Elimination kinetic of 17β-estradiol 3-benzoate and 17β-nandrolone laureate ester metabolites in calves’ urine

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Journal of Steroid Biochemistry & Molecular Biology 110 (2008) 30–38

Elimination kinetic of 17␤-estradiol 3-benzoate and 17␤-nandrolone laureate ester metabolites in calves’ urine Gaud Pinel a,∗ , Lauriane Rambaud a,1 , Giuseppe Cacciatore b , Aldert Bergwerff b , Chris Elliott c , Michel Nielen d , Bruno Le Bizec a,1 a

Laboratoire d’Etude des R´esidus et Contaminants dans les Aliments, Ecole Nationale V´et´erinaire de Nantes, Route de Gachet, BP 50707, 44307 Nantes cedex 3, France b Veterinary Public Health Division, Institute for Risk Assessment Sciences, Utrecht University, PO Box 80.175, 3508 TD Utrecht, The Netherlands c Institute of Agri-Food and Land Use, Queen’s University Belfast, BT5 9AY, Northern Ireland, United Kingdom d RIKILT – Institute of Food Safety, Wageningen, The Netherlands Received 22 May 2007; accepted 28 September 2007

Abstract Efficient control of the illegal use of anabolic steroids must both take into account metabolic patterns and associated kinetics of elimination; in this context, an extensive animal experiment involving 24 calves and consisting of three administrations of 17␤-estradiol 3-benzoate and 17␤-nandrolone laureate esters was carried out over 50 days. Urine samples were regularly collected during the experiment from all treated and non-treated calves. For sample preparation, a single step high throughput protocol based on 96-well C18 SPE was developed and validated according to the European Decision 2002/657/EC requirements. Decision limits (CC␣) for steroids were below 0.1 ␮g L−1 , except for 19-norandrosterone (CC␣ = 0.7 ␮g L−1 ) and estrone (CC␣ = 0.3 ␮g L−1 ). Kinetics of elimination of the administered 17␤-estradiol 3-benzoate and 17␤-nandrolone laureate were established by monitoring 17␤-estradiol, 17␣-estradiol, estrone and 17␤-nandrolone, 17␣-nandrolone, 19-noretiocholanolone, 19norandrostenedione, respectively. All animals demonstrated homogeneous patterns of elimination both from a qualitative (metabolite profile) and quantitative point of view (elimination kinetics in urine). Most abundant metabolites were 17␣-estradiol and 17␣-nandrolone (>20 and 2 mg L−1 , respectively after 17␤-estradiol 3-benzoate and 17␤-nandrolone laureate administration) whereas 17␤-estradiol, estrone, 17␤-nandrolone, 19noretiocholanolone and 19-norandrostenedione were found as secondary metabolites at concentration values up to the ␮g L−1 level. No significant difference was observed between male and female animals. The effect of several consecutive injections on elimination profiles was studied and revealed a tendency toward a decrease in the biotransformation of administered steroid 17␤ form. © 2008 Elsevier Ltd. All rights reserved. Keywords: Nandrolone; Estradiol; Metabolites; 96-Well SPE; Calves; Kinetic of elimination

1. Introduction Anabolic steroids have been widely used over the last past 50 years in cattle breeding practices with beneficial effects such as animal growth promotion and feed efficiency. They are banned in food producing livestock in Europe [1]. To enforce the prohibition on anabolic steroid abuse, effective monitoring, detection, identification and confirmation methods have been

∗ 1

Corresponding author. Tel.: +33 2 40 68 78 80; fax: +33 2 40 68 78 78. E-mail address: [email protected] (G. Pinel). Tel.: +33 2 40 68 78 80; fax: +33 2 40 68 78 78.

