Investigation of urinary steroid metabolites in calf urine after oral and intramuscular administration of DHEA

DHEA (3β-hydroxy-androst-5-en-17-one) is a natural steroid prohormone. Despite a lack of information on the effect, DHEA and other prohormones are frequently used as a food supplement by body-builders. DHEA is suspected for growth promoting abuse in cattle as well. Considering the latter, urine samples from a previous exposure study in which calves were exposed to 1 g DHEA per day for 7 days, were used. The calves were divided in three groups: one orally treated, one intramuscularly injected, and a control group. The effect of this treatment on the urinary profile of several precursors and metabolites of DHEA was investigated. Urine samples were collected several days before and during the 7 days of administration and were submitted to a clean-up procedure consisting of a separation of the different conjugates (free, glucuronidated, and sulfated forms) of each compound on a SAX column (Varian). An LC-MS/MS method was developed for the detection and quantification of several metabolites of the pathway of DHEA including 17α- and 17β-testosterone, 4-androstenedione, 5-androstenediol, pregnenolone, and hydroxypregnenolone. Elevated levels of DHEA, 5-androstenediol, and 17α-testosterone were observed in the free and sulfated fraction of the urine of the treated calves, thus indicating that the administered DHEA is metabolized mainly by the ∆⁵-pathway with 5-androstenediol as the intermediate. Sulfoconjugates of DHEA and its metabolites were found to constitute the largest proportion of the urinary metabolites. The free form was also present, but in a lesser extent than the sulfated form, while glucuronides were negligible.     

Pharmacokinetics of caffeine and its metabolites in horses after intravenous, intramuscular or oral administration.

The pharmacokinetics of caffeine (CAF) and its metabolites, dimethylxanthines, were examined in horses administered 2.5 mg/kg of CAF intravenously (i.v.), intramusculary (i.m.), or orally (p.o.). The plasma samples were extracted by Extrelut and the concentrations of CAF and metabolites were determined by high performance liquid chromatography (HPLC) with a short column. The pharmacokinetics of CAF after bolus i.v. injection were described by the assumption of a two-compartment model, and those of CAF after i.m. or p.o. administration were done by the assumption of a one-compartment model. The biologic half lives of CAF were 15.5, 18.6, and 16.4 h after administering i.v., i.m. and p.o., respectively. The extent of the bioavailability of the p.o. administration was determined as 1.04 times the dose. The differences in pharmacokinetic parameters were not statistically significant among administration routes. A straight correlation existed between the logarithms of body weights of different species of animals and those of their biologic half lives of CAF. Therefore, the biologic half life of CAF in an animal might be predictable as a function of its body weight.     

Excretion profile of boldenone and its metabolites after oral administration to veal calves

The residue profiles of boldenone (17β-Bol), its epimer (17α-Bol) and the related compound androsta-1,4-diene-3,17-dione (ADD), were investigated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in urine of male calves orally treated with boldenone, boldenone esters, and/or ADD. In all the experiments with the administered steroids residues of 17α-Bol decreased rapidly after end of treatment; detectable amounts of 17α-Bol were however noticed along the withdrawal observation period after end of treatment. Differently, residues of 17β-Bol were detectable only shortly after administration. This in vivo research concerning oral treatments of cattle with boldenone related substances proves ADD to be a very active boldenone precursor in bovine animals.     

Validation of the simultaneous determination of methylprednisolone and methylprednisolone acetate in human plasma by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic procedure was developed that accurately quantitates methylprednisolone (MP) and methylprednisolone acetate (MPA) in human plasma over the range 2.00-50.0 ng/ml. The internal standard, fluorometholone, was added to an aliquot of sodium fluoride-potassium oxalate-derived plasma. Samples were prewashed with hexane and extracted twice with methylene chloride. The extracts were dried with anhydrous sodium sulfate, centrifuged, and the organic layer separated and dried under nitrogen. The samples were reconstituted in mobile phase and washed an additional time with hexane before 100 microliters were injected onto a Beckman/Altex Ultrasphere ODS column with ultraviolet absorbance detection at 254 nm. Composition of the mobile phase was acetonitrile-water-glacial acetic acid (33:62:5, v/v/v). Calibration curves were obtained by unweighted, linear regression of peak-height ratios of MP (or MPA)/internal standard versus theoretical concentrations of MP or MPA using a Hewlett-Packard 3357 Laboratory Automation System. Extraction efficiencies for MP and MPA over the linear range were 86.4 and 84.7%, respectively. This method was successfully implemented for the analysis of specimens generated from a single-dose bioavailability and safety study for a new formulation of Depo-Medrol sterile aqueous suspension.     

Urinary metabolic profile of phenylketonuria in patients receiving total parenteral nutrition and medication

Nutrition and drugs are main environmental factors that affect metabolism. We performed metabolomics of urine from an 8‐year‐old patient (case 1) with epilepsy and an 11‐year‐old patient (case 2) with malignant lymphoma who was being treated with methotrexate. Both patients were receiving total parenteral nutrition (TPN). We used our diagnostic procedure consisting of urease pretreatment, partial adoption of stable isotope dilution, gas chromatography/mass spectrometry (GC/MS) measurement and target analysis for 200 analytes including organic acids and amino acids. Surprisingly, their metabolic profiles were identical to that of phenylketonuria. The neopterin level was markedly above normal in case 1, and both neopterin and biopterin were significantly above normal in case 2. Mutation analysis of genomic DNA from case 1 showed neither homozygosity nor heterozygosity for phenylalanine hydroxylase deficiency. The metabolic profiles of both cases were normal when they were not receiving TPN. TPN is presently prohibited for individuals who have inherited disorders that affect amino acid metabolism. Although the Phe content of the TPN was not the sole cause of the PKU profile, its effect, combined with other factors, e.g. specific medication or possibly underlying diseases, led to this metabolic abnormality. The present study suggests that GC/MS‐based metabolomics by target analysis could be important for assuring the safety of the treatments for patients receiving both TPN and methotrexate. Metabolomic profiling, both before and during TPN, is useful for determining the optimal nutritional formula not only for neonates, but also for young children who are known heterozygotes for metabolic disorders or whose status is unknown. Copyright © 2009 John Wiley & Sons, Ltd.     

3,4-Methylenedioxymethamphetamine (MDMA) and metabolites disposition in blood and plasma following controlled oral administration

3,4-Methylenedioxymethamphetamine (MDMA) is an illicit phenethylamine ingested for entactogenic and euphoric effects. Although blood is more commonly submitted for forensic analysis, previous human MDMA pharmacokinetics research focused on plasma data; no direct blood–plasma comparisons were drawn. Blood and plasma specimens from 50 healthy adult volunteers (33 males, 17 females, 36 African-American) who ingested recreational 1.0 and 1.6 mg/kg MDMA doses were quantified for MDMA and metabolites 4-hydroxy-3-methoxymethamphetamine (HMMA), 3,4-methylenedioxyamphetamine (MDA), and 4-hydroxy-3-methoxyamphetamine (HMA) by two-dimensional gas chromatography–mass spectrometry. Specimens were collected up to 3 h post-dose and evaluated for maximum concentration (C _max), first detection time (t _first), time of C _max (t _max), and 3-h area under the curve (AUC_0–3 h); as well as blood metabolite ratios and blood/plasma ratios. Median blood MDMA and MDA C _max were significantly greater (p?<?0.0005) than in plasma, but HMMA was significantly less (p?<?0.0005). HMA was detected in few blood specimens, at low concentrations. Nonlinear pharmacokinetics were not observed for MDMA or MDA in this absorptive phase, but HMMA C _max and AUC_0–3 h were similar for both doses despite the 1.6-fold dose difference. Blood MDA/MDMA and MDA/HMMA significantly increased (p?<?0.0001) over the 3-h time course, and HMMA/MDMA significantly decreased (p?<?0.0001). Blood MDMA C _max was significantly greater in females (p?=?0.010) after the low dose only. Low-dose HMMA AUC_0–3 h was significantly decreased in females’ blood and plasma (p?=?0.027) and in African-Americans’ plasma (p?=?0.035). These data provide valuable insight into MDMA blood–plasma relationships for forensic interpretation and evidence of sex- and race-based differential metabolism and risk profiles.

Urinary metabolites of cinobufagin in rats and their antiproliferative activities

Cinobufagin was one of the important cardenolidal steroids and a major component of Chan'Su, a famous traditional Chinese medicine. The urinary metabolites of cinobufagin after single oral doses of 25?mg?kg?1 in rats were investigated. Eleven metabolites were isolated and purified by liquid-liquid extraction, open-column chromatography, medium-pressure liquid chromatography, as well as semi-preparative high-performance liquid chromatography. Their structures were elucidated by chemical and various spectroscopic methods, which were identified as desacetylcinobufagin (M-1), 3-oxo-desacetylcinobufagin (M-2), 3-oxo-cinobufagin (M-3), 3-epi-desacetylcinobufagin (M-4), 3-epi-12β-hydroxyl desacetylcinobufagin (M-5), 5β-hydroxyl cinobufagin (M-6), 5β-hydroxyl desacetylcinobufagin (M-7), 12β-hydroxyl cinobufagin (M-8), 1β,12β-dihydroxyl cinobufagin (M-9), 12β-hydroxyl desacetylcinobufagin (M-10) and 1β,12β-dihydroxyl desacetylcinobufagin (M-11), respectively. Among them, M-1 was the main urinary metabolite of cinobufagin with a yield of 17.7%. Most metabolites were hydroxylated products of cinobufagin at C-1β, 5β and 12β positions, as well as deacetylated products at C-16. Except M-1, M-4 and M-7, the other eight metabolites were novel in vivo metabolites of cinobufagin. Some metabolites showed potential cytotoxicity against human hepatoma cells (HepG2) and human leukaemia (K562, HL-60) cells; however, their cytotoxicities generally decreased after metabolic conversion.     

A sensitive and specific precursor ion scanning approach in liquid chromatography/electrospray ionization tandem mass spectrometry to detect methylprednisolone acetate and its metabolites in rat urine

A new, simple, sensitive and specific liquid chromatography/electrospray ionization tandem mass spectrometric (LC/ESI‐MS/MS) method in precursor ion scanning (PIS) mode has been developed for the rapid detection of methylprednisolone acetate (MPA) and its metabolites in rat urine. A suitable product ion specific for methylprednisolone (MP) and MPA was selected after a fragmentation study on 20 (cortico)steroids at different collision energies (5–40 eV). Urine samples were simply treated with acetonitrile then dried in a SpeedVac system. The method was validated and compared with other PIS methods for detecting corticosteroids in human urine. It was more sensitive, with limit of detection (LOD) and lower limit of quantitation (LLOQ), respectively, of 5 and 10 ng mL^?1. The method was applied for the analysis of rat urine collected before and after (24, 48, 72 h) intra‐articular (IA) injection of a marketed formulation of MPA (Depo‐Medrol®). MS/MS acquisitions were taken at different collision energies for the precursor ions of interest, detected in PIS mode, to verify the MP‐related structure. Six different metabolites were detected in rat urine, and their chemical structures were assigned with a computational study. Copyright © 2010 John Wiley & Sons, Ltd.     

Urinary metabolic profile of 19-norsteroids in humans: glucuronide and sulphate conjugates after oral administration of 19-nor-4-androstenediol

19-Nor-4-androstenediol (NOL) is a prohormone of nandrolone (ND). Both substances are included in the WADA List of Prohibited Classes of Substances and their administration is determined by the presence of 19-norandrosterone (NA) with the urinary threshold concentration of 2 ng mL(-1). Routine analytical procedures allow the determination of NA excreted free and conjugated with glucuronic acid, but amounts of ND and NOL metabolites are also excreted in the sulphate fraction. The aim of this study is to determine the urinary metabolic profile after oral administration of a nutritional supplement containing NOL. Urine samples were collected up to 96 h following supplement administration and were extracted to obtain separately three metabolic fractions: free, glucuronide and sulphate. Extraction with tert-butyl methyl ether was performed after the hydrolysis steps and trimethylsilyl derivatives were analyzed by gas chromatography/mass spectrometry (GC/MS). After oral administration of NOL, the main metabolites detected were NA and noretiocholanolone (NE) in the glucuronide and sulphate fractions. The relative abundances of each metabolite in each fraction fluctuate with time; a few hours after administration the main metabolite was NA glucuronide whereas in the last sample (4 days after administration) the main metabolite was the NA sulphate and the second was the NE glucuronide. During the studied period almost half of the dose was excreted and the main metabolites were still found in urine after 96 h. Norepiandrosterone and norepietiocholanolone were also detected only in the sulphate fraction. Our results suggest that sulphate metabolites should be taken into consideration in order to increase the retrospectivity in the detection of 19-norsteroids after oral administration. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Quantification of cocaine and metabolites in exhaled breath by liquid chromatography-high-resolution mass spectrometry following controlled administration of intravenous cocaine

