Gas chromatographic/mass spectrometric analysis of 19-nortestosterone urinary metabolites in cattle Etienne Benoit, J.L. Guyot, D. Courtot, P. Delatour

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Etienne Benoit, J.L. Guyot, D. Courtot, P. Delatour. Gas chromatographic/mass spectrometric anal- ysis of 19-nortestosterone urinary metabolites in cattle. Annales de Recherches Vétérinaires, INRA Editions, 1989, 20 (4), pp.485-491. ￿hal-00901913￿

HAL Id: hal-00901913 https://hal.archives-ouvertes.fr/hal-00901913 Submitted on 1 Jan 1989

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Gas chromatographic/mass spectrometric analysis of 19-nortestosterone urinary metabolites in cattle

E. Benoît J.L. Guyot D. Courtot P. Delatour

1 INRA, Laboratoire No. 54i89, service de biochimie, Ecole Nationale Vétérinaire de Lyon, BP 31, 69752 Charbonnières Cedex; 2 INRA-ENVL, laboratoire de recherches sur le cheval de sport, Ecole Nationale Vétérinaire de Lyon, BP 31, 69752 Charbonnières Cedex, France (received 31 May 1988, accepted 18 January 1989)

Summary ― Gas chromatographic and mass spectrometric method is used to confirm the administration of 19-nortestosterone to cattle. The most abundant metabolites detected were 19- norepitestosterone and 5a-oestrane-3p,17a-diol.

gas chromatography ― mass spectrometry - 19-nortestosterone - metabolite - urine - cattle

Résumé ― Etude par chromatographie en phase gazeuse couplée à la spectrométrie de masse des métabolites urinaires de la 19-nortestostérone chez les bovins. La chromatographie en phase gazeuse couplée à la spectrométrie de masse permet de confirmer l’administration, actuellement interdite dans l’ensemble de la CEE, de 19-nortestostérone chez les bovins, par la mise en évidence de différents métabolites urinaires caractéristiques, tels que la 19- norépistestostérone et le 5 o’-œsffane-3j3,17o!-d/o/.

chromatographie en phase gazeuse ― spectrométrle de masse ― 19-nortestostérone - métabolite - urine - bovin

INTRODUCTION it has also been used for non-therapeutic purposes; in humans to improve athletic performance, or in the veterinary field as a 19-Nortestosterone (19-NT), and its growth promoting agent to accelerate and esters, are synthetic anabolic weight gain improve feeding in cattle. which are widely used as therapeutic efficiency agents. Since 19-NT promotes an in- On the basis of their toxic and creased formation of tissue protein, while hormone-like side effects, administration being less androgenic than , of anabolic steroids for zootechnical purposes in cattle has been banned Steroids throughout the EEC. However, it is generally agreed that the control of the Testosterone, , and 19-NT were illegal use of anabolic steroids is effective obtained from Sigma Chemical Co. (St Louis, only if sensitive and specific tests are MI, USA). 19-NT Undecanoate (Trophobo- used for the detection and identification of lene@) was kindly donated by Theramex (France). 5a-Estrane-30, 17-a-diol was kindly these drugs or their metabolites in urine. supplied from the Racecourse Security Service In the absence of such methods adapted Laboratory (Newmarket, England). 3,4-13C to cattle, it seems that 19-NT is widely testosterone was obtained from CEA (Gif-sur- used (Jansen et aL, 1985). Yvette, France). Methods based on radioimmunoassay (RIA) have been developed for the detection of 19-nortestosterone in man Animals, treatment and biological (Bosch, 1984; Belkien et aL, 1985), and in samples animal (Chapman et al., 1982; Benoit et aL, But due to their relative lack of 1985). Four adult friesian cows and four 2-month-old RIA methods can be used specificity, only calves were used in this study. They received a for screening purposes and positive single intramuscular injection of 1 mg of identification of steroids has to be undecanoate per kg body weight. Urine were obtained to performed by combined gas chromato- samples prior administration, and 15 days thereafter, by and mass graphy spectrometry. catheterisation of the bladder or following This paper describes an analytical natural micturition. In one adult cow, urine was methodology developed for the isolation collected three days after administration. Urines were stored frozen at - 30 °C, until analysed. and identification of different urinary metabolites of 19-NT undecanoate in cattle using high resolution gas chromatography combined with mass Isolation procedure of urinary steroids spectrometry. Urine (10 ml), added with testosterone !3C (0,25 wg) as an internal standard, was slowly passed through a Seppak C18, followed by a 10-ml wash with water. The free and MATERIALS AND METHODS conjugated steroids were recovered by elution with 10 ml of methanol. The methanolic eluate was evaporated to dryness at 40 °C under vacuum. The residues were dissolved in 5 ml of M sodium acetate buffer Chemical and solvents 0.2 (pH 5.2), containing 200 ml of Helix pomatia digestive juice. After incubation at 37 °C for 16 h, pH was Solvents were of analytical grade and were adjusted to 10 with NaOH N. The hydrolysate distilled before use. Heptafluorobutyric was then passed through an column, anhydride (HFBa) was purchased from Pierce followed by 5 ml wash with water. The neutral Chemical Co., Rockford, IL, USA. Helix steroids were eluted by 5 ml of methanol. The pomatia digestive juice was obtained from IBF methanolic eluate was evaporated to dryness (France). SepPack C18 and Estrogen Column then redissolved with 60 gl of methanol/ (AG1-X2) was obtained from Waters (Milford, diethyl!ther (1/1). The total solution was MS, USA) and BioRad (Spiral, Dijon, France), applied on a thin-layer chromatographic (TLC) respectively. Thin layer plate silica gel was plate, previously washed with the eluant purchased from Merck (Kieselgel 60 F254, [benzene/ethyl―acetate (70/30)]. Migration of Darmstadt, FRG). up to 15 cm was allowed. The silica gel zone, corresponding to the migration of a mixture of were maintained, respectively, at 180 °C and steroids such 17a-, epi- 290 °C, and the electron energy was set at 70 testosterone and dehydrotestosterone, was eV. Electron impact mass spectra were recuperated and eluted with 1 ml of acetone. recorded from 120 uma to 700 uma at a speed of 1 ms/uma.

