC-MS measurements on urine samples of humans exposed to different dietary interventions have been investigated. Eight volunteers consumed three high-fat meals (rich in saturated, monounsaturated and polyunsaturated fatty acids, respectively) in randomized order with a washout period in between. For each participant, urine samples were obtained prior to and at three timed intervals after each meal. Samples were processed either by dilution (1 : 4) or by liquid-liquid extraction and then run under two different gradient conditions. For each analysis method, a total of 96 observations (eight participants, four time points, three diets) were measured. The total ion count chromatograms were analyzed using partial-least-squares discriminant analysis. All three dietary classes could be discriminated irrespective of sample preparation and chromatographic method. However, the main discriminating metabolites varied according to sample preparation, indicating that sample treatment and chromatographic conditions influence the ability to extract biomolecular information. Diluted samples showed higher m/z compounds (ca 400 u) while liquid-liquid extraction samples showed low m/z at the same retention time span. Optimized methods for metabolite identification (e.g. organic acids) were statistically inferior to global screening for mixed compound identification, confirming that multiple compound class-based metabolic profiles are likely to give superior metabonomic (diagnostic) classification, although great care has to be taken in the interpretation in relation to matrix effects.

Ontogeny of valproic acid disposition and metabolism: a developmental study in postnatal lambs and adult sheep.

The ontogeny of valproic acid (VPA) disposition and metabolism was investigated in developing lambs and adult sheep (Dorset or Suffolk breed). Specifically, we wished to investigate the role of glucuronidation and beta-oxidation on VPA elimination during development. Catheters were implanted in a carotid artery, a jugular vein, and the urinary bladder in 10-day-old (10 d; n = 8), 1-month-old (1 M; n = 4), and 2-month-old lambs (2 M; n = 5). In adult sheep (n = 5), catheters were implanted in a femoral artery and vein. After the administration of a 10 mg/kg VPA i.v. bolus, serial blood samples and cumulative urine samples were collected for 36 h in the adult ewes and for 72 h in the lambs. Due to saturable protein binding, age-related differences in VPA clearance were more obvious when examining the total body clearance of unbound drug (Cl(u)tb). Mean Cl(u)tb increased significantly with age up to 2 months (10 d = 2.65 +/- 1.16 ml/min/kg; 1 M = 5.11 +/- 2.49 ml/min/kg; 2 M = 12.84 +/- 3.88 ml/min/kg) before decreasing to adult levels (7.73 +/- 2.64 ml/min/kg). Similarly, the urinary recovery of the major metabolite, VPA-glucuronide, was significantly less in 10 d lambs (29.2 +/- 16.0% of the dose) when compared with the adult and 2 M groups (both approximately 74% of the dose). No differences with age were observed in the portion of the dose excreted as the beta-oxidation metabolite, 2-n-propyl-3-oxopentanoic acid. The results suggest that alterations in Cl(u)tb with age may be attributable to postnatal development of enzymes involved in VPA glucuronidation.

A comparative investigation of 2-propyl-4-pentenoic acid (4-ene VPA) and its alpha-fluorinated analogue: phase II metabolism and pharmacokinetics.

An earlier study in rats revealed that alpha-fluorination of 2-propyl-4-pentenoic acid (4-ene VPA), a toxic metabolite of the anticonvulsant drug valproic acid, would avert its metabolism via beta-oxidation and eliminate the drug-related hepatotoxicity. This investigation was carried out to compare 4-ene VPA and the alpha-fluorinated analogue (alpha-fluoro-4-ene VPA) for their pharmacokinetic and protein binding properties. Male Sprague-Dawley rats were dosed with either 4-ene VPA or alpha-fluoro-4-ene VPA i.p. at 1.4 mmol/kg. Blood was collected from the tail vein at various time points and serum samples were prepared. Urine was collected for 24 hr. A second set of rats was treated the same but sacrificed 1 hr post dose, and the livers were homogenized in a Tris-buffer. Protein binding was assessed via ultrafiltration of the naive serum samples spiked with either of the drugs. The serum drug concentration-time profiles of 4-ene VPA and alpha-fluoro-4-ene VPA seemed to resemble one another during the initial 200 min within which differences were reported for their effects on mitochondrial GSH. A second serum peak concentration was observed for 4-ene VPA at approximately 300 min, which was attributed to the extensive glucuronidation of the drug and enterohepatic circulation. The alpha-fluoro-4-ene VPA, on the other hand, did not show these properties with its major phase II metabolite being the corresponding L-glutamine conjugate. The toxic metabolite (E)-2-propyl-2,4-pentadienoic acid and its N-acetylcysteine conjugate were detected only in 4-ene VPA treated rats. Liver concentrations of 4-ene VPA and alpha-fluoro-4-ene VPA were 0.96 +/- 0.11 and 0.89 +/- 0.19 micromol/g of wet liver, respectively, at 1 hr after the dose. Comparable and parallel serum free drug levels were apparent for the two drugs over a concentration range of 0.25 to 2.9 micromol/ml. Taken together, the data seem to suggest that the reported distinction in the ability of 4-ene VPA and alpha-fluoro-4-ene VPA to produce liver toxicity in the rat resides in differences in their metabolism rather than in their pharmacokinetic properties.

