Natural organic matter contained in clay rock pore water: Direct quantification at the molecular level using electrospray ionization mass spectrometry.

RATIONALE: Natural organic matter (NOM) is present in the environment and could influence the migration of heavy metals/radionuclides. The dissolved fraction of NOM (DOM) is usually quantified using total organic carbon analysis or UV-visible spectrometry. Nonetheless, analysis using pattern recognition cannot provide the full spectrum of organic molecules contained in waters, especially low-molecular-weight compounds. In the context of nuclear performance assessment studies, ground waters may contain DOM and a key aspect is to quantify different categories of NOM types in order to further evaluate the transport and fate of radionuclides in the environment. METHODS: Thus, a method for the quantification of DOM at the molecular level was developed, based on electrospray ionization mass spectrometry (ESI-MS). This method simultaneously gives structural information on DOM and the individual concentrations of these low-molecular-weight compounds without pretreatment and/or preconcentration of the samples. RESULTS: Several methods of quantification (internal calibration, calibrated addition of external standard, sequential tandem mass spectrometry) have been optimized and successfully applied to real natural samples. They are discussed in this paper with a focus on acidic compounds, which are the compounds that most probably could influence the migration of heavy metals and radionuclides in the clay rock pore water from the French Callovo-Oxfordian (COx) nuclear repository site. CONCLUSIONS: Quantification of in situ dissolved NOM from the COx has been performed using ESI-MS. For the first time to our knowledge, it was possible to give a quite exhaustive and quantitative inventory of the small organic compounds present without proceeding to any chemical treatment or sample crushing and for naturally occurring concentrations.

Comparison between liquid chromatography and supercritical fluid chromatography coupled to mass spectrometry for beta-agonists screening in feeding stuff.

ß-agonistic drugs have been forbidden as growth promoters in rearing animals in Europe since the late 1980s (Dir 96/22/EC). Specific and sensitive analytical methods based on UHPLC-MS/MS allow to monitor a large set of these substances. However, optimal performances are not observed for all the target analytes, especially for those exhibiting the highest polarities. We developed an SFC-MS/MS approach to cover the huge elution window of ß-agonists, from the most polar which are usually eluted in the void volume when using reversed phase chromatography in conventional HPLC to the most apolar ones. The objective was to reach performances in accordance with the European Union recommended level in feeding stuff, i.e. 50 µg kg-1. LC/MS and SFC/MS performances were thoroughly compared in terms of analytical validation data (linearity, selectivity, recovery rates, reproducibility, compounds identification, trueness, decision limit (CCα) and detection capability (CCß)) for 6 ß-agonistic drugs, namely bromobuterol, clenbuterol, isoxsuprine, ractopamine, salbutamol and zilpaterol. As a result, the SFC approach appeared complementary to the LC one because the elution order of compounds was totally different from the one obtained with a classical C18 stationary phase. Moreover, the UPLC-MS/MS approach gave a better response linearity and more accurate values, whereas SFC-MS/MS provided greater data for identification purposes, reproducibility and sensitivity. Both analytical approaches enabled the detection of targeted ß-agonists at a lower concentration than the recommended one (50 µg kg-1).

Fast and multiresidue determination of twenty glucocorticoids in bovine milk using ultra high performance liquid chromatography-tandem mass spectrometry.

