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.

N-methyl-N-alkylsilyltrifluoroacetamide-I2 as a new derivatization reagent for anabolic steroid control.

Analytical methods for the control of growth promoters have to be specific and sensitive. At low concentration levels, it is difficult to identify some molecules unambiguously even with the improved performance of analytical methods. GC-MS analysis of 17 beta-trenbolone and its major metabolite, 17 alpha-trenbolone, is a good example. A new derivatization agent has been developed which is based on silylation of the 3- and 17-oxygenated functions and nucleophilic substitution in the 4-position. The structure of the derivatized products was demonstrated using a simple model, cyclohex-2-en-1-one, by NMR and MS spectrometry. In contrast to data found in the literature, this derivative permitted specific mass spectra for trenbolone, sensitive signals for high mass ions and reproducible gas chromatograms to be obtained. The addition of an N(CH3)COCF3 radical to the steroid nucles allowed highly specific detection in GC-high resolution MS even following extraction from complex matrices; sensitive responses were also observed in the negative chemical ionization mode. Moreover, there are significant differences in the electron ionization mass spectra of the two stereoisomers, 17 alpha- and 17 beta-trenbolone. These preliminary results and those obtained for androsta-1,4-dien-3-one and pregna-4,6-dien-3-one indicate useful advances for the determination of steroids and potential applications for metabolism studies on such compounds.

Gas chromatographic-mass spectrometric identification of main metabolites of stanozolol in cattle after oral and subcutaneous administration.

An analytical method has been developed in order to control the illegal use of stanozolol as growth promoter in livestock. The procedure was based on enzymatic hydrolysis, purification on a Clean Screen DAU column and derivatization with heptafluorobutyric anhydride prior to GC-MS analysis. This method allowed us to study the metabolism of stanozolol in cattle after oral and subcutaneous administrations. Urinary metabolites were identified by mass spectrometry. Stanozolol and 16-hydroxystanozolol were detected after oral administration, while 16-hydroxystanozolol and 4,16-dihydroxystanozolol were found after subcutaneous administration.

Identification of endogenous 19-nortestosterone in pregnant ewes by gas chromatography-mass spectrometry.

Considered as a xenobiotic for many years, 19-nortestosterone has been extensively studied. Analyses developed to control the illegal use of this steroid in meat-producing animals led researchers to demonstrate the endogenous presence of 19-nortestosterone in several species. In this paper, the natural occurrence of 19-nortestosterone in its alpha form (epinandrolone) in the urine of pregnant sheep was demonstrated using gas chromatography-mass spectrometry. This reference method allowed 17 alpha-nandrolone to be detected and identified, and the absence of the beta epimer in the urine of pregnant sheep to be demonstrated. 17 alpha-Nandrolone was accurately determined at different stages of pregnancy. The analyses showed that epinandrolone, which was not detectable in the urine of non-pregnant sheep, was excreted in small amounts (leading to a < 0.5 ppb concentration) during the first 4 months of pregnancy. 17 alpha-Nandrolone concentrations then increased during the last month until parturition. The origin of this molecule was not determined.

Analysis of beta-agonists in urine and tissues by capillary gas chromatography-mass spectrometry: in vivo study of salbutamol disposition in calves.

The fate of salbutamol sulphate given orally has been investigated in calves. The urinary excretion rate and the tissue distribution of this beta-agonistic drug were studied by capillary gas chromatography coupled to low resolution mass spectrometry (GC-LRMS) under electron impact (EI) ionization mode, using an hexadeuterated salbutamol analogue as the internal standard. The parent drug and metabolites were extracted via solid phase extraction (SPE) mixed-phase-containing disposable columns and analysed as their trimethylsilyl derivatives. A more efficient clean-up had to be carried out for tissue samples. An acidic precipitation followed by a liquid-liquid extraction were therefore performed before the SPE. Moreover, the problem of tissue digestion was elucidated by means of an ultrasonic probe. Samples were also analysed before and after enzymic hydrolysis using purified beta-glucuronidase and a mixture of beta-glucuronidase and arylsulphatase, to obtain evidence of phase II conjugation mechanisms. Both free salbutamol and conjugated metabolites were detected in urine and tissue samples. Except for liver or kidney, salbutamol was rapidly cleared from most tissues after a withdrawal period. The possible excretion of some phase I metabolites was also investigated, using further analyses under positive chemical ionization LRMS and high resolution mass spectrometry (HRMS).

Developments in residue assay and metabolism study of growth-promoters by mass spectrometric analysis.

The French national reference laboratory, for the control of growth-promoter residues, has developed routine methods of multiresidue analysis. These are mainly for the determination of beta-agonistic and steroid compounds. Apart from this regulatory activity, some research into growth-premotor metabolism and assay has also been carried out. This research concerns the method development of assay and metabolism studies of new compounds, including in vitro metabolism studies on cell (mainly hepatocytes) cultures, as well as studying the organic synthesis of new compounds and metabolites. Three examples of the different areas of research are presented here: the metabolism study of 4-chlorotestosterone in cattle; a methodological study on beta-agonists assay by mass spectrometry; and a residue study of beta-agonists in edible tissues.