Revealing the metabolic sites of atazanavir in human by parallel administrations of D‐atazanavir analogs

Atazanavir (Reyataz^®) is an important member of the HIV protease inhibitor class. Because of the complexity of its chemical structure, metabolite identification and structural elucidation face serious challenges. So far, only seven non‐conjugated metabolites in human plasma have been reported, and their structural elucidation is not complete, especially for the major metabolites produced by oxidations. To probe the exact sites of metabolism and to elucidate the relationship among in vivo metabolites of atazanavir, we designed and performed two sets of experiments. The first set of experiments was to determine atazanavir metabolites in human plasma by LC‐MS, from which more than a dozen metabolites were discovered, including seven new ones that have not been reported. The second set involved deuterium labeling on potential metabolic sites to generate D‐atazanavir analogs. D‐atazanavir analogs were dosed to human in parallel with atazanavir. Metabolites of D‐atazanavir were identified by the same LC‐MS method, and the results were compared with those of atazanavir. A metabolite structure can be readily elucidated by comparing the results of the analogs and the pathway by which the metabolite is formed can be proposed with confidence. Experimental results demonstrated that oxidation is the most common metabolic pathway of atazanavir, resulting in the formation of six metabolites of monooxidation (M1, M2, M7, M8, M13, and M14) and four of dioxidation (M15, M16, M17, and M18). The second metabolic pathway is hydrolysis, and the third is N‐dealkylation. Metabolites produced by hydrolysis include M3, M4, and M19. Metabolites formed by N‐dealkylation are M5, M6a, and M6b. Copyright © 2013 John Wiley & Sons, Ltd.     

Screening flavonoid metabolites of naringin and narirutin in urine after human consumption of grapefruit juice by LC-MS and LC-MS/MS

The main flavonoids in grapefruit juice, naringin and narirutin, were quantified by LC-MS with structural differentiation by LC-MS/MS. After human consumption of grapefruit juice, urine samples were collected for 24 hours and screened for flavonoid metabolites by LC-MS. The metabolite structures (glucuronides, sulfates, and glucuronide sulfates) were then confirmed via their unique fragmentation patterns by LC-MS/MS. To further verify the identity of the common aglycon (naringenin) shared by the metabolites, enzymatic hydrolysis was performed and the resulting products were analyzed. This work demonstrates that LC-MS and LC-MS/MS techniques can be used for fast metabolite screening without extensive sample preparation.     

Metabolic studies of turinabol in horses

Turinabol (4-chloro-17alpha-methyl-17beta-hydroxy-1,4-androstadien-3-one) is a synthetic oral anabolic androgenic steroid. As in the case of other anabolic steroids, it is a prohibited substance in equine sports. The metabolism of turinabol in human has been reported previously; however, little is known about its metabolic fate in horses. This paper describes the studies of both the in vitro and in vivo metabolism of turinabol in racehorses with an objective to identify the most appropriate target metabolites for detecting turinabol administration. For the in vitro studies, turinabol was incubated with fresh horse liver microsomes. Metabolites in the incubation mixture were isolated by liquid-liquid extraction and analysed by gas chromatography-mass spectrometry (GC-MS) after trimethylsilylation. The results showed that the major biotransformation of turinabol was hydroxylation at the C6, C16 and C20 sites to give metabolites 6beta-hydroxyturinabol (M1), 20-hydroxyturinabol (M2), two stereoisomers of 6beta,16-dihydroxyturinabol (M3a, M3b) and 6beta,20-dihydroxyturinabol (M4). The metabolite 6beta-hydroxyturinabol was confirmed using an authentic reference standard. The structures of all other turinabol metabolites were tentatively identified by mass spectral interpretation. For the in vivo studies, two horses were administered orally with turinabol. Pre- and post-administration urine samples were collected for analysis. Free and conjugated metabolites were isolated using solid-phase extraction and analysed by GC-MS as described for the in vitro studies. The results revealed that turinabol was extensively metabolised and the parent drug was not detected in urine. Two metabolites detected in the in vitro studies, namely 20-hydroxyturinabol and 6beta,20-dihydroxyturinabol, these were also detected in post-administration urine samples. In addition, 17-epi-turinabol (M5) and six other metabolites (M6a-M6c and M7a-M7c), derived from D-ring hydroxylation and A-ring reduction, were also detected. Except for 17-epi-turinabol, none of these metabolites has ever been reported in any species. All in vivo metabolites were detected within 48 h after administration.     