Glucocorticoids constitute a class of molecules widely used in animal husbandry. Some of these compounds are licensed for veterinary practices while their use for growth promoting purposes is prohibited within the European Union. In order to ensure the respect of the legislation and consumers safety, several methodologies have been proposed to monitor these substances in various products, including edible matrices for which a regulatory limit has been set up (MRL). An extended range of targeted analytes together with reduced time of analysis and cost are however still current challenges regularly revisited according to the continuous technological improvements. In this context, the aim of the present study was to develop and implement a new fast and multi-residue method based on UHPLC-MS/MS for the determination of twenty glucocorticoids in bovine milk, included the screening of the three regulated MRL compounds (dexamethasone, betamethasone and prednisolone). This validated method authorises such multi-analyte measurement within a 10min runtime while the signal specificity is ensured through the SRM acquisition mode. Decision limits and detection capabilities were calculated in the range of 0.001-0.363µgL(-1), which allows a very efficient control at low trace level for a potential illegal use of these substances. The performances obtained in terms of application range, selectivity and sensitivity were found to be significantly improved in comparison to other reported approaches either for screening or confirmation purposes: regarding linearity, correlation coefficients were above 0.98 within the range of 0.01-5.0µgL(-1), repeatability and reproducibility parameters ranged from 1 to 30% with the maximum relative standard deviation (RSD) observed for cortisone (30.1%). Stability of the stock solutions and minor changes in the standard operating procedure have been included for the determination of ruggedness of the method. Identification was systematically ensured according to 4 identification points, RSD of transitions ratio (T2/T1) ranged from 3.2% and 19.3% and the RSD of the retention time was lower than 0.25%.

Improvement of estradiol esters monitoring in bovine hair by dansylation and liquid chromatography/tandem mass spectrometry analysis in multiple reaction monitoring and precursor ion scan modes.

RATIONALE: The control of forbidden anabolic practices in cattle in the European Union has become challenging since endogenous compounds such as estradiol derivatives can potentially be used as growth promoters. Due to the great difficulty in establishing a reference threshold value for endogenous steroids, the direct detection of steroid esters in hair is an efficient strategy for the detection of 'natural' steroid abuse in cattle. METHODS: The present study aimed to develop and validate according to the current European standards a specific liquid chromatography/tandem mass spectrometry (LC/MS/MS) analytical strategy to monitor estrogen esters in bovine hair. The analysis was performed by positive ion electrospray ionisation (ESI+) after dansylation. Two acquisition modes were then assessed: single reaction monitoring and precursor ion scanning. RESULTS: The results showed that the introduction of a dansylation step strongly improves the sensitivity of the detection of estradiol-17-esters by LC/(ESI+)-MS/MS. The CCα values are in the range 1-10 ng g(-1) after optimisation, except for estradiol decanoate for which the derivatisation is not efficient. In addition, this LC/MS/MS approach makes it possible to carry out a precursor ion scan to screen for the presence of these estradiol 17-esters in hair samples. CONCLUSIONS: Based on the specific product ions, i.e. m/z 255 in native conditions or m/z 171 after dansylation, this strategy has the advantage of detecting any (un)known estradiol ester and of giving access to the [M + H](+) ion of the suspected ester through only a single analysis.

Comparative study of low- versus high-resolution liquid chromatography-mass spectrometric strategies for measuring perfluorinated contaminants in fish.

Perfluorinated compounds (PFCs) are usually monitored by high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) on triple quadrupole instruments. Although not yet widely implemented in the field, high-resolution mass spectrometry (HRMS) today appears as a valuable alternative for these halogenated chemicals due to their significant mass defect. Indeed, this second approach offers a way to cope with particular matrix effects caused by co-eluting and isobaric interferences affecting the measurement of some PFCs in fish. The present study compares three different LC-MS-related instruments and various signal acquisition modes, from low-resolution full-scan and selected ion-monitoring (SIM) mode on a triple quadrupole (QqQ) instrument to high-resolution full-scan or product ion-scan mode on orbital trap (LTQ-Orbitrap) or quadrupole-time-of-flight (Q-TOF) devices. Performances are compared for seven model compounds belonging to seven PFCs subclasses: perfluoralkylsulfonate, perfluoroalkylcarboxylate, perfluoroalkylsulfinate, perfluoroalkyl-sulfonamide, fluorotelomer saturated acid, fluorotelomer unsaturated acid and perfluoroalkylphosphonic acid. Low-resolution MS/MS was found to be unsurprisingly reliable for extended multi-residue monitoring. However, the high stability of PFCs leads to a relatively poor and non-specific fragmentation pathway in MS/MS. In addition, biliary acid-interfering compounds (e.g. taurochenodeoxycholic acid), which where encountered in the present case in fish samples but that may be present in other biological samples, were found particularly disturbing in low-resolution MS/MS. Indeed, these interferences presented the same retention time and diagnostic signals as PFOS, leading to a possible overestimation of the PFOS quantification in LC-MS/MS. On the other hand, high-resolution MS and MS/MS (LTQ-Orbitrap and Q-TOF) provided better results in terms of signal specificity and sensitivity. For instance, the estimated limits of detection (LOD) reached for PFOS on QqQ, Q-TOF and LTQ-Orbitrap instruments were 3.8, 0.7 and 0.5 pg injected, respectively.