0960-0760/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2007.09.024

developed. For screening and confirmation of anabolic steroids, gas chromatography coupled to mass spectrometry (GC–MS) is the technique commonly used for the analysis of urine samples by anti-doping or analytical laboratories [2–5]. Improved analytical sensitivity, leading to increased periods of detection post drug administration, can be achieved in urine using gas chromatography–tandem mass spectrometry (GC–MS/MS) [6–9] or liquid chromatography–tandem mass spectrometry (LC–MS/MS) [10–15]. Efficient control of the illegal use of anabolic steroids must both take into account metabolic patterns and associated kinetics of elimination [5,8,16]. This knowledge is available from in vivo experiments and from the scientific literature, most studies have been performed on human subjects

G. Pinel et al. / Journal of Steroid Biochemistry & Molecular Biology 110 (2008) 30–38

[5,8,9,17–20] but few deal with bovine animals [21–23] and in particular calves [21,24], although this population has been linked with illegal anabolic treatment in EU member states. The present study aims at supplying incurred calves’ urine samples from a large group of animals (12 treated calves) after administration of a mixture of 17␤-estradiol 3-benzoate and 17␤-nandrolone laureate esters according to a protocol reflecting likely illegal practices and consisting in three administrations during the 45 days before the animals are slaughtered. Urine samples obtained during the experiment have been used to establish efficiency of the treatment, to study the elimination patterns of administered compounds as well as potential metabolism pathway adjustment upon several administrations and to provide confirmatory data to be compared in a further work to modifications in plasma protein profiles as indicators of steroid administration. 2. Materials and methods 2.1. Reagents and chemicals Most of the reagents and solvents were of analytical grade quality and provided by Solvants Documentation Synthesis (SDS, Peypin, France). The derivatisation reagents N-methyl-N(trimethylsilyl)-trifluoroacetamide (MSTFA), trimethyliodosilane (TMIS) were purchased from Fluka (Buchs, Switzerland) and dithiothreitol (DTE) was from Sigma–Aldrich (St. Quentin Fallavier, France). ␤-Glucuronidase (Helix pomatia) was provided by Sigma–Aldrich (St. Quentin Fallavier, France). The reference steroids were from Interchim (Montluc¸on, France), Sigma–Aldrich (St. Quentin Fallavier, France), AGAL/NARL (Australia) and RIVM (Bilthoven, The Netherlands). The internal standards used were 17␤-nandrolone-d3 (17␤-NTd3 ), 19-norandrosterone-d4 (NA-d4 ), 19-noretiocholanolone-d3 (NE-d3 ), and 17␤-estradiol-d3 (17␤-E2-d3 ). 2.2. Materials

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OV-1 (30 m × 0.25 mm i.d., film thickness 0.25 ␮m) (Ohio Valley, USA). Injection conditions were as follows: splitless (1 min), initial temperature (120 ◦ C), injected volume (2 ␮L). Temperature programme was set as follows: 120 ◦ C to 250 ◦ C (15 ◦ C min−1 ). Steroids were analysed on a triple quadrupole mass spectrometer (Quattro II, Micromass, UK) after electron ionisation (EI). The reactions monitored for the different steroids of interest as well as the detection conditions are reported in Table 1. 2.3. Animal experiment An animal experiment has been conducted within the department of Veterinary Animal Health of the Faculty of Veterinary Medicine of the Utrecht University (The Netherlands). The protocol has been approved by the ethical committee from Utrecht University. Twenty-four calves (Breed: HolsteinFriesen × Fries-Holland), 12 male and 12 female animals, were obtained from identified sources at an age of two weeks (10 days minimum and 3 weeks maximum) and subjected to a sevenweek acclimatization period. Twelve healthy calves (six females and six males) were selected randomly for the study after the acclimatization period and assigned to the treatment group. At the same time, another 12 healthy calves (6 females and 6 males) were selected randomly and assigned to the control group. Over the study period, each animal from the treatment group received three intramuscular doses of a mixture of 5 mL 17␤oestradiol benzoate (Oestradiol Benzoaat® , 5 mg/mL, Intervet, Boxmeer, The Netherlands) and 3 mL 17␤-nortestosterone laureate (Decadurabolin® 50 mg/mL, Intervet, Boxmeer, The Netherlands) at day 0, day 14 and day 28. The first dose was administered at the average age of 10 weeks. Urine samples were collected at regular time points (20 and 11 collection points for treated and non-treated calves, respectively) starting seven days before administration (average age 9 weeks) and stored prior to analysis below −18 ◦ C. At day 42 (average age 16 weeks) treated animals were euthanized. 2.4. Methods

