RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2008; 22: 1863–1870 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.3563 Detection of the administration of 17b-nortestosterone in boars by gas chromatography/mass spectrometry
Rosa Ventura1,2*,y, Meritxell Roig1,2*,y,Bele´nPe´rez3, Sergi Lo´ pez4, Mireia Medina5, Jaume Bosch5 and Jordi Segura1,2 1Unitat de Recerca en Farmacologia, Institut Municipal d’Investigacio´ Me`dica, IMIM, Barcelona, Spain 2Departament de Cie`ncies Experimentals i de la Salut, Universitat Pompeu Fabra, UPF, Barcelona, Spain 3Departament de Farmacologia i Terape`utica, Facultat de Veterina`ria, Universitat Auto`noma de Barcelona, UAB, Bellaterrra, Spain 4RCC CIDA S.A., Barcelona, Spain 5Laboratori Agroalimentari, Departament d’Agricultura Ramaderia i Pesca, Generalitat de Catalunya, Cabrils, Spain Received 29 August 2007; Revised 26 March 2008; Accepted 27 March 2008
17b-Nortestosterone (17bN) is illegally used in livestock as a growth promoter and its endogenous production has been described in some animals, such as adult boars. In this paper, the metabolism of 17bN in boars has been studied by gas chromatography/mass spectrometry (GC/MS) in order to identify markers of the exogenous administration. Administration studies of intramuscular 17bN laurate to male pigs were performed. Free, sulphate and glucuronide fractions of the urine samples were separated and the steroids present were quantified by GC/MS. 17bN was detected in some pre-administration samples. After administration, 17bN, norandrosterone, noretiocholanolone (NorE), norepiandrosterone, 5b-estrane-3a,17b-diol and 5a-estrane-3b,17b-diol were detected in different fractions, being the most important metabolites, 17bN excreted as a sulphate and free NorE. Samples collected in routine controls were also analyzed by GC/MS to identify endogenous compounds. 17bN, norandrostenedione and estrone were detected in almost all the samples. No other 17bN metabolites were detected. According to these results, the detection by GC/MS of some of the 17bN metabolites described above, different from 17bN, could be indicative of the exogenous administration of 17bN to boars. Copyright # 2008 John Wiley & Sons, Ltd.
Anabolic androgenic steroids have been widely used in in humans; norepiandrosterone (NorEpiA) is also excreted in human and in veterinary medicine either for therapeutic for the sulphate fraction.1 In horses, isomers of estranediol fraudulent purposes, to increase muscular mass in sports or (mainly 5a-estrane-3b,17a-diol (ABA) and 5a-estrane-3b, to accelerate weight gain and to improve feeding efficiency in 17b-diol (ABB)) and the metabolite resulting from epimer- meat-producing animals. The use of anabolic androgenic ization at the C17 position, 17a-nortestosterone (17aN), have steroids is banned in sports and in horseracing, as well as been detected as main metabolites, 17a isomers mainly in growth promoters in livestock in the European Union. the glucuronide fraction and 17b isomers mainly in the Because of its similarity to testosterone, 17b-nortestosterone sulphate fraction.7–13 In bovines, 17aN, ABA and NorE (17bN) is one of the most common anabolic steroids used. As have also been detected as main metabolites in cows and a result of its extensive metabolism and the possibility of calves.14–18 17bN, isomers of 3-hydroxyestran-17-one (NorA, interference from endogenous compounds, which are NorE, NorEpiA) and isomers of estrane-3,17-diol (BAB and species-dependent, detection of the illegal use of 17bNis ABB) have been identified in miniature pigs after the difficult. administration of 17bN laurate.19,20 The metabolism of 17bN and 19-norsteroids has been In recent years, different authors have reported the extensively studied in humans1–6 and in different animal endogenous production of 17bN or its metabolites in species.7–20 The structures of 17bN and its metabolites different species.21 There is clear evidence for the biosyn- identified in different species are presented in Fig. 1. thesis of NorA in humans, during pregnancy or the ovulation Norandrosterone (NorA) and noretiocholanolone (NorE) period of the menstrual cycle.22–24 Low concentrations of excreted in the glucuronide fraction are the main metabolites NorA have also been reported in humans.25 In horses, ABA and 17bN were detected as endogenous compounds in the *Correspondence to: R. Ventura and M. Roig, Unitat de Recerca en urine of normal stallions.11–13,26,27 17aN was identifed Farmacologia, Institut Municipal d’Investigacuo´ Me`dica, Parc 28 29,30 de Recerca Biome`dica de Barcelona, Dr. Aiguader, 88, 08003 during pregnancy in ewes and in goats and mares. Barcelona, Spain. In non-treated calves, ABA and traces of NorE were E-mail: [email protected]; [email protected] detected.15,21 17bN was also detected in non-castrated and yThese authors are first co-authors as they contributed equally to the manuscript. cryptorchid male pigs, in testicular tissue and in urine from Contract/grant sponsor: Direccio´n General de Investigacio´n boars,32–34 at very low concentrations in barrow and female (Ministerio de Educacio´n y Ciencia, Spain); Contract/grant pigs,19,20,35–37 and in the follicular fluid of sows.38 number: AGL-2001-0439-C02-01.