Determination of naturally occurring oestrogens and androgens in retail samples of milk and eggs.

The occurrence of the main steroid hormones (oestrone, 17alpha-oestradiol, 17beta-oestradiol, 17alpha-testosterone, 17beta-testosterone, dehydroepiandrosterone, 4-androstenedione), especially in milk and eggs, was investigated. An analytical method based on GC-MS/MS was developed for steroid measurement at an ultra-trace level in food products. The limits of detection for oestrogens were about 5 and 30 ng kg(-1) in milk and eggs, respectively. For androgens, the limits of detection were around 10 and 50 ng kg(-1) in milk and eggs, respectively. The method was applied to milk and egg samples collected in a French supermarket. In milk, oestrone was found at levels between 100 and 300 ng l(-1), while 17beta-oestradiol levels were estimated to be near 20 ng l(-1). 17alpha-testosterone was found to be from 50 ng l(-1) in skimmed milk to 85 ng l(-1) in whole milk. In egg samples, oestrone and 17beta-oestradiol were found at 1.5 and 0.9 microg kg(-1), respectively, while 17alpha-oestradiol was found to be in lower concentrations (i.e. around 0.55 microg kg(-1)). Regarding androgens, 17alpha- and 17beta-testosterone were estimated at 1.9 and 1.3 microg kg(-1), respectively. These results represent a first attempt to estimate the food exposure to steroid hormones. In the future, the collection of additional data should permit the comparison between this exogenous dietary intake and the daily endogenous production in pre-pubertal children as a basis of risk assessment regarding endocrine disruption linked to these molecules for this critical population.

Development and application of stable carbon isotope analysis to the detection of cortisol administration in cattle.

The use of anabolic agents in food-producing animals has been prohibited within the EU since 1988. The control of the illegal use of natural steroid hormones in cattle is still an exciting analytical challenge as no definitive method and nonambiguous analytical criteria are available. We have used gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) to demonstrate the administration of cortisol to cattle. The method consisted of an efficient combination between OASIS HLB solid-phase extraction (SPE), oxidation, SiOH SPE and semi-preparative high-performance liquid chromatography (HPLC) for glucocorticoid purification. By comparison of the (13)C/(12)C isotopic ratio of the oxidised product of cortisol, i.e. 5 beta-androstane-3,11,17-trione (5 beta AAT), with an endogenous reference compound (ERC), dehydroepiandrosterone (DHEA), the differentiation of cortisol metabolite origin, either endogenous or exogenous, has been achieved. After treatment of an animal, the delta(13)C(VPDB) values of 5 beta AAT reached -30 to -32 per thousand, whereas the delta(13)C(VPDB) values of DHEA remained at -25 per thousand. A significant difference in the delta(13)C(VPDB) values between DHEA and 5 beta AAT was measurable over a period of 3 days after a single administration of cortisol to the animal.

Milk and urine excretion of polycyclic aromatic hydrocarbons and their hydroxylated metabolites after a single oral administration in ruminants.