96-Well SPE filled with 50 mg C18 phase were purchased from Interchim (Montluc¸on, France). Separation of steroid compounds was performed on non-polar capillary column

To a quantity of 2 mL urine was added 2 ng 17␤-NT-d3 , NAd4 , NE-d3 and 17␤-E2-d3 , 0.5 mL acetate buffer (2 M, pH 5.2)

Table 1 Monitored reactions and conditions of detection for each steroid of interest and internal or external standards Compounds

Reaction 1

Collision R1 (eV)

NA-d4 NA NE-d3 NE 17␣-NT NAED 17␣-E2 17ß-NT-d3 17ß-NT E1 17ß-E2-d3 17ß-E2 Norgestrel

424.4 > 409.4 420.4 > 405.4 423.4 > 408.4 420.4 > 405.4 418.4 > 182.1 416.4 > 234.2 416.4 > 285.2 421.4 > 194.1 418.4 > 182.1 414.4 > 399.3 419.4 > 285.2 416.4 > 285.2 456.4 > 301.3

12 12 12 12 15 20 15 20 15 10 20 15 25

Reaction 2

Collision R2 (eV)

Reaction 3

Collision R3 (eV)

420.4 > 315.3

20

420.4 > 169.1

20

420.4 > 315.3 418.4 > 194.1 416.4 > 220.2 416.4 > 129.1

20 20 20 25

420.4 > 169.1 418.4 > 287.2 416.4 > 207.2 416.4 > 232.2

20 15 25 15

418.4 > 194.1 414.4 > 309.3

20 20

418.4 > 287.2 414.4 > 231.2

15 25

416.4 > 129.1

25

416.4 > 232.2

15

TR (min) 13.87 13.89 14.29 14.31 15.20 15.42 15.53 15.62 15.64 15.70 15.92 15.94 17.85

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and 200 ␮L ␤-glucuronidase from Helix pomatia. Hydrolysis was performed over 15 h at 52 ◦ C. Urine samples were centrifuged (5 min, 1000 × g) before purification on 96-well SPE C18 . The reversed-phase wells were conditioned with 2 mL methanol then 2 mL water. The extract was applied into the well. The phase was washed with 2 mL water then 2 mL cyclohexane.

Steroids were eluted with 2 mL diethylether which was evaporated to dryness under a gentle stream of nitrogen. After addition of 5 ng norgestrel (Sigma–Aldrich) as internal standard, the samples were derivatised 40 min at 60 ◦ C with MSTFA-TMIS-DTE (1000:5:5, v/v/w). Of this extract, 2 ␮L was injected onto the GC-column.

Fig. 1. Example, for one calf, of ion chromatograms of urine samples collected on three points: before treatment (day −7), after the first administration (day 2) and at the end of the experiment (day 39). Monitored signals corresponds to 17␤-NT, 17␣-NT, NE and NAED. GC–EI(+)-MS/MS analysis and SRM acquisition mode.