Copyright # 2008 John Wiley & Sons, Ltd. 1864 R. Ventura et al.
Figure 1. Molecular structures of 17b-nortestosterone, metabolites and related steroids identified in different species in urine samples.
Detection of the consumption of anabolic agents is Gas chromatography coupled to isotope ratio mass spec- performed by the identification of specific metabolites using trometry via a combustion interface (GC/C/IRMS) has been gas chromatography or liquid chromatography coupled to demonstrated to be useful in detecting the administration of mass spectrometry (GC/MS, LC/MS) or to tandem mass hormones naturally present in urine, due to the difference in spectrometry (MS/MS).39–41 These techniques are effective in the 13C/12C ratio between endogenous and synthetic detecting the administration of exogenous compounds. compounds. GC/C/IRMS has been useful in detecting However, they are not able to distinguish the exogenous the exogenous administration of testosterone42–44 and origin of compounds normally present in urine, as is the case 17b-estradiol45 in cattle urine, and the administration of with 17bN metabolites in different species. The only testosterone and precursors in human samples.46–51 possibility of detecting the exogenous administration of a The aim of the present study was to investigate the compound by GC/MS and LC/MS is to find specific metabolism of 17bN after intramuscular administration in metabolites, only present after exogenous administration. pigs in order to detect specific metabolites of exogenous
Copyright # 2008 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2008; 22: 1863–1870 DOI: 10.1002/rcm Detection of 17bN in boars by GC/MS 1865 administration. First, administration studies of 17bNto norandrosterone-d4, used as internal standards, and adjusted young male pigs were performed to identify and quantify the to pH 9.5 with ammonium chloride buffer (100 mL). Free urinary metabolites of the compound. Secondly, the steroids were extracted with 5 mL of TBME by shaking at compounds endogenously present in adult boar urines were 40 mpm for 20 min. After centrifugation (3500 rpm, 5 min), the studied. These studies allowed us to select the target organic layers were evaporated to dryness under a stream of metabolites to detect the exogenous administration of 17bN. nitrogen in a water bath at 508C. The extracts were kept in a
desiccator containing P2O5 and maintained under vacuum for at least 30 min before derivatization. EXPERIMENTAL Reagents and reference compounds Extraction of the glucuronide fraction 5a-Estrane-3a-ol-17-one (norandrosterone, NorA), 5b- The small volume of ether still present on top of the aqueous estrane-3a-ol-17-one (noretiocholanolone, NorE), methyltes- phase was evaporated under a stream of nitrogen. The tosterone, 4-estren-17a–ol-3-one (17a-nortestosterone, 17aN), aqueous phase was applied to Sep Pak C18 cartridges norandrosterone-d4, testosterone, and epitestosterone were previously conditioned with methanol (2 mL) and water supplied by NARL, Reference Materials (Cottesloe, Australia). (2 mL). The cartridges were washed with Milli Q water 5a-Estrane-3b-ol-17-one (norepiandrosterone, NorEpiA), (2 mL), dried for 2 min, and eluted with methanol (2 mL). estra-1,3,5(10)-triene-3,17a-diol (17a-estradiol), 5a-estran-3b, The methanolic extracts were evaporated to dryness under 17b-diol (ABB), 5b-estran-3a,17b-diol (BAB), and 5a-estran- a stream of nitrogen in a bath at 508C. The residues were 3b,17a-diol (ABA) were purchased by Steraloids Inc. reconstituted in 1 mL of sodium phosphate buffer (0.2 M, (Newport, RI, USA). Estra-1,3,5(10)-triene3,17b-diol (17b- pH 7), and subjected to enzymatic hydrolysis with 50 mLof estradiol) and estrone were supplied by Sigma (St. Louis, b-glucoronidase from E. coli and incubation in a water bath at