Imatinib metabolite profiling in parallel to imatinib quantification in plasma of treated patients using liquid chromatography-mass spectrometry

Besides affecting the systemic bioavailability of the parent drug, drug metabolizing enzymes (DMEs) may produce bioactive and/or toxic metabolites of clinical interest. We have investigated the capability to analyze simultaneously the parent drug and newly identified metabolites in patients' plasma by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The anticancer drug, imatinib, was chosen as a model drug because it has opened a new area in cancer therapy and is given orally and chronically. In addition, resistance and rare but sometimes severe side effects have been reported with this therapy. The quantification of imatinib and the profiling of its metabolites in plasma were established following three steps: (1) set-up of a generic sample extraction and LC-MS/MS conditions, (2) metabolite identification by LC-MS/MS using either in vitro incubations performed with human liver microsomes (HLMs) or patient plasma samples, (3) the simultaneous determination of plasma levels of imatinib and 14 metabolites in the plasma samples of 38 patients. Partial or cross method validation has been done and revealed that precise determinations of metabolite levels can be performed whereas pure standards are not available. Preliminary results indicate that the disposition of imatinib and its metabolites is related to interindividual variables and that outlier metabolite profiles can be revealed. This article underscores that, in addition to usual therapeutic drug monitoring (TDM), LC-MS/MS methods can simultaneously record a complete drug metabolic profile enabling various correlation studies of clinical interest.     

Quantification of azoxystrobin and identification of two novel metabolites in lettuce via liquid chromatography–quadrupole-linear ion trap (QTRAP) mass spectrometry

Pesticide metabolite identification is gaining increased attention because of the interest in potential metabolite toxicity. Azoxystrobin is one of the most prevalent pesticide residues in foods in Europe. The majority of azoxystrobin metabolites have been identified using radiolabelled standards, which are either expensive or not readily available. Thus, alternative approaches for metabolite identification are desirable. Here, an LC-MS/MS method for quantifying azoxystrobin and identifying its metabolites using quadrupole-linear ion trap mass spectrometry is reported. Seven metabolites of azoxystrobin were identified 2 and 4 weeks after spraying lettuce with azoxystrobin. Among them, two metabolites are reported for the first time. The hydrolysis, reduction, hydroxylation, photoisomerisation and hydrolytic cleavage of ether bonds are identified as biotransformation processes involved in azoxystrobin metabolism in lettuce.     

In vivo biotransformation of 17 alpha-methyltestosterone in the horse revisited: identification of 17-hydroxymethyl metabolites in equine urine by capillary gas chromatography/mass spectrometry

The in vivo phase I biotransformation of 17 alpha-methyltestosterone in the horse leads to the formation of a complex mixture of regio- and stereoisomeric C(20)O(2), C(20)O(3) and C(20)O(4) metabolites, excreted in urine as glucuronide and sulphate phase II conjugates. The major pathways of in vivo metabolism are the reduction of the A-ring (di- and tetrahydro), epimerisation at C-17 and oxidations mainly at C-6 and C-16. Some phase I metabolites have been identified previously by positive ion electron ionisation capillary gas chromatography/mass spectrometry (GC/EI + MS) mainly from the characteristic fragmentation patterns of their methyloxime-trimethylsilyl ether (MO-TMS), enol-TMS or TMS ether derivatives. Following oral administration of 17 alpha-methyltestosterone to two castrated thoroughbred male horses, the glucuronic acid conjugates excreted in post-administration urine samples were selectively hydrolysed by E. coli beta-glucuronidase enzymes. Unconjugated metabolites and the steroid aglycones obtained after enzymatic deconjugation were isolated from urine by solid-phase extraction, derivatised as MO-TMS ethers and analysed by GC/EI + MS. In addition to some of the known metabolites previously identified from the characteristic mass spectral fragmentation patterns of 17 alpha-methyl steroids, some isobaric compounds exhibiting a diagnostic loss of 103 mass units from the molecular ions with subsequent losses of trimethylsilanol or methoxy groups and an absence of the classical D-ring fragment ion were detected. From an interpretation of their mass spectra, these compounds were identified as 17-hydroxymethyl metabolites, formed in vivo in the horse by oxidation of the 17-methyl moiety of 17 alpha-methyltestosterone. This study reports on the GC/EI + MS identification of these novel 17-hydroxymethyl C(20)O(3) and C(20)O(4) metabolites of 17 alpha-methyltestosterone excreted in thoroughbred horse urine.     

