Detectability of testosterone esters and estradiol benzoate in bovine hair and plasma following pour-on treatment

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1 Anl Bionl Chem (2009) 395:75 87 DOI.07/s ORIGINAL PAPER Detectility of testosterone esters nd estrdiol enzote in ovine hir nd plsm following pour-on tretment A. A. M. Stolker & M. J. Groot & J. J. P. Lsroms & A. W. J. M. Nijrolder & M. H. Bloklnd & I. Riedmier & C. Becker & H. H. D. Meyer & M. W. F. Nielen Received: 4 June 2009 /Revised: 30 July 2009 /Accepted: 3 August 2009 /Pulished online: 26 August 2009 # The Author(s) This rticle is pulished with open ccess t Springerlink.com Astrct The use of synthetic esters of nturl steroids such s testosterone nd estrdiol in cttle fttening nd sports is hrd to detect vi routine urine testing. The esters re rpidly hydrolysed in vivo into sustnces which re lso endogenously present in urine. An interesting lterntive cn e provided y the nlysis of the dministered synthetic steroids themselves, i.e., the nlysis of intct steroid esters in hir y liquid chromtogrphy tndem mss spectrometry (LC/MS/MS). However, retrospective estimtion of the ppliction dte following noncomplint finding is hindered y the complexity of the kinetics of the incorportion of steroid esters in hir. In this study, the incorportion of intct steroid esters in hir following pour-on tretment hs een studied nd criticlly compred with results from intrmusculr tretment. To this end nimls were pour-on treted with hormone cocktil contining testosterone cypionte, testosterone decnote nd estrdiol enzote in different crriers. The nimls A. A. M. Stolker (*) : M. J. Groot : J. J. P. Lsroms : A. W. J. M. Nijrolder : M. W. F. Nielen RIKILT Institute of Food Sfety, P.O. Box 230, 6700 AE Wgeningen, The Netherlnds e-mil: lind.stolker@wur.nl M. H. