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Methenolone (17β-hydroxy-1-methyl-5α-androst-1-en-3-one) misuse in doping control is commonly detected by monitoring the parent molecule and its metabolite (1-methylene-5α-androstan-3α-ol-17-one) excreted conjugated with glucuronic acid using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS) for the parent molecule, after hydrolysis with β-glucuronidase. The aim of the present study was the evaluation of the sulfate fraction of methenolone metabolism by LC-high resolution (HR)MS and the estimation of the long-term detectability of its sulfate metabolites analyzed by liquid chromatography tandem mass spectrometry (LC-HRMSMS) compared with the current practice for the detection of methenolone misuse used by the anti-doping laboratories. Methenolone was administered to two healthy male volunteers, and urine samples were collected up to 12 and 26 days, respectively. Ethyl acetate extraction at weak alkaline pH was performed and then the sulfate conjugates were analyzed by LC-HRMS using electrospray ionization in negative mode searching for [M-H](-) ions corresponding to potential sulfate structures (comprising structure alterations such as hydroxylations, oxidations, reductions and combinations of them). Eight sulfate metabolites were finally detected, but four of them were considered important as the most abundant and long term detectable. LC clean up followed by solvolysis and GC/MS analysis of trimethylsilylated (TMS) derivatives reveal that the sulfate analogs of methenolone as well as of 1-methylene-5α-androstan-3α-ol-17-one, 3z-hydroxy-1β-methyl-5α-androstan-17-one and 16β-hydroxy-1-methyl-5α-androst-1-ene-3,17-dione were the major metabolites in the sulfate fraction. The results of the present study also document for the first time the methenolone sulfate as well as the 3z-hydroxy-1β-methyl-5α-androstan-17-one sulfate as metabolites of methenolone in human urine. The time window for the detectability of methenolone sulfate metabolites by LC-HRMS is comparable with that of their hydrolyzed glucuronide analogs analyzed by GC-MS. The results of the study demonstrate the importance of sulfation as a phase II metabolic pathway for methenolone metabolism, proposing four metabolites as significant components of the sulfate fraction., (Copyright © 2015 John Wiley & Sons, Ltd.)

3,4-Methylenedioxymethamphetamine (MDMA) is a racemic drug of abuse and its R- and S-enantiomers are known to differ in their dose-response curve. The S-enantiomer was shown to be eliminated at a higher rate than the R-enantiomer most likely explained by stereoselective metabolism that was observed in various in vitro experiments. The aim of this work was the development and validation of methods for evaluating the stereoselective elimination of phase I and particularly phase II metabolites of MDMA in human urine. Urine samples were divided into three different methods. Method A allowed stereoselective determination of the 4-hydroxy-3-methoxymethamphetamine (HMMA) glucuronides and only achiral determination of the intact sulfate conjugates of HMMA and 3,4-dihydroxymethamphetamine (DHMA) after C18 solid-phase extraction by liquid chromatography-high-resolution mass spectrometry with electrospray ionization. Method B allowed the determination of the enantiomer ratios of DHMA and HMMA sulfate conjugates after selective enzymatic cleavage and chiral analysis of the corresponding deconjugated metabolites after chiral derivatization with S-heptafluorobutyrylprolyl chloride using gas chromatography-mass spectrometry with negative-ion chemical ionization. Method C allowed the chiral determination of MDMA and its unconjugated metabolites using method B without sulfate cleavage. The validation process including specificity, recovery, matrix effects, process efficiency, accuracy and precision, stabilities and limits of quantification and detection showed that all methods were selective, sensitive, accurate and precise for all tested analytes., (Copyright © 2011 John Wiley & Sons, Ltd.)

Packed capillary liquid chromatography/mass spectrometry (LC/MS) using electrospray ionization (ESI) was used to study the human biotransformation of the anti-emetic drug dolasetron. Urine from subjects given a single 100 mg intravenous dose, containing 14C-labeled dolasetron (50 microCi), was de-salted and concentrated for LC/MS with minimal loss of radioactivity (97% recovery). Aliquots of the de-salted material were injected directly onto a C8 packed capillary column (25 cm x 0.32 mm i.d.) and eluted with an acetonitrile-water gradient, buffered with 1% acetic acid, at a flow rate of 2 microliters min-1. Five metabolites were detected by LC ESI-MS which, yielded molecular mass information but no fragmentation. The identity of each metabolite was confirmed in a subsequent analysis using product ion scans in conjunction with collisionally induced dissociation. Precursor ion scanning was also employed and did not reveal any new biotransformation products. In addition to defining the major routes of biotransformation, the data obtained were compared with a 14C radioprofile prepared in a separate experiment. Qualitative agreement in the two chromatographic profiles enabled the major clusters of radioactivity to be assigned to specific metabolites of dolasetron. An important observation in this comparison was that the signal obtained by ESI did not provide an accurate assessment of the quantity of each metabolite. This was especially true for acidic conjugates (i.e. glucuronides, sulfates), which in the case of dolasetron can exist as zwitterions (no net charge). The results demonstrate the power of packed capillary LC ESI-MS for use in drug biotransformation studies and suggest that caution should be exercised when interpreting relative metabolite abundances from ESI data in the absence of actual reference standards.

Erythrocyte and plasma antioxidant enzyme activities and antioxidants as well as concentrations of total sulfate and thiocyanate were estimated in a group of healthy subjects and three groups of smokers (cigarette smokers, mixed tobacco smokers, and miscellaneous tobacco smokers). Plasma vitamin E, uric acid, ascorbic acid, ceruloplasmin, and urinary total sulfate concentrations were decreased, whereas dehydroascorbate and urinary thiocyanate concentrations were elevated in the three groups of smokers in comparison to the corresponding levels of the control subjects. On the other hand, erythrocyte superoxide dismutase and catalase as well as plasma superoxide dismutase activities were elevated in subjects of the three groups of smokers compared with the corresponding activity in subjects of the control group. Plasma catalase activity is statistically unaffected by smoking, but blood glutathione peroxidase activities were decreased in the three groups of smokers in comparison with the corresponding levels of the control group. There were also statistically meaningful differences between mean values of the antioxidant concentrations and the activities of the antioxidant enzymes in most of the smokers groups.

The pharmacokinetics and renal excretion of a trichothecene mycotoxin, verrucarol, were studied in six mongrel dogs following IV administration (0.4 mg kg-1). The fraction of verrucarol excreted intact in the urine ranged from 0.9% to 2.7% of the administered dose. The fraction of verrucarol metabolites excreted in the urine was 32-60% for verrucaryl glucuronides and 32-47% for verrucaryl sulphates. These urinary conjugated metabolites were analysed quantitatively following their enzymatic hydrolysis. The half-life of verrucarol calculated from the urinary data of its conjugated metabolites was not significantly different from the half-life calculated from the plasma data of the parent compound.

Eight healthy subjects were fed a high-protein-low-carbohydrate diet and, after a 3-day washout period, an isocaloric low-protein-high-carbohydrate diet. They received acetaminophen and oxazepam, drugs metabolized primarily by conjugation, on days 11 and 13, respectively, of each diet. Changing the diets of subjects from the high-protein-low-carbohydrate diet to the low-protein-high-carbohydrate diet resulted in a 14% increase in urinary recovery of acetaminophen glucuronide and a 32% increase in urinary recovery of oxazepam glucuronide (p less than 0.05). The increases in glucuronidation were at the expense of other pathways of metabolism, and there were no significant changes in the metabolic clearance rates of acetaminophen and oxazepam. Mean renal clearances of acetaminophen glucuronide, acetaminophen sulfate, and oxazepam glucuronide decreased 45%, 32%, and 54%, respectively (p less than 0.05), when the subjects were switched to the low-protein-high-carbohydrate diet.