Mass spectrometric characterization of urinary toremifene metabolites for doping control analyses

Toremifene is a selective estrogen receptor modulator included in the list of prohibited substances in sport by the World Anti-doping Agency. The aim of the present study was to investigate toremifene metabolism in humans in order to elucidate the structures of the most abundant urinary metabolites and to define the best marker to detect toremifene administration through the analysis of urine samples. Toremifene (Fareston) was administered to healthy volunteers and the urine samples were subjected to different preparation methods to detect free metabolites as well as metabolites conjugated with glucuronic acid or sulphate. Urinary extracts were analyzed by LC-MS/MS with triple quadrupole analyzer using selected reaction monitoring mode. Transitions for potential metabolites were selected by using the theoretical [M+H](+) as precursor ion and m/z 72 or m/z 58 as product ions for N,N-dimethyl and N-desmethyl metabolites, respectively. Toremifene and 20 metabolites were detected in excretion study samples, excreted free or conjugated with glucuronic acid or sulphate. Structures for most abundant phase I metabolites were proposed using accurate mass measurements performed by QTOF MS, based on fragmentation pattern observed for those metabolites available as reference standards. Several metabolic pathways including mono- and di-hydroxylation, N-desmethylation, hydroxymethylation, oxidation, dehalogenation and combinations were proposed. All metabolites were detected up to one month after toremifene administration; the most abundant metabolites were detected in the free fraction and they were metabolites resulting from dehalogenation. Several of the metabolites elucidated in this work have not been reported until now in the scientific literature.     

Metabolites in feces can be important markers for the abuse of anabolic steroids in cattle.

In Belgium, to control the abuse of anabolic steroids in cattle, urine samples have been gradually replaced by feces samples, because the latter can be obtained more easily from living animals. Urine and feces samples were collected from heifers after administration of boldenone, norethandrolone or ethylestrenol. Metabolites present in feces or urine were determined by GC-MS. Large qualitative and quantitative differences in the metabolic profiles were observed. In feces, in contrast to urine, the parent compounds or their major metabolites were detectable only shortly after administration. On the other hand, metabolites resulting from the reduction of the 3-oxo group and the unsaturated carbon-carbon bonds, present on the A-ring, allow for long-term detection in feces. A-ring reduced metabolites have been identified in samples found positive for norgestrel, boldenone, methylboldenone and methyltestosterone, respectively. These results are in agreement with concomitant in vivo experiments.     

Synthesis of heparin saccharides III. Synthesis of derivatives of D-glucosamine as starting materials for disaccharides.

Derivatives of benzyl 2-[1-(benzyloxy)formamido]-2-deoxy-α-D-glucopyranoside with various protecting groups at C(3) (benzoyl, benzyl and N-phenylcarbamoyl) and C(6) (benzoyl, benzylsulfonyl, N-phenylcarbamoyl and tosyl) have been synthesized as starting materials for disaccharides. The C(4) and C(6) hydroxyl groups of the amino sugar were initially blocked by an acetal group. After introduction of the protecting group at C(3), the acetal group was removed by acid hydrolysis, and the C(6) hydroxyl group was selectively acylated or sulfonylated. The 3,6-di-O-benzoate has also been prepared by dimolar benzoylation of the amino sugar, whereby the 4,6-isomer was obtained as a by-product.     

Determination of the metabolites of gestrinone in human urine by high performance liquid chromatography, liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry

Gestrinone was studied by high performance liquid chromatography (HPLC) for screening and by gas chromatography/mass spectrometry (GC/MS) for confirmation. When the chromatograms of blank, spiked urine and dosed urine were compared by HPLC, two unknown metabolites were found and these were excreted as the conjugated forms. Metabolites 1 and 2 were tested by LC/MS and LC/MS/MS and both had parent ions at m/z 325. The fragment ion of metabolite 1 was at m/z 263 and ions for metabolite 2 were m/z 307 [MH - H(2)O](+), 289, 279 and 241. LC/MS/MS of m/z 263 as the parent ion of metabolite 1 gave fragment ions at m/z 245 and 217, which were assumed to be [263 - H(2)O](+) and [235 - H(2)O](+), respectively. The trimethylsilyl (TMS)-enol-TMS ether derivative of gestrinone displayed three peaks in its GC/MS chromatogram, formed by tautomerism.     

Metabolism of a novel antiangiogenic agent KR-31831 in rats using liquid chromatography-electrospray mass spectrometry

KR-31831 ((2S,3R,4S)-4-(((1H-imidazol-2-yl)methyl)(4-chlorophenyl)amino)-6-amino-2-(dimethoxymethyl)-2-methyl-3,4-dihydro-2H-chromen-3-ol) is a novel antiangiogenic agent. In vitro and in vivo metabolism of KR-31831 in rats has been investigated using LC-MS and LC-MS/MS analysis. Incubation of rat liver microsomes and hepatocytes with KR-31831 produced three metabolites (M1-M3). M1, M2, and M3 were identified as N-((1H-imidazol-2-yl)methyl)-4-chlorobenzenamine, (2R,3R,4S)-4-(((1H-imidazol-2-yl)methyl)(4-chlorophenyl) amino)-6-amino-2-(hydroxymethyl)-2-methyl-3,4-dihydro-2H-chromen-3-ol, and N-((2S,3R,4S)-4- (((1H-imidazol-2-yl)methyl)(4-chlorophenyl)amino)-2-(dimethoxymethyl)-3-hydroxy-2-methyl-3,4-dihydro-2H-chromen-6yl)acetamide, respectively, by co-chromatography with the authentic standards and by comparison with product ion spectra of the authentic standards. Those in vitro metabolites were also detected in bile, plasma, or urine samples after an intravenous administration of KR-31831 to rats. The metabolic routes for KR-31381 included the metabolism of acetal group to hydroxymethyl group (M2), N-dealkylation to M1, and N-acetylation at the 6-amino group (M3).     

Development of a method which discriminates between endogenous and exogenous beta-boldenone

One potential explanation for the presence of beta-boldenone in calf urine is contamination of the sample with feces containing beta-boldenone. It has been demonstrated that after oral and intramuscular administration of beta-boldenone esters, several metabolites are formed and excreted in urine. One of the (minor) metabolites is 6beta-hydroxy-17alpha-boldenone. This paper describes an analytical method that can discriminate between unconjugated boldenone, its glucuronide- and sulphate-conjugates, 6beta-hydroxy-17alpha/beta-boldenone and coprostanol, a marker for fecal contamination. The method was applied to all samples suspected to contain boldenone within the Dutch National Residue Control Plan. Approximately 10,000 samples of urine were screened (LC-MS) in 2004-2005 by VWA-East, one of the official Dutch control laboratories, from which 261 samples were suspected to contain boldenone. These samples were all analyzed for their conjugation state, 6beta-hydroxy-17alpha/beta-boldenone and for the presence of coprostanol. Alfa-boldenone, the major metabolite in bovine urine after boldenone-ester administration, was found in a large number of these samples. The presence of alpha-boldenone was proven also to be a result of fecal contamination. None of the samples tested contained residues of the metabolite 6beta-hydroxy-17alpha/beta-boldenone. Not finding this metabolite indicates that the origin of alpha-boldenone-conjugates is endogenous. The results confirm that the presence of unconjugated beta-boldenone and alpha-boldenone conjugates next to alpha-boldenone are no indicators for illegal administration of boldenone-esters. No indications were obtained that conjugated beta-boldenone can be of endogenous origin.     

6alpha-Methylandrostenedione: gas chromatographic mass spectrometric detection in doping control

In recent years products containing 6alpha-methylandrost-4-ene-3,17-dione have appeared on the sport supplement market. Scientific studies have proven aromatase inhibition and anabolic and mild androgenic properties; however, no preparation has been approved for medical use up to now. In sports 6alpha-methylandrost-4-ene-3,17-dione has to be classified as a prohibited substance according to the regulations of the World Anti-Doping Agency (WADA). For the detection of its misuse the metabolism was studied following the administration of two preparations obtained from the Internet (Formadrol and Methyl-1-Pro). Several metabolites as well as the parent compounds were synthesized and the structures of 3alpha-hydroxy-6alpha-methyl-5beta-androstan-17-one, 6alpha-methylandrost-4-ene-3,17-dione, and 5beta-dihydromedroxyprogesterone were confirmed by nuclear magnetic resonance (NMR) spectroscopy. The main metabolite, 3alpha-hydroxy-6alpha-methyl-5beta-androstan-17-one, was found to be excreted as glucuronide and was still detectable in microg/mL amounts until urine collection was terminated (after 25 h). Additionally, samples from routine human sports doping control had already tested positive for the presence of metabolites of 6alpha-methylandrost-4-ene-3,17-dione. Screening analysis can be easily performed by the existing screening procedure for anabolic steroids using 3alpha-hydroxy-6alpha-methyl-5beta-androstan-17-one as target substance (limit of detection <10 ng/mL). Its discrimination from the closely eluting drostanolone metabolite, 3alpha-hydroxy-2alpha-methyl-5alpha-androstan-17-one, is possible as the mono-TMS derivative.     

Quantitative monitoring of tamoxifen in human plasma extended to 40 metabolites using liquid-chromatography high-resolution mass spectrometry: new investigation capabilities for clinical pharmacology

Liquid-chromatography (LC) high-resolution (HR) mass spectrometry (MS) analysis can record HR full scans, a technique of detection that shows comparable selectivity and sensitivity to ion transitions (SRM) performed with triple-quadrupole (TQ)-MS but that allows de facto determination of “all” ions including drug metabolites. This could be of potential utility in in vivo drug metabolism and pharmacovigilance studies in order to have a more comprehensive insight in drug biotransformation profile differences in patients. This simultaneous quantitative and qualitative (Quan/Qual) approach has been tested with 20 patients chronically treated with tamoxifen (TAM). The absolute quantification of TAM and three metabolites in plasma was realized using HR- and TQ-MS and compared. The same LC-HR-MS analysis allowed the identification and relative quantification of 37 additional TAM metabolites. A number of new metabolites were detected in patients’ plasma including metabolites identified as didemethyl-trihydroxy-TAM-glucoside and didemethyl-tetrahydroxy-TAM-glucoside conjugates corresponding to TAM with six and seven biotransformation steps, respectively. Multivariate analysis allowed relevant patterns of metabolites and ratios to be associated with TAM administration and CYP2D6 genotype. Two hydroxylated metabolites, α-OH-TAM and 4′-OH-TAM, were newly identified as putative CYP2D6 substrates. The relative quantification was precise (<20 %), and the semiquantitative estimation suggests that metabolite levels are non-negligible. Metabolites could play an important role in drug toxicity, but their impact on drug-related side effects has been partially neglected due to the tremendous effort needed with previous MS technologies. Using present HR-MS, this situation should evolve with the straightforward determination of drug metabolites, enlarging the possibilities in studying inter- and intra-patients drug metabolism variability and related effects.

Identification of in vitro metabolites of indinavir by “intelligent automated LC-MS/MS” (INTAMS) utilizing triple quadrupole tandem mass spectrometry

In an effort to improve the efficiency of the TSQ 7000 LC-MS/MS system for identification of drug metabolites in biological matrices in support of drug discovery programs, a combination of instrument control language procedures for the Finnigan MAT TSQ 7000 mass spectrometer, referred to as INTAMS, were composed. INTAMS was designed to conduct unattended, automatic liquid chromatography/mass spectrometry (LC-MS) and LC-MS/MS analyses of drugs and metabolites in commonly encountered in vitro biological matrices. A novel peak detection algorithm was developed to automatically detect and record the pseudomolecular ions and retention times of chromatographic components, even if not fully resolved. This algorithm was used in combination with an automated technique for predicting the molecular weights of metabolites based on incremental changes of the molecular weight of the parent drug resulting from well-known biotransformation processes. When applied to a sample of an incubation mixture of the HIV protease inhibitor Indinavir with a rat liver S9 preparation, the results obtained by the automatic metabolite detection procedures for LC-MS and LC-MS/MS analyses in real time were the same as those which were determined manually, by a knowledgeable operator.     

Identification of phase I and phase II metabolites of benfluron and dimefluron in rat urine using high-performance liquid chromatography/tandem mass spectrometry

Biotransformation products of two potential antineoplastic agents, benfluron and dimefluron, are characterized using our integrated approach based on the combination of high-performance liquid chromatography (HPLC) separation of phase I and phase II metabolites followed by photodiode-array UV detection and electrospray ionization tandem mass spectrometry (MS/MS). High mass accuracy measurement allows confirmation of an elemental composition and metabolic reactions according to exact mass defects. The combination of different HPLC/MS/MS scans, such as reconstructed ion current chromatograms, constant neutral loss chromatograms or exact mass filtration, helps the unambiguous detection of low abundance metabolites. The arene oxidation, N-oxidation, N-demethylation, O-demethylation, carbonyl reduction, glucuronidation and sulfation are typical mechanisms of the metabolite formation. The interpretation of their tandem mass spectra enables the distinction of demethylation position (N- vs. O-) as well as to differentiate N-oxidation from arene oxidation for both phase I and phase II metabolites. Two metabolic pathways are rather unusual for rat samples, i.e., glucosylation and double glucuronidation. The formation of metabolites that lead to a significant change in the chromophoric system of studied compounds, such as the reduction of carbonyl group in 7H-benzo[c]fluorene-7-one chromophore, is reflected in their UV spectra, which provides valuable complementary information to MS/MS data.     

Metabolism of androsta‐1,4,6‐triene‐3,17‐dione and detection by gas chromatography/mass spectrometry in doping control

The urinary metabolism of the irreversible aromatase inhibitor androsta‐1,4,6‐triene‐3,17‐dione was investigated. It is mainly excreted unchanged and as its 17β‐hydroxy analogue. For confirmation, 17β‐hydroxyandrosta‐1,4,6‐trien‐3‐one was synthesized and characterized by nuclear magnetic resonance (NMR) in addition to the parent compound. In addition, several reduced metabolites were detected in the post‐administration urines, namely 17β‐hydroxyandrosta‐1,4‐dien‐3‐one (boldenone), 17β‐hydroxy‐5β‐androst‐1‐en‐3‐one (boldenone metabolite), 17β‐hydroxyandrosta‐4,6‐dien‐3‐one, and androsta‐4,6‐diene‐3,17‐dione. The identification was performed by comparison of the metabolites with reference material utilizing gas chromatography/mass spectrometry (GC/MS) of the underivatized compounds and GC/MS and GC/tandem mass spectrometry (MS/MS) of their trimethylsilyl (TMS) derivatives. Alterations in the steroid profile were also observed, most obviously in the androsterone/testosterone ratio. Even if not explicitly listed, androsta‐1,4,6‐triene‐3,17‐dione is classified as a prohibited substance in sports by the World Anti‐Doping Agency (WADA) due to its aromatase‐inhibiting properties. In 2006 three samples from human routine sports doping control tested positive for metabolites of androsta‐1,4,6‐triene‐3,17‐dione. The samples were initially found suspicious for the boldenone metabolite 17β‐hydroxy‐5β‐androst‐1‐en‐3‐one. Since metabolites of androst‐4‐ene‐3,6,17‐trione were also present in the urine samples, it is presumed that these findings were due to the administration of a product like ‘Novedex Xtreme’, which could be easily obtained from the sport supplement market. Copyright © 2008 John Wiley & Sons, Ltd.     

Collision energy alteration during mass spectrometric acquisition is essential to ensure unbiased metabolomic analysis

Metabolomics entails identification and quantification of all metabolites within a biological system with a given physiological status; as such, it should be unbiased. A variety of techniques are used to measure the metabolite content of living systems, and results differ with the mode of data acquisition and output generation. LC-MS is one of many techniques that has been used to study the metabolomes of different organisms but, although used extensively, it does not provide a complete metabolic picture. Recent developments in technology, for example the introduction of UPLC-ESI-MS, have, however, seen LC-MS become the preferred technique for metabolomics. Here, we show that when MS settings are varied in UPLC-ESI-MS, different metabolite profiles result from the same sample. During use of a Synapt UPLC-high definition MS instrument, the collision energy was continually altered (3, 10, 20, and 30?eV) during MS acquisition. PCA and OPLS-DA analysis of the generated UPLC-MS data of metabolites extracted from elicited tobacco cells revealed different clustering and different distribution patterns. As expected, ion abundance decreases with increasing collision energy, but, more importantly, results in unique multivariate data patterns from the same samples. Our findings suggest that different collision energy settings should be investigated during MS data acquisition because these can contribute to coverage of a wider range of the metabolome by UPLC-ESI-MS and prevent biased results.     

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.     

Der Metabolismus des Retinoids Ro 10-9359. Isolierung und Identifizierung der Hauptmetaboliten aus Plasma,Urin und Faeces des Menschen sowie aus der Galle der Ratte Synthese von drei Urinmetaboliten

The Metabolism of the Retinoid Ro 10-9359. Isolation and Identification of the Major Metabolites in Human Plasma, Urine and Feces Synthesis of Three Urinary Metabolites After oral administration of therapeutic doses of the ³H-labelled aromatic retinoic acid analog (retinoid) Ro 10-9359 (ethyl all-trans-9-(4-methoxy-2,3,6-trimethyl-phenyl)-3,7-dimethyl-2,4,6,8-nonatetraenoate) to humans 75 and 15% of the ³H-dose were excreted within the first five days in the feces and the urine, respectively. Using chromatographic procedures including high pressure liquid chromatography 18 metabolites could be isolated from human urine. Their structures were elucidated by mass spectrometry and FT–¹H-NMR. spectroscopy. In these urinary metabolites the tetraene side chain of the parent compound Ro 10-9359 is shortened. The radioactivity of the identified urinary metabolites accounted for about 11% of the dose. Three urinary metabolites were synthesized. The main part of the radioactivity excreted within the first five days in the feces consisted of unchanged drug (60% of the dose). A smaller (amount 15% of the dose) could not be identified. The unchanged drug and a major metabolite, the corresponding acid, were found in human plasma. In an experiment with bile-duct cannulated rats the radioactively labelled retinoid Ro 10-9359 was injected intravenously. About 70% of the ³H-dose was excreted in the bile, within the first 48 hours. The whole radioactivity of the rat bile consisted of polar metabolites. No unchanged drug could be found. After enzymatic hydrolysis of the bile conjugates three metabolites were isolated. The main metabolite (49% of the i.v. dose) was a conjugate of the corresponding acid of the parent drug, already found as free compound in human plasma. The other bile metabolites (9 and 7% of the i.v. dose) had an intact side chain, too. An enterohepatic recycling of the bile metabolites was observed in the rat.