MO, USA). Norandrostenedione was purchased by Research 558C for 1 h. A volume of 250 mL of 5% solution of K2CO3 was Plus Inc. (Manasquan, NJ, USA). 4-Estren-17b-ol-3-one then added and the samples were extracted with TBME (17b-nortestosterone, 17bN) standards were obtained from (5 mL) by shaking at 40 mpm for 20 min. After centrifugation Steraloids. (3500 rpm, 5 min), the organic layers were evaporated to Methanol and ethyl acetate (both of HPLC grade), dryness under a stream of nitrogen in a bath at 508C. The tert-butyl methyl ether (TBME), 25% ammonia, ammonium extracts were kept in a desiccator with P2O5 and maintained chloride, sodium hydroxide, disodium hydrogen phosphate, under vacuum for at least 30 min, until derivatization. sodium hydrogen phosphate, potassium carbonate, ammonium iodide, 2-mercaptoethanol, and sulphuric acid Extraction of the sulphate fraction (extra pure grade) were purchased from Merck (Darmstadt, For the analysis of the sulphate fraction, the free fraction was Germany). b-Glucuronidase from E. coli K12 was supplied by extracted from urine samples (aliquots of 10 mL), as Roche Diagnostics GmbH (Mannheim, Germany). described before. The remaining organic residue in the N-Methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) was aqueous phase was evaporated under a stream of nitrogen. purchased from Macherey-Nagel (Du¨ ren, Germany). C18 The aqueous phases were extracted with Sep Pak C18 cartridges (Sep Pak Vac RC, 500 mg) were supplied by cartridges as described previously. The resulting organic Waters (Milford, MA, USA). Milli Q water was obtained from extracts were then evaporated to dryness under a stream of a Milli Q purification system (Millipore, Ibe´rica, Barcelona, nitrogen in a bath at 508C. Spain). The dried residues were reconstituted with 4 mL of The solid-phase extraction (SPE) was performed on a a mixture of ethyl acetate/methanol/sulphuric acid (80:20: vacuum manifold (Biochem Diagnostics, Edgewood, NY, 0.06, v/v/v) and incubated in a water bath at 558C for 2 h. USA). Organic layers were evaporated to dryness under a After incubation, samples were neutralized with 1 M NaOH stream of nitrogen with a Turbo-Vap LV evaporator from (60 mL) and evaporated to dryness under a stream of Zymark Corporation (Hopkinton, MA, USA). nitrogen. The residues were reconstituted in 1 mL of sodium
phosphate buffer (0.2 M, pH 7) and 250 mLof5%K2CO3 Standard solutions solution, and then extracted with TBME (5 mL). The organic Stock standard solutions (1 mg mL 1) of each analyte were layers were evaporated to dryness under a stream of nitrogen prepared by dissolving 10 mg of the free base in 10 mL of at 508C and kept in a desiccator containing P2O5 under methanol. Working solutions of 100, 10 and 1 mgmL 1 were vacuum for at least 30 min before derivatization. prepared by 1:10, 1:100 and 1:1000 dilutions of the 1 mg mL 1 stock solutions with methanol, respectively. All solutions Derivatization were stored at 208C. The dry extracts were derivatized by using 50 mL of a mixture
of MSTFA/NH4I/2-mercaptoethanol (1000:2:6, v/w/v), and Sample preparation heating at 608C for 20 min. After incubation, the derivatized A procedure previously described was used to prepare extracts were transferred to injection vials and analyzed by samples for GC/MS analysis.52 GC/MS.