Sex differences in the metabolism of (+)-S-145, a novel thromboxane A2 receptor antagonist in rat.

1. After the oral administration of 5 mg/kg S-1452 to rat, the plasma levels of (+)-S-145 were similar between the male and female, but there were sex differences in the profiles of its beta-oxidized and hydroxylated metabolites in plasma. 2. beta-Oxidation of (+)-S-145 determined in vitro was slightly higher in the female than in the male, and agreed with the plasma levels of the beta-oxidized metabolites. 3. 5-Hydroxylation activities of (+)-S-145 and beta-oxidized metabolites by rat liver microsomes were significantly higher in the male than in the female, but marked sex differences were not observed in 6-hydroxylation activities. These results revealed that differences in monooxygenase activities directly account for the sex differences in the plasma level of 5-hydroxylated metabolites, and that the peroxisomal beta-oxidation enzyme system also affected the plasma level of 6-hydroxylated metabolites. 4. Biliary excretion was higher in the male than in the female, and quantitative identification of metabolites in bile indicated that this was based on the prominent excretion of taurine conjugates in the male rat. This conclusion was supported by the fact that taurine conjugation activity was higher in male liver homogenates than in the female.

Biotransformation of fluvastatin sodium in humans.

The metabolic pathways of fluvastatin sodium (FV; Lescol, Sandoz compound XU 62-320), [R*,S*-(E)]-(+-)-sodium-3,5-dihydroxy-7-[3- (4-fluorophenyl)-1-(1-methylethyl)-1H-indole-2-yl]-hept-6-enoate, a potent inhibitor of hydroxy-methylglutaryl-CoA reductase (HMG-CoA reductase)--the rate-limiting enzyme in cholesterol biosynthesis--were determined in normal male volunteers at steady state. The metabolite profiles were determined in pooled human blood/plasma, urine, and feces obtained from healthy male volunteers after a single dose of 2 and 10 mg of [3H]FV and at steady state after a single 40 mg daily dose of [3H]FV for 6 sequential days utilizing HPLC coupled with radioactivity monitoring. The two major components in plasma were FV and the desisopropylpropionic acid (4) derivative of FV, the latter a result of oxidative removal of the N-isopropyl group and beta-oxidation of the side chain. Minor amounts of the 4,5-pentenoic acid derivative of FV, the threo-isomer of FV, the trans-lactone of FV, and conjugates of 5-hydroxy FV and 6-hydroxy FV were also present in plasma. Parent FV was not present in feces, the major excretory route, or in urine. In urine, 4 and conjugates of 5-hydroxy FV, and 6-hydroxy FV were present, and each represented < 1% of the dose. In feces 5-hydroxy FV, 6-hydroxy FV, and desisopropyl-FV represented the only peaks of significance. The metabolism of FV leading to the 5-hydroxy FV and 6-hydroxy FV was not stereospecific.(ABSTRACT TRUNCATED AT 250 WORDS)

Capillary gas chromatographic method for the determination of the thromboxane A2 receptor antagonist S-1452 and its metabolites in human urine.

A capillary gas chromatographic method using a sulphur-specific detector (Hall's electrolytic conductivity detector) was established to determine the thromboxane A2 antagonist S-1452 and its metabolites in human urine. The target species were the free acid (+)-S-145 of the drug and its nine metabolites: the three hydroxyl forms of (+)-S-145 (I, II and III), bis-nor-(+)-S-145 (IV) the hydroxylated forms of IV (V and VI), tetranor-(+)-S-145 (VII) and the hydroxylated forms of VII (VIII and IX). These ten compounds, which have the same sulphur-containing functional group in common, were determined simultaneously. Their conjugated forms, which were assumed to be glucuronides, were also assayed after hydrolysis. The first derivatization was esterification with diazomethane. The second, for the hydroxylated compounds, was trimethylsilylation with bis(trimethylsilyl)trifluoroacetamide. The ten analytes appeared as separate peaks without mutual interference during 5 min. Hall's detector distinguished the ten analytes selectively from the other urinary components, which removed the need for complex clean-up procedures and led to higher sensitivity with a lower noise level. The method is sensitive enough for the assay of substances present at more than 0.1 micrograms/ml of urine. All the compounds could be determined with a high level of precision and accuracy, with 2-5% relative standard deviation and within +/- 5% deviation from the actual value. Day-to-day measurements verified the reproducibility of the method. Recovered substances were quantified by following the time course, and the analytical data together with previously obtained plasma data clarified the metabolism pharmacokinetically.