The aim of this study was to establish the transfer of phenanthrene, pyrene, and benzo[a]pyrene and their major hydroxylated metabolites to milk and to urine after a single oral administration (100 mg per animal of each compound) in 4 lactating goats. Detection and identification of the analytes (native compounds, 1-OH pyrene, 3-OH phenanthrene, 3-OH benzo[a]pyrene) were achieved using gas chromatography-mass spectrometry. Benzo[a]pyrene, phenanthrene, and pyrene were rapidly detected in the plasma stream, whereas 1-OH pyrene and 3-OH phenanthrene appeared later in plasma. These data suggest that pyrene and phenanthrene are progressively metabolized within the organism. Recovery rates of pyrene and phenanthrene in milk over a 24-h period appeared to be very low (0.014 and 0.006%, respectively), whereas the transfer rates of their corresponding metabolites were significantly higher: 0.44% for 1-OH pyrene and 0.073% for 3-OH phenanthrene. Recovery rates in urine were found to be higher (1 to 10 times) than recovery rates in milk. The 1-OH pyrene was found to be the main metabolite in urine as well as in milk. Thus, as has been established for humans, 1-OH pyrene could be considered as a marker of ruminant exposure to polycyclic aromatic hydrocarbons. Because 1-OH pyrene and 3-OH phenanthrene were measured in milk (unlike their corresponding native molecules), metabolites of polycyclic aromatic hydrocarbons should be taken into consideration when evaluating the safety of milk. Benzo[a]pyrene and 3-OH benzo[a]pyrene were (less than 0.005%) transferred to milk and urine in very slight amounts. This very limited transfer rate of both compounds suggests a low risk of exposure by humans to benzo[a]pyrene or its major metabolite from milk or milk products.

Simultaneous measurement of plasma concentrations and 13C-enrichment of short-chain fatty acids, lactic acid and ketone bodies by gas chromatography coupled to mass spectrometry.

A new method has been developed for the simultaneous measurement, in a reduced plasma sample, of concentration and 13C-isotopic enrichment of acetic, propionic, butyric, lactic, acetoacetic and beta-hydroxybutyric acids by gas chromatography coupled to mass spectrometry. After plasma deproteinisation, a diethylic extraction and a N-tert.-butyldimethylsilyl-N-methyltrifluoroacetamide derivatisation were performed. Both diethyl extraction and derivatisation procedures were optimised using the central composite designs methodology. The optimised method provides good linearity, intra-day and within-day repeatability. Except for beta-hydroxybutyric (49 microM) and acetoacetic acid (5 microM), detection limits were ranging between 0.2 and 0.7 microM allowing uses of this method for colonic metabolism studies.

Gas chromatography/combustion/isotope ratio mass spectrometry to control the misuse of androgens in breeding animals: new derivatisation method applied to testosterone metabolites and precursors in urine samples.

A new derivatisation reaction applied to the analysis of steroids by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) was studied. The trimethylsilylated steroids were characterised by well-resolved chromatographic signals, no peak tailing, reproducible 13C/12C measurements (0.32 per thousand, n = 28), good signal-to-noise ratio and absolute intensity (5 x 10(-9) A, 20 ng), and a slow degradation of copper oxide pellets in the combustion furnace. In addition, two new metabolites and one precursor of testosterone in bovine have been brought into consideration and used for GC/C/IRMS measurements, namely, 3beta-hydroxy-5alpha-androstan-17-one (epiandrosterone), 3beta,17alpha-dihydroxy-5alpha-androstane, and 3beta,17alpha-dihydroxy-5-androstene. The new findings have been applied to an elimination study in bovine of testosterone metabolites after an intramuscular injection of testosterone enanthate. Significant differences (up to 4 per thousand) between testosterone metabolites and precursor were detectable at least three weeks after administration.

Multi-residue extraction-purification procedure for corticosteroids in biological samples for efficient control of their misuse in livestock production.