Preparation of derivatives

The acetone eluate was evaporated to dryness, RESULTS dissolved in 50 wl of acetone, and then 10 III of HFBa was added. The mixture was left for 155 min at room temperature, then evaporated to We were interested only in steroids which dryness. The derivatives were recovered with were not observed in urine sample 50 wl of anhydrous benzene. collected prior to administration of the anabolic . The presence of steroids derived by a reductive process, or by Gas chromatographylmass spectro- epimerisation, from the structure of metry nandrolone were investigated (i.e. 3-oxo- estr-4-ene-17-ol, estr-4-ene-3,17-diol and Gas chromatographic/mass spectrometric estrane-3,17-diol). analyses were carried out using a NERMAG R The total chromatogram and 10.10 B with a GIRDEL instrument, equipped ion current chromatograms for 31 gas chromatograph. The analyses were run fragment at m/z 666 ion of 19-NT on a SE-30 fused silica capillary column (25 m (molecular x 0.32 mm). Helium was used as the carrier diHFB derivative), corresponding to gas at a speed of 50 cm/s. Injection port was steroids extracted from a urine sample held at 250 °C. A sample volume of 2 pl was collected three days after administration into the column a solid injected through injector. of 19-NT undecanoate, is presented in The temperature was programmed from 180-230 °C at 3 °C/min. The ion source tem- Figure 1. Fragment ion current chromato- perature and the interface oven temperature gram for ions at m/z 666 shows the presence of two characteristic peaks. found with the other characteristic ions of Structural identification of compound B estranediol di HFB derivatives. was ascertained by comparison of its Nevertheless, in the mass spectrum of mass spectrum and retention time with compound C and D, the relative intensity those of authentic 19-NT di HFB of ion at m/z 670 seems to be too derivative. Mass spectrum, but not important in comparision with reference retention time, was identical to 19-NT di estranediol; as a result, we are of the HFB derivative. However, the relative opinion that only compounds E-I are retention time of compound A, in isomeric steroids. Relative retention time comparison to that of compound B, (19- of compoundI in comparision to NT) (0.89) was similar to relative retention compound G (0.868) is consistent with a time of authentic epitestosterone di HFB hypothetical structure 5a-estrane-3p,17p- derivative, in comparison to authentic di HFB derivative but its intensity is too testosterone di HFB derivative (0.89). In weak to permit us to observe a complete consequence, compound A is believed to mass spectrum; the same problem occurs be 19-norepitestosterone di HFB. Relative with compound I. From quantitative point concentration of 17a derivative was more of view, compound G (5a-estrane-3I3,17a- important (x 7) than 17P derivative. diol) appears to be the most important. Fragment ion current chromatograms The presence of A4-estrene-3,17-diol for ions at m/z 456 (most prominent ion of di HFB derivative was also examined, but estranediol diHFB derivative), corres- the standard of these isomeric steroids is ponding to steroids extracted from the not commercially available. Molecular same urine sample, is presented in Figure weight of a di HFB derivative of such a 1. It shows the presence of seven peaks molecule is 668. By analogy, with the (C-I). Retention time and mass spectrum fragmentation of other 19-norsteroids, we of compound G are identical to those of suspect the presence of different ions at authentic 5a-estrane-30,17 a-di HFB m/z 454 (loss of HFB), 240 or 241 or 242 derivative, i.e. minor ion at m/z 670, most (loss of two HFB = estrene cycle), and prominent ion at m/z 456, and different after the loss of the methyl 18 from the typical ions at m/z 441, 430, 415, 401, previous fragments, the ions at m/z 653, 347, 243 (corresponding to the estrane 439, 225 or 226 or 227. Fragment ion cycle). In the mass spectrum of com- current chromatogram for ions at m/z 668 pound C, D, E and H, ion at m/z 670 was and 454 (Figure 2), led us to observe four interesting peaks (J, K, L and M). The principal steps : partition chromatography mass spectra of these four compounds (C18 SepPak), ion exchange chromato- include all the presupposed