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3. Results and discussion 3.1. Evaluation of 96-well SPE Based on the high number of urine samples which had to be analysed (n > 300), a high sample throughput preparation protocol was developed using 96-well SPE filled with 50 mg C18 reversed-phase. This simplified sample preparation was possible because of the nature of urine samples which were obtained from the calves, i.e. clear from pigment and dilute. Furthermore, the urine samples to be analysed were known to come from treated animals and steroid concentrations were expected to be high so a reduced sample size (2 mL urine), in comparison to 10 mL usually reported, was used. For all these reasons, a simplified, rapid and fit for purpose one-step sample preparation protocol was developed when protocols usually reported for extraction/purification of such compounds in urine comprise at least two different SPE steps and liquid/liquid extractions [5,17]. Validation of the developed protocol was performed according to the requirements of the European Decision 2002/657/EC [25]. For this purpose 20 blank calf urine samples, 20 spiked (1 ␮g L−1 ) calf urine samples and 5 urine samples spiked at different levels were analysed according to the protocol described previously. First, analysis of 20 blank samples was performed to check the selectivity of the method. The specificity was found to be satisfactory with no interferences being detected for the analyte diagnostic chromatograms. A pool of blank samples was then used to determine the calibration curve. The coefficient of determination (R2 ) was better than 0.99 for all steroids, proving the excellent linearity of the method. Furthermore, 20 blank samples were spiked at 1 ␮g L−1 to determine the repeatability of the method. The standard deviations of the relative retention time were lower than the minimum required performance limit of 0.5% for all steroids. The standard deviations of signal intensity were below 20% for all analytes and for the two SRM traces, except for 17␣-E2, which presented higher variability (34.6%). This endogenous hormone showed higher variability because of its natural presence in the blank samples. The standard deviations of the ratio between the two SRM traces (for each analyte) varied from 5.0% to 18.0%. The

Fig. 2. Kinetics of elimination over 40 days of 17␤-NT, 17␣-NT, NE and NAED for the 12 17␤-NT laureate/17␤-E2 benzoate treated calves. Concentrations have been calculated from GC–EI(+)-MS/MS analysis of collected urine samples.

Table 2 Decision limits (CC␣) and detection capabilities (CC␤) obtained with the developed protocol for the compounds of interest Compounds

NA NE NAED 17␣-NT 17␤-NT E1 17␣-E2 17␤-E2

CC␣ (␮g L−1 )

CC␤ (␮g L−1 )

MRPL (␮g L−1 )

Screening

Confirmation

Screening

Confirmation

0.17 0.02 0.05 0.03 0.03 0.21 0.04 0.04

0.68 0.06 0.07 0.03 0.03 0.27 0.11 0.08

0.21 0.02 0.10 0.03 0.04 0.38 0.13 0.05

1.00 0.09 0.11 0.04 0.04 0.40 0.46 0.11

Minimum required performances level (MRPL) suggested by CRL or LABERCA are indicated. a LABERCA proposition. b CRL proposition.

1a 1a 1a 2b 2b 1a 1a 1a

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repeatability regarding relative retention time, transitions ratio and relative response of analytes versus internal standards was judged acceptable. CC␣ and CC␤ are reported in Table 2. The decision limit CC␣ is defined as the limit at and above which it can be concluded with an error probability of ␣ that a sample is noncompliant. Decision limits were based on the analysis of the 20 blank samples. The detection capability CC␤ is defined as the lowest concentration at which a method is able to detect contaminated samples with a statistical certainty of 1-␤ (error probability = 5%). Detection capability was based on the analysis of the 20 spiked urine samples. CC␣ and CC␤ were calculated for the different steroids of interest. Screening and confirmation values were obtained considering the highest and lowest intense associated transitions, respectively. As acceptation criteria, performances of the method were compared to Minimum Required Performance Limits (MRPL) as proposed by the Community Reference Laboratory or by our laboratory (LABERCA) when no proposition was available (Table 2). Detection capabilities obtained for confirmatory purposes were equal or below the MRPLs which is satisfactory for such a method, especially when taking into account the small urine sample size (2 mL) used.