A fast and efficient multi-residue extraction-purification procedure was developed for 12 corticosteroids in biological matrices (hair, urine and meat), in order to control their illegal use as growth promoters in cattle. Detection and identification of the analytes were achieved using a previously described LC-MS-MS method based on negative electrospray ionisation and a triple quadrupole analyser. The presented procedures included acid (hair) or enzymatic (urine and meat) hydrolysis, C18 reversed-phase SPE, Na2CO3 liquid-liquid clean-up and SiOH normal-phase SPE. The detection limits of the developed methods were between 2.9 and 9.3 pg/mg (ppb) for hair samples and in the 40 - 70 pg/g (ppt) range for the urine or meat samples. The acid hydrolysis used for corticosteroid extraction in hair was optimised using an experimental design and response surface methodology. Achieved performances were linked to a physico-chemical approach based on the corticosteroids specific C17 side-chain. This original multi-residue and multi-matrices analytical methodology will be used for further metabolism studies.

Biosynthesis of 6beta-hydroxymethyltestosterone using bovine hepatocyte cultures.

Usually performed to investigate biotransformations of xenobiotics, in vitro liver models could become useful tools for the synthesis of not commercially available compounds. In this study, bovine hepatocyte cultures were used to biosynthesise, on the laboratory scale, one major metabolite of methyltestosterone: 6beta-hydroxymethyltestosterone. After incubation of bovine hepatocytes with methyltestosterone for 24 h, culture medium was removed and stored at -20 degrees C until analysis. The sample was extracted and purified on a reversed-phase HPLC system. The metabolite of interest was then analysed in LC-MS and GC-MS for structural identification. The purity and the isomery of the 6 and 17 positions were confirmed by NMR analyses. This first success in producing purified 6beta-hydroxymethyltestosterone from bovine hepatocyte cultures allowed us to consider that in vitro liver models could be reliable tools for standard biosynthesis.

Consequence of boar edible tissue consumption on urinary profiles of nandrolone metabolites. I. Mass spectrometric detection and quantification of 19-norandrosterone and 19-noretiocholanolone in human urine.

For the first time in the field of steroid residues in humans, demonstration of 19-norandrosterone (19-NA: 3alpha-hydroxy-5alpha-estran-17-one) and 19-noretiocholanolone (19-NE: 3alpha-hydroxy-5beta-estran-17-one) excretion in urine subsequent to boar consumption is reported. Three male volunteers agreed to consume 310 g of tissues from the edible parts (meat, liver, heart and kidney) of a boar. The three individuals delivered urine samples before and during 24 h after meal intake. After deconjugation of phase II metabolites, purification and specific derivatisation of target metabolites, the urinary extracts were analysed by mass spectrometry. Identification was carried out using measurements obtained by gas chromatography/high resolution mass spectrometry (GC/HRMS) (R = 7000) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) (positive electrospray ionisation (ESI+)). Quantification was realised using a quadrupole mass filter. 19-NA and 19-NE concentrations in urine reached 3.1 to 7.5 microg/L nearby 10 hours after boar tissue consumption. Levels returned to endogenous values 24 hours after. These two steroids are usually exploited to confirm the exogenous administration of 19-nortestosterone (19-NT: 17beta-hydroxyestr-4-en-3-one), especially in the antidoping field. We have thus proved that eating tissues of non-castrated male pork (in which 17beta-nandrolone is present) might induce some false accusations of the abuse of nandrolone in antidoping.

Characterization of exogenous testosterone in livestock by gas chromatography/combustion/isotope ratio mass spectrometry: influence of feeding and age.

The detection of exogenous testosterone in bovine urine was investigated by using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). The carbon isotopic ratio measurement of epitestosterone, etiocholanolone (testosterone metabolite) and DHEA (testosterone precursor) in female bovine urines after testosterone enanthate administration was carried out. An important modification in the 13C/12C ratio of testosterone metabolites was observed, such that significant differences between precursor and metabolites of testosterone occurred until three weeks after intramuscular administration of testosterone enanthate. The factors influencing the 13C/12C of endogenous steroids were studied especially through cattle feeding and age. The DHEA mean delta13C value was found to vary between -25 and -26/1000 when hay and concentrate diet were used for fattening. On the other hand the delta13C value observed when maize silage was used increased to -20/1000. Testosterone metabolites showed the same delta13C increase as their precursor. Moreover, we observed a clear relationship between age and efficiency of misuse determination. Indeed, because of the lower concentration of natural hormones in young animals, the contribution of exogenous molecules increases significantly compared with older subjects. Consequently, demonstration of administration is easier to achieve in calves than in mature animals.