Depletion of endogenous inorganic sulfate can have pronounced effects on the elimination kinetics and metabolic fate of phenolic drugs. Our purpose was to determine the effects of acetaminophen (which is partly metabolized to acetaminophen sulfate), ascorbic acid (subject to more limited sulfation than acetaminophen), and sodium sulfate (useful for sulfate repletion by the oral route) on the serum concentration and renal excretion of inorganic sulfate in healthy adults. Six men and two women, 26 to 35 yr old, were studied on four occasions that were at least 4 days apart. They received no medication, 1.5 gm acetaminophen, 6 gm ascorbic acid, or 9 gm sodium sulfate decahydrate orally, in aqueous solution. A blood sample was obtained 2 hr later and urine was collected from 1 to 3 hr. Serum inorganic sulfate concentrations (mean +/- SD), 0.410 +/- 0.043 mM in the control period, were decreased after acetaminophen (0.311 +/- 0.043 mM, P less than 0.001), increased after sodium sulfate (0.513 +/- 0.055 mM, P less than 0.001), and apparently unchanged after ascorbic acid (0.417 +/- 0.059 mM). The urinary excretion of inorganic sulfate was decreased after acetaminophen and increased after sodium sulfate. The renal clearance of endogenous creatinine was not affected by any of the treatments. The renal tubular reabsorption of inorganic sulfate is capacity limited, as evidenced by the decrease of the reabsorbed fraction with increasing glomerular filtration rate of the anion (r = -0.54, P less than 0.005). This saturable reabsorption facilitates sulfate homeostasis.

The role of a low intracellular glutathione concentration in sulphur amino-acid degradation was investigated in two sisters (10 and 13 years old) with glutathione-synthetase deficiency, by determination of the urinary excretion of sulphur amino-acids and their main degradation products. The urinary excretion of total sulphur and inorganic sulphate was normal, indicating adequate dietary intake of sulphur amino-acids and normal oxidation to inorganic sulfate. Decreased excretion of N-acetylcysteine was found in the younger sister, which could be due to a reduced intracellular availability of cyst(e)ine. Increased excretion of mercaptolactate was found in the older sister, probably featuring a more catabolic condition in this subject. She also had an increased excretion of thiosulphate. Low excretion of thiocyanate was observed in both subjects, probably a consequence of a reduced dietary intake of thiocyanate or its precursors. Neither glutathione nor gamma-glutamylcysteine were detectable in urine. The excretion of cysteinylglycine was clearly reduced, reflecting that this dipeptide mainly is a degradation product of glutathione.

The elimination of acetaminophen (APAP) following an oral dose of 10 mg/kg in newborn infants, children, and adults was compared. Urinary excretion of unchanged APAP, APAP-sulfate, and APAP-glucuronide was complete within 30 hr at all ages. Higher percentages of the dose were excreted in the urine as APAP-sulfate in neonates (0-2 days old) and children (3-9 yr old) than in 12-yr old children and adults. A pharmacokinetic analysis indicated that the higher rate of APAP-sulfate formation in younger age groups apparently compensated for a deficiency in glucuronide formation. No dramatic age-related differences in the overall elimination rate constant for APAP were observed despite the quantitative changes in the metabolic pathways during early childhood.

A group of eleven subjects with Gilbert's syndrome was characterized by conventional tests and determination of bilirubin and its conjugates in plasma by alkaline methanolysis and thin layer chromatography. After a 1 g dose of paracetamol h.s. the drug and its metabolites were measured by high performance liquid chromatography (HPLC) in the overnight 8-h urine sample. The amounts of paracetamol and of its metabolites recovered in urine were almost identical with those found in the control group (n = 10). The glucuronide:paracetamol ratio, which is considered to be an index of glucuronidation, was not correlated with the fraction of bilirubin present in plasma as glucuronides. These data do not suggest that in subjects with Gilbert's syndrome therapeutic doses of paracetamol are associated with an increased risk for hepatic or systemic toxicity.