Free fraction extraction GC/MS instrumental conditions Aliquots of urine samples (10 mL) were spiked with a Analyses were performed on a Hewlett-Packard 5890 II GC concentration of 20 ng mL 1 of methyltestosterone and model fitted with a HP 7673 A autosampler and connected to
Copyright # 2008 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2008; 22: 1863–1870 DOI: 10.1002/rcm 1866 R. Ventura et al. Table 1. Analytes under study, relative retention times (RRT) and diagnostic ions used for identification and for quantitation
Analyte RRT Diagnostic ions (m/z) Quantitation ion (m/z)
Norandrosterone-d4 0.582 409, 424, 319 409 Norandrosterone (NorA) 0.583 405, 420, 315 405 5b-Estrane-3a,17a-diol 0.585 242, 332, 407 242 5(10)-Estrene-3b,17a-diol 0.621 330, 240, 405 330 5a-Estrane-3a,17b-diol 0.624 242, 332, 407 242 Norepiandrosterone (NorEpiA) 0.635 405, 420, 315 405 5a-Estrane-3b,17a-diol (ABA) 0.641 242, 332, 407 242 Norepietiocholanolone 0.647 405, 420, 315 405 Noretiocholanolone (NorE) 0.648 405, 420, 315 405 5b-Estrane-3a,17b-diol (BAB) 0.676 242, 332, 407 242 5a-Estrane-3b,17b-diol (ABB) 0.695 242, 332 407 242 5(10)-Estrene-3a,17b-diol 0.733 330, 240, 405 330 17a-Nortestosterone (17aN) 0.762 418, 403, 182 418 Norandrostenedione 0.787 416, 401, 311 416 Epitestosterone 0.808 432, 417 432 17a-Estradiol 0.813 285, 416, 325 285 17b-Nortestosterone (17bN) 0.813 418, 403, 182 418 Estrone 0.839 414, 399 414 17b-Estradiol 0.860 285, 416 325 285 Testosterone 0.882 432, 417 432 Methyltestosterone 1.000 446, 301 446
a HP 5970 mass-selective detector (Agilent Technologies, Urine samples were collected daily before the administration Palo Alto, CA, USA). The separation was carried out in a of the drug (n ¼ 2) and after the administration up to 19 days. 100% methylsilicone fused-silica capillary column (HP Urine samples from the second administration day were Ultra-1; 17 m 0.2 mm i.d.; film thickness 0.11 mm). collected after administration of the drug. The urine samples The oven temperature was programmed as follows: initial were stored at 208C until analysis. temperature 1818C; increase at 38Cmin 1 to 2308C, then increase at 408Cmin 1 to 3108C, at which temperature it was Study 2 maintained for 3 min. Helium was used as the carrier gas at a Study 2 was designed to quantify the urinary metabolites flow rate of 0.8 mL min 1 (measured at 1808C). The injector of 17bN in boars after intramuscular administration. The and detector temperatures were maintained at 2808C. The administration study was performed in RCC CIDA S.A. injection volume was 2 mL and a split mode of 10:1 was used. (Santa Perpe`tua de la Mogoda, Barcelona, Spain) using The compounds were monitored as their bis-O-TMS protocol CD05/9603FC. Six healthy male non-castrated derivatives. The mass spectrometer was operated in electron Landrace boars participated in the study. They were between ionization (EI) selected ion monitoring (SIM) acquisition 8 and 10 weeks old and their weights were in the range from mode using three diagnostic ions to monitor each compound 20.8 to 24.6 kg when the study started. of interest. The ions used for identification and quantitation A period of 10 days was needed to allow the adaptation of of the compounds have previously been described and are the animals in