Finally, six samples were used to assess the yield of the procedure; they were blank samples spiked just before the derivatisation step. Most recoveries were found to be in the range 12–36% with an average of 23%. 3.2. Nandrolone and its main metabolites profiles of elimination All urine samples taken during the experiment (240 and 132 samples for administered and non-treated calves, respectively) have been analysed with the developed and validated method. Elimination of 17␤-nortestosterone laureate ester after its administration to calves was studied in urine by monitoring 17␤-NT and focussing on some pertinent metabolites: 17␣-NT, NE, NA and norandrostenedione (NAED). For each of these compounds, except for NA which has never been identified in the samples, Fig. 1 shows two ion chromatograms corresponding to three collection points: before administration (day −7), after the first administration (day 2) and at the end of the experiment (day 39). When none of the monitored compounds were detected before administration, they could be identified in urine samples of treated animals immediately after the first injection. Urinary excretion has already

Fig. 3. Proposition of nandrolone (17␤-NT) metabolic pathway.

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been identified in bovine species, as the main metabolic path for 17␤-NT [23]. Intensities of the signals and length of detection post steroid were dependant on the compound of interest. For each treated calf and for each compound, kinetics of elimination is shown in Figure 2. In the case of all compounds and for all animals, profiles of elimination are similar. After each injection the urinary elimination of 17␤NT and its metabolites increase but not proportionally to the injected dose. The excretion rates reached the highest value 24–48 h after each administration. However, calculated concentrations are different depending on the metabolites with high concentrations reached for 17␣-NT (>2 mg L−1 ) whereas 17␤-NT, NE and NAED mean highest concentration values were 10, 60 and 7 ␮g L−1 , respectively. These results are in agreement with the metabolism of 17␤NT in bovine species: C17 epimerization is a major pathway. Thus, the urinary concentration of 17␣-NT is far more important than that of other metabolites. Similar metabolic trends have already been reported in cattle with 17␣-NT being the most abundant metabolite after 17␤-NT administrations [21-24]. In horses [26–29], main reported metabolites observed after 17␤NT administrations are17␣-NT, NE but also NA, which has rarely been observed in cattle. These metabolic pathways are slightly different from that observed in human [5,8,17,20,30]

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and pig [29,31,32] where no epimerization exists and main observed metabolites are NA and NE. Most of the previous studies have also reported in different animal species estranediols as important metabolites resulting from the complete reduction of 17␤-NT (4-3-oxo reduction), 5␣-estrane-3␤,17␣-diol being the predominant one in cattle [21,22,24]; these particular metabolites were not investigated in the present study. No significant difference could be observed between male and female animals in term of urinary excretions profiles as in term of steroid concentrations detected, excepted for the 17␣NT that was excreted in amounts on average twice as high in female urines than in male samples. A number of trends were observed during the course of the experiment, in particular following the third injection, it was noticed that the concentrations in 17␤-NT increased slightly whereas those in 17␣-NT and NE relatively decreased or remained stable for NAED. An explanation of this phenomenon might be found in hepatic enzymes induction or repression of nandrolone metabolism. According to the specific pathway (Fig. 3), 17␤-NT is firstly metabolised to NAED through the oxidation of the alcohol group in position 17 (17␤-hydroxysteroid-deshydrogenase). NAED is further reduced to give 17␣-NT and NE (3␣/␤-hydroxysteroiddeshydrogenase and 4-reductase). It is then possible to theorise

Fig. 4. Example, for one calf, of ion chromatograms of urine samples collected on three points: before treatment (day −7), after the first administration (day 2) and at the end of the experiment (day 39). Monitored signals correspond to 17␤-E2, 17␣-E2 and E1. GC–EI(+)-/MS analysis and SRM acquisition mode.