Glucuronides of lithocholate, chenodeoxycholate and cholate were synthesized enzymatically and characterized by thin layer chromatography, column chromatography and specific enzymatic hydrolysis. Bile salt glucuronides were quantitatively analysed in thr urine of patients with intra- and extra- hepatic cholestasis and were found to be present in 19 out of 20 patients studied. Our patients with intrahepatic cholestasis excreted 8.9 mg non-sulphated and non-glucoronidated bile salts, 18.2 mg sulphated bile salts and 7.2 mg glucuronidated bile salts. The patients with extrahepatic cholestasis excreted 14.7 mg non-sulphated and non-glucuronidated bile salts, 20.7 mg sulphated bile salts and 4.7 mg glucuronidated bile salts. These findings indicate that glucuronidation of bile salts occurs in man and represents a metabolic pathway in patients with cholestasis.

Melatonin is metabolised by hydroxylation to form 6-hydroxy-melatonin and by demethylation to form N-acetyl-serotonin, which are excreted as sulphate and glucuronide conjugates. We required these metabolites as pure powders and therefore undertook their isolation and characterisation. Three volunteers ingested 1 g each of melatonin, and their urine was collected and pooled. For the sulphate conjugates, a Lichoprep column was used to concentrate the metabolites and to remove most of the urea. The sulphate conjugates were separated from the glucuronides on a Florisil column and further purified on a fractogel column. They were separated by high-performance liquid chromatography (HPLC) resulting in white powders of 6-hydroxy-melatonin sulphate (SaMT) and N-acetyl-serotonin sulphate (SNAS). For the glucuronide conjugates, an aliquot of the pooled urine was taken to dryness, the residue was dissolved in methanol, and the solution was filtered. The methanol filtrate was taken to dryness, and the residue was applied to a Florisil column. The isolated glucuronide conjugates were recrystallized prior to separation by HPLC, which gave pure white powders of N-acetyl-serotonin glucuronide (GNAS) and 6-hydroxy-melatonin glucuronide (GaMT). Characterisation was achieved by using infrared and ultraviolet spectroscopy, thin-layer chromatography (TLC), and gas chromatography-mass spectrometry (GCMS). These techniques unambiguously confirmed the assigned structures for SaMT and SNAS and fully supported the assigned structures for GNAS and GaMT. Three TLC solvent systems were used, and in each case the individual conjugated metabolite appeared as a discreet spot. Purity, as assessed by GCMS, was shown to be greater than 95% for SNAS, SaMT, and GaMT and to be 88% for GNAS.

Ritodrine is a beta 2-adrenergic agonist that is used clinically for the management of preterm labor. The beta 2 activity of ritodrine produces the relaxation of smooth muscles and is believed to act directly on the beta 2-receptors of the myometrium. Reports in the literature suggest that ritodrine is inactivated by sulfate and glucuronide conjugation, but this has not been verified in humans. Studies on animal models indicate that the sulfate conjugate is a major urinary metabolite of ritodrine. Recent investigations of maternal and neonatal urinary excretion of ritodrine indicate that 80% to 90% of the drug is in the form of conjugates. The purpose of this study was to determine the nature of these conjugates. Our study indicates that both the mother and neonate excrete glucuronide and sulfate conjugates of ritodrine. The sulfate conjugate accounts for 45% of maternal excretion and 66% of neonatal excretion; the glucuronide conjugate accounts for 38% and 23% of maternal and neonatal excretion, respectively. Significantly different metabolic profiles suggest that the neonate may be capable of forming conjugated metabolites of ritodrine.

1. 5-Hydroxytryptamine-O-glucuronide has been extracted from sheep urine and characterized by its chemical reactions and R(F) values on paper chromatograms.2. 5-Hydroxytryptamine-O-glucuronide in urine was separated from an inhibitor of beta-glucuronidase using an anion exchange resin, and then estimated by measurement of the 5-hydroxytryptamine (5-HT) liberated on incubation with beta-glucuronidase.3. About 20% of the 5-hydroxyindoles found in sheep urine was 5-HT conjugated with glucuronic acid (1.18+/-0.08 mg 5-HT/24 h, thirty experiments).4. Sheep urine also contained free 5-HT (0.35+/-0.04 mg/24 h, thirty experiments) and 5-hydroxyindolylacetic acid (4.43+/-0.33 mg/24 h, thirty experiments).5. 5-Hydroxytryptamine-O-sulphate was not found in sheep urine.