the experimental farm and to ensure the listed in Table 1.52 absence of some interference from other treatments. The animals were individually housed in metabolic cages during Administration studies of 17bN laurate the study. Two doses of 2 mg kg 1 of 17bN laurate (Erton 1 Two different administration studies of 17bN laurate were Fortificante , Laboratorios Ovejero S.A, Leon, Spain) were performed in boars. administered intramuscularly on two consecutive days. Urine samples were collected after the first dose (day 1) Study 1 and daily up to 15 days. The volume of each urine sample Study 1 was designed to investigate the urinary metabolites was registered. Urine samples were stored at 208C until of 17bN in boars after intramuscular administration. The analysis. During the study, boars were fed daily with 1 kg of study was performed on an experimental farm authorized to growth fodder and 3–3.5 L of water. perform studies in animals (SYBA, Bigues i Riells, Barcelona, The administration study samples were quantified using Spain; register number B 9900040). calibration curves of five points in duplicate. The concen- Two healthy non-castrated male boars 14 weeks old with a tration ranges were those described previously.52 Quality weight of 28.5 kg were used for the study. After a period of 10 control samples at two concentration levels were analyzed in days of adaptation of the animals in the experimental farm, triplicate in each analytical batch. they were housed in individual metabolic cages during the 1 study. Two doses of 2 mg kg 1 of 17bN laurate (Laurabolin , Samples from non-treated animals Laboratory Intervet, Boxmeer, The Netherlands) were Urine samples from boars (n ¼ 60) were taken from the administered intramuscularly on two consecutive days. routine controls performed by the Government of Catalonia
Copyright # 2008 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2008; 22: 1863–1870 DOI: 10.1002/rcm Detection of 17bN in boars by GC/MS 1867 Table 2. Concentrations of 17b-nortestosterone metabolites obtained after administration of 17b-nortestosterone laurate to six boars
FREE FRACTION
NorA (ng mL 1) NorE (ng mL 1) NorEpiA (ng mL 1) BAB (ng mL 1) ABB (ng mL 1)
Day na mean sd range na mean sd range na Mean sd range na mean sd range na mean sd range
1— nd — nd — nd — nd — nd 2— nd — nd — nd — nd — nd 3— nd 5 18.8 10.4 8.2–36.2 5 7.8 5.0 2.9–15.8 1 15.4 1 5.4 4 35.1 4.7 3.1–10.4 5 30.4 22.0 6.6–59.3 5 16.9 13.2 3.6–17.8 1 44.2 — nd 5 47.2 4.0 1.8–11.5 6 25.9 15.7 6.9–50 6 18.1 13.4 4.5–43.8 2 55.2 61.7 11.5–98.8 1 35.3 6 54.5 3.1 1.5–9.2 6 20.8 12.5 6.5–37.2 6 13.6 5.9 6.0–19.9 2 15.4 10.1 8.2–22.5 1 10.5 7 45.2 6.8 0.5–15.2 5 20.8 19.0 3.6–48.0 6 9.6 9.2 1.9–24.3 3 14.1 17.3 3.4–34.1 2 8.1 8.5 2.1–14.1 8 55.4 5.3 1.5–14.3 6 27.7 16.6 8.6–49.2 6 15.9 8.2 6.8–25.3 3 17.4 10.0 9.4–28.6 2 6.4 4.8 3.0–9.8 9 54.4 2.7 2.0–8.8 6 23.2 17.7 7.5–47.6 6 16.0 8.0 7.4–27.7 2 29.8 34.2 5.5–53.9 2 18.5 17.1 6.4–30.6 10 64.0 1.8 2.3–6.4 6 24.6 19.3 7.8–60.5 6 17.5 11.2 10.9–39.9 2 19.0 17.6 6.6–31.4 2 10.6 5.2 6.9–14.3 11 44.8 3.0 2.0–8.8 6 22.7 19.9 4.3–57.9 6 15.4 7.8 6.6–29.8 2 35.3 38.7 7.9–62.9 2 19.1 13.1 9.8–28.4 12 53.6 2.6 1.8–7.9 6 18.2 12.2 6.3–37.7 6 14.4 8.2 7.2–29.6 3 16.5 13.4 8.5–32.0 3 7.8 3.9 4.3–11.9 13 54.5 2.6 2.0–8.7 5 24.0 13.5 10.4–44.2 6 12.8 7.7 8.5–24.7 3 13.7 6.6 9.7–21.3 2 8.4 0.7 7.8–8.9 14 44.3 2.8 2.3–8.3 5 24.3 15.7 12.1–47.8 6 12.5 6.3 5.7–22.6 3 15.4 6.1 11.6–22.3 2 10.2 4.3 7.1–13.2 15 34.9 2.5 2.3–7.3 5 28.4 17.6 9.6–53.6 5 17.1 5.0 10.7–20.9 3 12.8 4.9 9.3–18.4 2 7.0 2.7 5.1–8.9