Ultra trace detection of a wide range of anabolic steroids in meat by gas chromatography coupled to mass spectrometry.

The control on use of anabolic agents in meat producing animals is generally based on urine, faeces or hair analysis. This exercise, which is usually performed in slaughterhouses or on farms, is not relevant to imported carcasses or retail meat. A single sensitive method for a wide range of anabolic steroids was developed. After extraction of the lyophilised meat, enzymatic hydrolysis was used for deconjugation. Solid-phase extraction on a polymeric stationary phase was performed prior to hydrolysis of ester residues under alkaline conditions. Liquid-liquid partitioning was used to separate the analytes into two main categories: phenol containing molecules, such as phenolic steroids, resorcylic acid lactones and stilbenes, and delta4-3-one containing molecules, such as most androgens and progestagens. Solid-phase extraction on silica columns was performed before applying a specific derivatisation for each compound sub-group. The combination of high-resolution chromatography with a quadrupole mass spectrometer permitted detection of 23 steroids in the 5-100 ng/kg range. Ion chromatograms for residue positive samples are shown and discussed.

Collision-induced dissociation of corticosteroids in electrospray tandem mass spectrometry and development of a screening method by high performance liquid chromatography/tandem mass spectrometry.

A screening method based on liquid chromatography/electrospray tandem mass spectrometry was developed in order to control the illegal use of corticosteroids as growth promoters in cattle. The objective was the detection of low residue levels of corticosteroids or metabolites in biological matrices. Relative to other studies published on this subject, the present work focused on enhancing specificity and sensitivity. Firstly, fragmentation of corticosteroids by collision-induced dissociation was studied. In positive mode, the losses of H(2)O for each hydroxyl group fixed on the molecule, as well as the loss of HF or HCl for halogenated compounds, were observed. For higher collision energy, fragmentations in the B, C and D rings were induced. The negative mode was found to be more specific, inducing a cleavage of the C(20)-C(21) bond with concomitant loss of formaldehyde (CH(2)O). Secondly, three acquisition methods in the negative mode were studied and evaluated, recorded signals being the parent ion [M + acetate](-) and the two daughter ions, [M - H](-) and [M - H - CH(2)O](-). For dexamethasone, MS/MS instrumental detection limits of fragment ion and neutral loss scans, and of multiple reaction monitoring (MRM), were 250, 20 and 5 pg injected, respectively. The MRM method was then evaluated with the objective of use for the detection of corticosteroid residues in biological samples (urine, hair, muscle) and for a metabolism study.

Enzymatic hydrolysis of conjugated steroid metabolites: search for optimum conditions using response surface methodology.

The optimum conditions for hydrolysing conjugated metabolites of steroid hormones in bovine urine were performed with Helix pomatia juice, beta-glucuronidase from bovine liver and preparations of limpets and abalone entrails using response surface methodology. The experimental design and empirical modelling used allowed us to assess the main effects of factors (time, temperature, pH and enzyme quantity) and to predict the optimum conditions for each enzyme preparation. Confirmatory experiments were applied to check the predicted values and to validate the model. The comparison of the enzyme preparation efficiency for various conjugate steroids and the study of possible by-product synthesis led us to select abalone entrails to hydrolyse natural dehydroepiandrosterone, etiocholanolone, epitestosterone; 17 alpha-estradiol and estrone in bovine urine. The optimum conditions were found to be 20 h at 42 degrees C with the pH adjusted to 5.2 and using 12,000 units of enzyme preparation.

Evidence for the presence of endogenous 19-norandrosterone in human urine.

In 1997, in the scope of antidoping control in sport, a not inconsiderable number of urine analysed by official laboratories revealed the presence of 19-nortestosterone (19-NT: 17beta-hydroxyestr-4-en-3-one) metabolites: 19-norandrosterone (19-NA: 3alpha-hydroxy-5alpha-estran-17-one) and 19-noretiocholanolone (19-NE: 3alpha-hydroxy-5beta-estran-17-one). These repeated results on a short period of time generated some investigations and especially the verification of the possible production of these metabolites by an unknown endogenous route in adult entire male. Some experiences were led on different persons known to be non-treated with steroids and more precisely with nandrolone. Extractive methods were developed focusing on their selectivity, i.e. searching to eliminate at best matrix interferences from the target analytes. Gas chromatography coupled to mass spectrometry (quadrupole and magnetic instruments) was used to detect, identify and quantify the suspected signals. Two types of derivatization (TMS and TBDMS), a semi-preparative HPLC as well as co-chromatography proved unambiguously the presence, in more than 50% of the analysed urine (n = 40), of 19-NA at concentrations between 0.05 and 0.60 ng/ml. 19-NE was not detected with the developed methods (LOD<0.02 ng/ml). Experiments led on athletes showed that after a prolonged intense effort, the 19-NA concentration can be increased by a factor varying between 2 and 4. Even if some complementary researches have to be done in order to determine the maximal physiological level of 19-NA and 19-NE, these results should considerably change the strategy of antidoping laboratories.

4-Chlorotestosterone acetate metabolites in cattle after intramuscular and oral administrations.

The use of 4-chlorotestosterone acetate by farmers for cattle fattening was recently demonstrated although the use of this anabolic steroid is strictly forbidden in the European Union. We investigated the metabolism of 4-chlorotestosterone acetate in the bovine species after intramuscular and oral administration. Nineteen metabolites were detected in urine after intramuscular injection, and eight metabolites were identified. For this purpose, preparative HPLC, mass spectrometry with different ionization modes (electronic impact and chemical ionization), and different acquisition techniques were used (high resolution, selected ion monitoring, and scan measurement). Metabolite stereoisomerism was determined on the basis of retention time and organic synthesis. 4-Chloroepitestosterone (M2), 4-chloroandrost-4-en-3alpha-ol-17-one (M3), and 4-chloroandrost-4-ene-3,17-dione (M4) were identified as the main urinary markers of intramuscular administration. On the other hand, 4-chloroandrost-4-ene-3alpha,17beta-diol (M7), 4-chloroandrostan-3beta-ol-17-one (M5), and M2 were the primary indicators of an oral administration. In addition, we have shown that 95% of the metabolites were sulfo-conjugated, except for M3, which was partially conjugated to glucuronic acid. Finally, the main metabolites (M2, M3, and M4) were easily identified for 1.5 months after intramuscular administration.

Calf primary hepatocyte culture as a tool for anabolic steroid metabolism studies.

Calf hepatocyte cultures were developed for the predictive analysis of in vivo xenobiotic biotransformations. The goal of this work was to show the feasibility of a system based on calf primary hepatocyte cultures using radiolabelled molecules to study xenobiotic metabolism rather than to study exhaustively the metabolism of a particular steroid. 19-Nortestosterone was chosen as a model substrate because of its relatively well known metabolism in the bovine species. Incubation of a mixture of tritiated and non-tritiated 19-nortestosterone was investigated in cell culture in order to target the biosynthesized metabolites. After extraction and purification, some of the in vitro metabolite structures were elucidated by GC-MS and compared with the main urinary metabolites detected in vivo after intramuscular injection of 19-nortestosterone into a calf. 19-Norepitestosterone, the main in vivo metabolite, was identified in vitro. However, the main in vitro metabolites were mostly in an oxidized form (4-estrene-3,17-dione, hydroxy-4-estrene-3,17-dione).