Breath has been investigated as an alternative matrix for detecting recent cocaine intake; however, there are no controlled cocaine administration studies that investigated the drug’s disposition into breath. Breath was collected from 10 healthy adult cocaine users by asking them to breathe into a SensAbues device for 3 min before and up to 22 h following 25 mg intravenous (IV) cocaine dosing on days 1, 5, and 10, and assayed with a validated liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method to quantify breath cocaine, benzoylecgonine (BE), ecgonine methyl ester (EME), and norcocaine. The assay was linear from 25 to 1,000 pg/filter, extraction efficiencies were 83.6–126 %, intra- and inter-assay imprecision was <10.6 %, and bias was between ?8.5 and 16.8 %. No endogenous or exogenous interferences were observed for more than 75 tested. Analytes were generally stable under short-term storage conditions. Ion suppression was less than 46 %. Of breath specimens collected after controlled cocaine administration, 2.6 % were positive for cocaine (26.1–66 pg/filter, 1–9.5 h), 0.72 % BE (83.3–151 pg/filter, 6.5–12.5 h), and 0.72 % EME (50–69.1 pg/filter, 6.5–12.5 h); norcocaine was not detected. Methanolic extraction of the devices themselves, after filters were removed, yielded 19.2 % positive cocaine tests (25.2–36.4 pg/device, 10 min–22 h) and 4.3 % positive BE tests (26.4–93.7 pg/device, 10 min–22 h), explaining differences between the two extraction techniques. These results suggest that the device reflects the drug in oral fluid as well as lung microparticles, while the filter reflects only drug-laden microparticles. A sensitive and specific method for cocaine, BE, EME, and norcocaine quantification in breath was developed and validated. Cocaine in breath identifies recent cocaine ingestion, but its absence does not preclude recent use.

Open image in new window

Graphical abstract ?

     

Urinary metabolites of nobiletin orally administered to rats

During the course of our systematic investigation of the metabolism of flavonoids, the polymethoxyflavone nobiletin, occurring in the fruits of Citrus depressa, was orally administered to rats. The urinary metabolites were separated and identified by three-dimensional HPLC equipped with a photodiode array detector and the structure was determined by spectroscopic methods to be 4'-hydroxy-3',5,6,7,8-pentamethoxyflavone (1).     

Urinary metabolites of genistein administered orally to rats.

In a study on the metabolism of flavonoids, the isoflavone genistein was administered orally to rats. Urine samples were collected and treated with beta-glucuronidase and arylsulfatase. Genistein and its metabolites, 4',5,7-trihydroxyisoflavanone (M1), 4',7-dihydroxyisoflavan (M2), and p-ethylphenol (M3) were isolated from the urine following treatment with enzymes. The structures of M1, M2, and M3 were determined on the basis of chemical and spectral data.     

Characterisation of putative pentose-containing conjugates as minor metabolites of 4-bromoaniline present in the urine of rats following intraperitoneal administration

The metabolic fate of 4-bromoaniline (4-BrA) was investigated following intraperitoneal administration to the rat at 50 mg kg(-1), using high-performance liquid chromatography/time-of-flight tandem mass spectrometry (HPLC/TOF-MS/MS). Up to five metabolites were detected in urine that correspond to isomeric pentose conjugates (possibly ribosides) of a hydroxysulphate of 4-BrA. This identification is supported by further studies where the water used in the reversed-phase solvent system was replaced with deuterated water in order to confirm that the number of exchangeable protons present in the metabolites was consistent with the proposed structures.     

Identification of budesonide metabolites in human urine after oral administration

Budesonide (BUD) is a glucocorticoid widely used for the treatment of asthma, rhinitis, and inflammatory bowel disease. Its use in sport competitions is prohibited when administered by oral, intravenous, intramuscular, or rectal routes. However, topical preparations are not prohibited. Strategies to discriminate between legal and forbidden administrations have to be developed by doping control laboratories. For this reason, metabolism of BUD has been re-evaluated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with different scan methods. Urine samples obtained after oral administration of 3?mg of BUD to two healthy volunteers have been analyzed for metabolite detection in free and glucuronide metabolic fractions. Structures of the metabolites have been studied by LC-MS/MS using collision induced dissociation and gas chromatography-mass spectrometry (GC/MS) in full scan mode with electron ionization. Combination of all structural information allowed the proposition of the most comprehensive picture for BUD metabolism in humans to this date. Overall, 16 metabolites including ten previously unreported compounds have been detected. The main metabolite is 16α-hydroxy-prednisolone resulting from the cleavage of the acetal group. Other metabolites without the acetal group have been identified such as those resulting from reduction of C20 carbonyl group, oxidation of the C11 hydroxyl group and reduction of the A ring. Metabolites maintaining the acetal group have also been identified, resulting from 6-hydroxylation (6α and 6β-hydroxy-budesonide), 23-hydroxylation, reduction of C6-C7, oxidation of the C11 hydroxyl group, and reduction of the C20 carbonyl group. Metabolites were mainly excreted in the free fraction. All of them were excreted in urine during the first 24?h after administration, and seven of them were still detected up to 48?h after administration for both volunteers.