GLUCURONIDE FRACTION
NorA (ng mL 1) NorE (ng mL 1) NorEpiA (ng mL 1)17bN (ng mL 1)
Day na mean sd range na mean sd range na mean sd range na mean sd range
1 — nd — nd — nd 4 13.4 9.5 5.2–23.4 222.0 1.6 0.8–3.1 4 9.6 3.0 6.2–12.9 4 5.9 3.2 3.3–10.6 2 9.0 6.7 4.3–13.7 345.6 2.7 1.8–8.1 6 18.5 19.0 2.3–52.5 4 13.8 10.4 2.4–22.8 464.5 2.4 1.4–8.2 6 10.1 6.6 3.4–21.9 6 8.7 4.7 2.3–16.7 553.4 2.1 0.8–6.4 5 9.8 6.5 2.7–16.9 6 6.3 3.7 2.6–11.7 646.9 4.2 1.9–11.3 6 15.8 6.7 6.6–20.0 6 11.7 5.7 5.1–21.4 764.4 2.5 2.0–8.3 6 15.2 6.7 6.5–24.6 6 10.6 3.9 5.4–16.6 863.8 2.6 1.6–8.4 6 11.3 4.1 6.3–15.1 6 8.5 2.4 5.3–11.6 954.2 4.4 0.9–10.7 5 11.5 6.6 3.0–18.4 5 8.1 6.4 1.5–18.1 10 4 3.6 4.3 0.7–10.1 5 7.2 3.5 2.0–11.3 5 5.3 1.9 2.2–6.8 11 5 2.3 1.8 0.5–4.9 5 6.1 3.6 3.0–11.2 6 5.8 2.1 2.5–8.9 12 3 3.3 2.8 0.5–6.1 4 5.7 4.3 1.8–11.8 5 5.4 2.8 2.9–9.9 13 4 4.9 3.5 0.8–9.3 4 11.9 3.6 8.2–16.7 5 9.5 5.7 3.3–17.4 14 3 4.7 0.9 3.9–5.7 4 7.9 3.4 6.2–12.9 6 7.2 3.9 2.8–12.5 15 4 6.1 2.5 2.8–8.9 5 17.6 10.0 4.4–30.9 6 13.5 9.0 4.7–28.2
SULPHATE FRACTION
NorA (ng mL 1) NorE (ng mL 1) NorEpiA (ng mL 1)17bN (ng mL 1)
Day na mean sd range na mean sd range na mean sd range na mean sd range
1— nd — nd — nd 6 157.7 62.6 75.8–234.0 2 11.6 — nd — nd 6 72.2 46.4 27.9–135.6 3 44.6 2.3 2.9–7.9 1 7.8 1 2.7 6 42.9 41.2 10.7–104.7 4 56.3 3.2 2.4–11.1 4 6.8 4.4 3.5–13.3 3 4.0 2.6 2.1–7.0 6 17.4 17.9 4.2–50.4 5 68.2 4.7 4.0–16.5 4 7.2 4.5 2.6–11.8 3 4.4 1.5 2.8–5.5 6 8.9 7.5 2.0–21.9 6 6 10.0 5.0 3.9–15.5 2 12.8 6.5 8.2–17.4 3 7.1 4.1 3.2–11.3 5 11.7 10.8 1.3–29.4 7 57.3 2.6 4.0–10.1 2 10.5 2.9 8.4–12.5 2 5.5 0.2 5.3–5.6 6 5.7 4.8 2.8–15.4 8 47.7 4.1 3.9–13.5 2 13.7 6.4 9.2–18.3 3 6.5 6.2 1.8–13.6 5 8.7 7.7 2.0–21.8 9 67.9 5.1 3.6–17.4 2 8.7 1.2 7.8–9.5 3 4.4 1.8 2.8–6.40 5 9.0 10.7 1.5–27.7 10 56.6 1.6 5.0–9.2 2 13.1 4.1 10.2–16.0 3 6.8 3.1 4.2–10.1 5 8.5 13.2 2.1–32.1 11 66.2 1.5 4.2–7.6 2 11.2 4.0 8.4–14.0 3 6.2 0.2 6.1–6.4 6 3.8 3.2 1.1–8.5 12 65.7 3.0 2.8–8.9 2 5.9 0.8 5.3–6.5 3 4.7 2.3 3.3–7.3 6 3.5 4.2 0.6–11.6 13 57.2 2.3 5.0–10.0 2 9.5 0.1 9.4–9.7 3 5.4 1.7 3.4–6.6 5 2.9 1.3 0.8–4.6 14 56.8 2.3 4.1–9.6 2 8.1 1.4 7.2–9.1 3 4.3 2.2 1.8–6.1 3 5.0 2.5 3.5–7.9 15 68.8 3.0 5.4–10.0 2 5.6 1.5 4.5–6.6 3 4.1 1.8 3.1–6.2 3 10.8 13.7 2.6–26.6 NorA: norandrosterone; NorE: noretiocholanolone; NorEpiA: norepiandrosterone; BAB: 5b-estrane-3a,17b-diol; ABB: 5a-estrane-3b,17b-diol; 17bN: 17b-nortestosterone. na: number of boars in which the metabolite was detected.
Copyright # 2008 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2008; 22: 1863–1870 DOI: 10.1002/rcm 1868 R. Ventura et al.
(Departament d’Agricultura Ramaderia i Pesca, Generalitat de Catalunya, Spain). These samples belonged to theoreti- cally non-treated animals. These animals were older than 6 months and information on their gender was not available. All samples were stored at 208C until analysis. The estimation of the concentration of steroids in the different fractions was performed using response factors calculated from a calibration sample.