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that when faced with high concentrations of 17␤-NT due to the exogenous administration the hepatic enzymes are less available for 17␤-NT transformation leading to an increased observed concentration of this particular compound. According to the observed concentrations and associated tendencies, a diminution of phase I metabolism seems to be induced following several administrations of nandrolone. 3.3. Estradiol and its main metabolite profiles of elimination Elimination of 17␤-estradiol 3-benzoate after the three administrations to calves was studied in urine by monitoring 17␤-E2 and its main metabolites, 17␣-E2 and estrone (E1). For each of these compounds, Fig. 4 shows two ion chromatograms corresponding to three collection points: before administration (day −7), after the first administration (day 2) and at the end of the experiment (day 39). As can be observed on the chro-

matograms (and as expected), small quantities of 17␣-E2 were detected in calf urine samples before any treatment has been administered, whereas neither 17␤-E2 nor E1 were detected at or above the limit of detection. In males, 17␣-E2 was found to be present at day −7 and day 0 at a concentration in the 0–1 ␮g L−1 range. Urine samples from females were found to contain approximately 1.5–2 times more steroid than samples from the male calves. All three estrogenic compounds were detected in urine with intense associated signals immediately after the first injection and were still identified in urine samples collected at the end of the experiment For all treated calves and for each compound, kinetics of elimination are shown in Fig. 5. As previously observed for nandrolone and its metabolites, the time–concentration profiles followed similar elimination kinetics for all estrogenic compounds in all treated animals. Their concentrations peaked 24 h post each administration then declined. Calculated concentrations varied greatly between the measured compounds. Highest concentrations were found in the case of 17␣-E2 (>20 mg L−1 ) as opposed to maximum detected concentrations of E1 and 17␤E2 were around 1 and 0.15 mg L−1 , respectively. At the end of the experiment (D39), 17␣-E2, 17␤-E2 and estrone were still detected and identified in a number of the urine samples. As with previous results reported, no significant difference could be observed amongst male and female animals in term of residue concentration post steroid administration, with the exception of 17␣-E2 that was excreted in concentrations, on average, twice as high in female urines compared to their male counterparts. These results confirm that the epimerisation step, as already noted for nandrolone catabolism, is the important metabolic pathway for oestradiol in bovine [33,34]. Furthermore, a tendency toward a general decrease in 17␤-E2, 17␣-E2 and E1 concentrations upon three consecutive injections was observed as for nandrolone. 4. Conclusion

Fig. 5. Kinetics of elimination over 40 days of 17␤-E2, 17␣-E2 and E1 for the 12 17␤-NT laureate/17␤-E2 benzoate treated calves. Concentrations have been calculated from GC–EI(+)-MS/MS analysis of collected urine samples.

Analysis of all samples arising from the large animal study in order to establish elimination kinetics and study the effects of repeated injections on the elimination profiles of the steroids could be performed in a reasonable time period due to the development of a high sample preparation throughput protocol involving the use of SPE 96-well plates. The performance of the developed method was established by means of a rigorous validation according to the requirements of the European Decision 2002/657/EC and was in accordance with proposed MRPL values for these compounds. The results showed efficiency of the treatment since the anticipated compounds were found in urine samples analysed for absorption, transformation and elimination of administered compounds. All animals showed homogeneous elimination profiles which were in strong correlation with the pattern of steroid administrations. The effect of several consecutive injections on elimination profiles was studied and revealed a tendency toward a decrease in phase I metabolism. This experiment also provided a wide range of reference residue incurred samples such as urine as well as tissues (gonads, kidney, kidney fat, liver,

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muscle, injection sites), hair and retina which are still under intensive investigation. Further work is currently being performed to attempt to correlate the steroids concentration pattern found with modifications in plasma protein profiles as indicators of steroid administration. Acknowledgment This work has been supported by the European Commission under the specific research and technological development of the 6th Framework Programme “Integrating and strengthening the European Research Area within the BioCop project “New technologies to Screen Multiple Chemical Contaminants in Foods”. Contract number: FOOD-CT-2004-06988. We gratefully thank Dr. Suzanne W.F. Eisenberg who was responsible for the animal experiment and coordination of the sample collection.

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