ions. The graphy (Estrogen Column) and adsorption mass spectrum of M is presented in chromatography (TLC on Kieselgel G 60). Figure 3. Ion at m/z 454, corresponding to This procedure is necessary to obtain the loss of one HFB, was the most sufficiently pure urinary extracts so that prominent (as in the estrane compound) capillary column overloading and loss of in all compounds. These products were resolution are avoided. This is of supposed to be isomeric forms of estr-4- particular importance to achieve the ene-3,17-diol. Nevertheless the concen- separation of 19-NT isomeric metabolites tration of these compounds appears to be and their separation from endogenous very little, in comparison to the different steroids which are also extracted from estranediols. Compound J appears to be urinary specimens. With this method, the most abundant. Fifteen days after the phenolsteroids were eliminated by ion administration of 19-NT undecanoate to exchange chromatography, so that adult cows and calves it was still possible phenolsteroid catabolites of nandrolone to detect in urine the two following could not be observed. compounds : 19-norepitestosterone and HFB derivatives were used for 5a-estrane-3p, 1 7cc-diol. gas chromatography and mass spectrometry. This method afforded, for diol compounds or 44-3oxo steroids 17 OH, di HFB deri- DISCUSSION vatives with relatively high molecular weight derivatives and stable molecular ions, increasing specificity of detection. Extraction and derivatization of This method is particularly well adapted to steroids the study of 17 OH catabolites of 19-NT In previous studies on the catabolism of The rather complex extraction and 19-NT in human or in horses, trimethylsilyl purification procedure consists of three ether (TMS), O-methyloxime-trimethylsilyl ether (MO-TMS) or t-butyldimethysilyl An analogous metabolic pathway is ether (TBDMS) (Houghton et al., 1986) observed in horses (Houghton, 1977; were generally used. Houghton et aL, 1986), but some 16 oxygenated metabolites of 19-nortesto- sterone were also identified in this species (Houghton and Dumasia, 1980). Metabolism Due to the fact that HFB derivatives are not adapted to the detection of 17 to notice the of It is of interest importance cetosteroids, the presence of these the epimerisation of 19-NT. The urinary molecules has not been investigated. In concentration of 19-norepitestosterone is humans, the detection of urinary 19- far more important than that of 19-NT and norandrosterone, major metabolite, 19- 5a-estrane-313,17a-diol is the most noretiocholanolone and 19-norepiandro- abundant 17-OH catabolite of this steroid. sterone has been reported by Bjorkhem The importance of this metabolic pathway and Hakan (1982). Masse et al. (1985), is also observed for testosterone in cattle, reported the presence of 19-norepietio- (Rico, 1983), (Dunn et al., 1977; cholanolone as an urinary metabolite. In Ivie et al., 1986) or (Pottier et the rabbit, different 19-norcetosteroids al., 1981). were observed in urine by Both-Miedema Reduction of the 3-oxo function of et al. (1972). nandrolone to obtain different permits Thus, gas chromatography and mass A4estrenediols. In urine of cattle, we have spectrometry can be used to confirm the four of these observed the presence of administration of this in that in isomeric steroids. It is worth noting bovine. Using the repetitive scanning two urine of non castrated stallions, mode, the characterization of 19-NT, 19- are but as A5(10) estrenediols observed, norepitestosterone, 5a-estrane-3p, 17a- our naturally occuring compounds. Using diol can be carried out. Selected ion gas chromatographic and mass spectro- monitoring of the prominent ions of the metric method with urine of stallions, the last compound can also be used to these two retention times of A5(10) increase the sensitivity of the method. estrenediols are different from estrene- diols observed in cattle, but ions at m/z 668, 454, 439, 241 or 242 and 225, were common to all these compounds. Complete reduction of A4-3oxo group REFERENCES produces estranediols. In cattle and in the most abundant of these horses, Belkien L., Schurmeyer T., Hano R., Gunnars- is In steroids 5a-estrane-3fi,17a-diol, son P.E. & Nieschlag E. 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