RESULTS Urinary metabolites of 17bN in boars Identification and quantitation of 17bN metabolites in urines collected after the administration of the compound to boars were performed using a GC/MS method already Figure 2. Percentage of the excreted dose corresponding to 52 described. The efficiency of the hydrolysis procedures for each metabolite. Mean values and standard deviation (n ¼ 6). glucuronide and sulphate conjugates was estimated as 91.7% FF: free fraction; GF: glucuronide fraction; SF: sulphate frac- for NorE glucuronide and 90.7% for dehydroepiandroster- tion. one-3-sulphate.52 The stabilities of NorA and NorE glucur- onides in the solvolysis procedure were above 95% (n ¼ 4). The following metabolites were identified up to 16 to NorE and NorEpiA appeared from day 3 of the study, mainly 18 days after intramuscular administration of 17bN laurate to in the free and glucuronide fractions, and the concentrations two boars (study 1): NorA, NorE, NorEpiA, BAB and after day 4 of the study were higher than those obtained 17bN. In pre-administration samples, 17bN was detected in for 17bN. one of the animals. The percentage of the excreted dose corresponding to each The concentrations of urinary metabolites detected in the metabolite is presented in Fig. 2. NorE in the free fraction free, the glucuronide and the sulphate fractions after and 17bN in the sulphate fraction were the most important intramuscular administration of 17bN laurate to six Land- metabolites. NorEpiA in the free fraction was also an race boars (study 2) are summarized in Table 2. 17bN was important metabolite. A mean of 2.6% of the dose was detected in the sulphate fraction of all animals and in the recovered in urine up to 15 days after the first administration. glucuronide fraction of some of them on the first days after In addition to the 17bN metabolites, estrogen compounds administration. NorE and NorEpiA were detected in all the (such as estrone) and testosterone and its precursors or animals in the free and glucuronide fractions, and in some of metabolites (such as norandrostenedione, epitestosterone, them in the sulphate fraction. In contrast, NorA was detected androsterone, etiocholanolone, androstenedione, dihydro- in all animals in the sulphate fraction and in some of them in testosterone, and dehydroepiandrosterone) were detected in the free and glucuronide fractions. Finally, BAB and ABB some urine sample at low concentrations. were detected in the free fractions of the urines of some animals. Other related steroids, such as 17aN or ABA, were not detected in any of the fractions studied. Results from non-treated animals All the metabolites appeared from the second and third To investigate the steroidal compounds naturally present in day after administration, except 17bN that appeared from boar urine, urine samples from 60 adult boars were analyzed. day 1, and nearly all of them were detected for at least Information on the gender and the exact ages of the animals 15 days after administration. The highest concentrations in generating the samples was not available. The results are urine were obtained for 17bN in the sulphate fraction until presented in Table 3. As can be observed, 17bN was detected day 3 of the study. Then, the concentrations of 17bN in most samples in all three fractions. Norandrostendione decreased to values lower than 10 ng mL 1 for most of the was detected in the free fraction. Small concentrations of the animals, except for one boar in which the concentration was compound were also detected in the glucuronide and maintained at between 20 and 30 ng mL 1 for up to 15 days. sulphate fractions, probably due to the non-complete
Table 3. Concentration (ng mL 1) of different compounds in samples from non-treated adult boars (n ¼ 60)
FREE FRACTION GLUCURONIDE FRACTION SULPHATE FRACTION
Compound n mean sd range n mean sd range n mean sd range
17bN 48 306.7 527.7 1.2–3257.1 41 113.9 216.7 2.2–1165.9 44 191.1 215.1 2.0–872.4 Norandrostenedione 45 38.1 54.9 0.6–285.2 24 13.2 5.4 3.6–53.3 14 31.5 26.8 8.6–78.5 Estrone 41 61.3 177.5 2.3–1087.8 53 454.1 1423.1 1.7–10269.8 37 48.8 46.3 7.6–522.2 17b-Estradiol 16 42.8 112.6 0.4–461.6 4 608.0 933.2 35.1–1989.3 1 119.4 n: number of samples where the compound was detected.