Valproate metabolites in serum and urine during antiepileptic therapy in children with infantile spasms: abnormal metabolite pattern associated with reversible hepatotoxicity.

The purpose of this study was to identify abnormal metabolite patterns of valproate (VPA) as possible early indicators of VPA-induced liver toxicity. In a prospective study, we determined serum and urine levels of VPA metabolites by gas chromatography-mass spectrometry (GC-MS) during the course of therapy in 25 children treated for infantile spasms with high VPA doses (less than or equal to 100 mg/kg body weight/day). Most patients had similar metabolite profiles: The main metabolites in serum were the beta-oxidation products (2-en-VPA and 3-keto-VPA) and the major diunsaturated metabolite 2,3'-dien-VPA. Glucuronide conjugates and the oxidation products represent the most abundant metabolites in urine. Other metabolites, including the potential hepatotoxin 4-en-VPA, were detected only in low concentrations. Two children had transiently aberrant metabolite profiles, indicating altered beta-oxidation, (levels of 2-en-VPA, 2,3'-dien-VPA, and 3-en-VPA were markedly increased) in connection with hepatomegaly and increased liver enzyme activities at a time when both had febrile infections and were receiving dexamethasone comedication. At no time were increased levels of 4-en-VPA or its derivatives detected. Establishing the VPA metabolite profile may aid in evaluation of patients who show signs and symptoms of liver dysfunction during VPA therapy. The present study shows that initial stages of hepatotoxicity reactions to VPA may be accompanied by characteristic changes in VPA metabolism; early detection of such abnormal metabolite patterns might decrease the risk of severe hepatic injury.

Effects of polytherapy with phenytoin, carbamazepine, and stiripentol on formation of 4-ene-valproate, a hepatotoxic metabolite of valproic acid.

The incidence of valproic acid hepatotoxicity has been reported to increase in patients who are receiving polytherapy. A minor valproic acid metabolite, 2-propyl-4-pentenoic acid (4-ene-VPA), formed by a cytochrome P450-mediated reaction, has been shown to be a potent inducer of microvesicular steatosis in rats. This study tested the hypothesis that formation of 4-ene-VPA would be increased in patients taking valproic acid with carbamazepine or with phenytoin but decreased with coadministration of an inhibitor of cytochrome P450 (the antiepileptic drug stiripentol in 300 to 1200 mg daily doses) in healthy subjects. Blood and urine samples in the studies were collected during a dosing interval at steady state. Valproic acid was assayed in plasma by capillary gas chromatography; valproic acid and 15 metabolites were measured in urine by gas chromatography/mass spectrometry. The formation clearance (CLf) of 4-ene-VPA was increased twofold in the valproic acid-carbamazepine and valproic acid-phenytoin groups. In the valproic acid/stiripentol studies, the CLf of 4-ene-VPA decreased by 32% in the 1200 mg/day stiripentol study. Similar findings were obtained at 600 and 300 mg/day stiripentol. These findings provide evidence supporting a role for cytochrome P450 in the formation of the hepatotoxic metabolite, 4-ene-VPA, in humans. The increased formation of 4-ene-VPA associated with carbamazepine and phenytoin is striking in relation to the epidemiologic finding of increased incidence of valproic acid-related hepatotoxicity during polytherapy with P450 inducers.

The covalent binding to protein of valproic acid and its hepatotoxic metabolite, 2-n-propyl-4-pentenoic acid, in rats and in isolated rat hepatocytes.

The covalent binding of radioactivity to protein following administration of 14C-labeled analogs of valproic acid (VPA) and a hepatotoxic metabolite thereof, 2-n-propyl-4-pentenoic acid (delta 4-VPA), was investigated in male rats. Covalent binding occurred in a number of tissues, the level of binding being greatest to proteins in liver for each compound. Moreover, the binding of radioactivity from delta 4-VPA to hepatic macromolecules was higher than the corresponding value for VPA. When radiolabeled VPA and delta 4-VPA were incubated with rat hepatocytes, radiolabel again became bound to cellular proteins, the time-course of which suggested the existence of at least two underlying mechanisms. Thus, after initial rapid binding of both substrates, a secondary slow phase was evident, which favored binding of delta 4-VPA. Although phenobarbital pretreatment of rats had little effect on the covalent binding of either substrate to isolated hepatocytes, clofibrate pretreatment markedly enhanced the covalent binding of both VPA and delta 4-VPA to these cells. In contrast, the covalent binding of VPA and delta 4-VPA was suppressed strongly by 4-pentenoic acid, a potent inhibitor of beta-oxidation, but was not affected by metyrapone, an inhibitor of cytochrome P-450 activity. (-)-Borneol and 8-bromo-cAMP, two inhibitors of glucuronidation, acted to decrease the binding of both substrates, although this inhibition was evident only in the early stages of incubation. A similar effect was seen with valeric acid, the saturated analog of 4-pentenoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)