Copyright # 2008 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2008; 22: 1863–1870 DOI: 10.1002/rcm Detection of 17bN in boars by GC/MS 1869 extraction of the free compound. In spite of the high of 17bN. The analysis of urine samples collected from adult concentration of 17bN in some samples, no other pigs in routine controls demonstrated the presence of 17bN metabolites were detected. Other analytes such endogenous 17bN in agreement with data described by as 17aN, ABA, or 5(10)-estrene-3b,17a-diol were not detected other authors.19,20,35–37 The wide range of concentrations in any of the urine sample analyzed. obtained for 17bN and its precursor, norandrostendione Testosterone and some precursors or metabolites (Table 3), may be explained by the gender and the age of (norandrostenedione, epitestosterone, androsterone, etio- the animals providing the samples, in agreement with data cholanolone, epiandrosterone, androstenedione, dehydroe- recently published by other authors.37 Information about the piandrosterone) were detected in some of the samples. gender and the ages of the animals providing our samples Estrogen compounds were also identified in non-treated was not available. animals (Table 3). Other compounds detected in non-treated boars were estrone and, to a lesser extent, 17b-estradiol mainly excreted in the glucuronide fraction. The presence of high concen- DISCUSSION trations of estrogenic compounds of endogenous origin Urinary metabolites of 17bN in boars have been extensively (mainly estrone and 17b-estradiol) in boar urine has been studied in this work to identify suitable target analytes for described previously.54 As for the other steroidal com- the detection of exogenous 17bN administration. For the first pounds, the concentrations of these analytes were higher time, quantitative results of the metabolites excreted in the than those obtained in samples of animals participating in free, glucuronide and sulphate fractions are presented for up the administration studies of 17bN laurate due to the to 15 days after intramuscular administration. Identification difference in the ages of the animals. Other related analytes of the metabolites excreted in urine was first performed in a detected in other species, such as 17aN, ABA and 5(10)- pilot study of 17bN administration to two boars (study 1), estrene-3b,17a-diol,15,26–23,31 were not detected in samples and the quantitative study of the metabolites was performed from non-treated animals. with six young male pigs in study 2. Young male boars were The wide concentration range obtained for 17bN due to the selected to perform the administration studies, because of different factors (gender, age, interindividual variability) their low endogenous production of 17bN. makes it difficult to establish reference concentration ranges The percentage of the dose recovered in urine is relatively for non-treated animals, that allow discrimination based on low, accounting for a mean of 2.6%. This could be because concentration between the abuse of 17bN and its natural only metabolites recovered in urine have been considered. presence. Other excretion routes, such as biliar excretion, that have to In spite of the high concentrations of 17bN detected in be considered when a complete mass balance is performed, some animals, other 17bN metabolites such as NorA, NorE, have not been studied. Biliar excretion has been demon- NorEpiA, BAB and ABB were not detected in any of the strated to be a quantitatively much more important route of samples belonging to adult non-treated animals nor in elimination of some metabolites than urine in cattle.31,53 samples collected before the administration of 17bN laurate. The most important metabolites detected in urine Thus the detection of these metabolites in an urine sample were 17bN, mainly excreted in the sulphate fraction, and could be indicative of the exogenous administration NorE, mainly excreted in the free fraction. Other metabolites of 17bN to pigs. Taking into account the concentrations such as NorEpiA, NorA, BAB and ABB were also detected. detected, the detection window and their presence in all the These metabolites were also identified by other workers in animals participating in the study, NorE and NorEpiA in single urine samples collected after 2 weeks of intramuscular the free fraction are proposed as the best markers administration of 17bN laurate to three pigs.20 Similarly to of 17bN administration. They also offer the highest retro- what was described in horses, 17bN was excreted mainly in spectivity because the concentrations in urine remain higher the sulphate fraction.9,10 than those of 17bN for a longer period of time. 17aN was not detected in either the pre- or the post-administration samples, in agreement with data pre- 19,20 CONCLUSIONS sented by other authors. C17 epimerization is not an important metabolic pathway of 17bN in boars, in contrast to In this study, the feasibility of GC/MS analysis for the data reported for bovines14–18 and equines.8,9 ABA, another detection of the exogenous administration of 17bN in boars important metabolite of 17bN in other species,9–18 was not has been investigated. After administration of 17bN to young detected in boars. pigs, the following metabolites were detected in different It is worth noting the variability in the concentrations metabolic fractions: 17bN, NorE, NorEpiA, NorA, BAB and obtained for the different animals. From a qualitative point of ABB, in agreement with the results obtained by other view the variability is also shown because some metabolites authors. In studies carried out with adult animals, NorE in were only detected in some of the animals participating in the the free fraction and 17bN in the sulphate fraction were the study. most important metabolites. Among all the metabolites The objective of the analysis of samples from non-treated obtained after administration of 17bN, only 17bN was boars was to determine the compounds endogenously detected as an endogenous compound in samples from present, as well as the range of concentrations normally non-treated animals. Thus, the detection by GC/MS of some present in urine, in adult animals to help in the identi- of the metabolites described above could be used as a marker fication of target analytes of the exogenous administration of the exogenous administration of 17bN to boars.
Copyright # 2008 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2008; 22: 1863–1870 DOI: 10.1002/rcm 1870 R. Ventura et al.
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Copyright # 2008 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2008; 22: 1863–1870 DOI: 10.1002/rcm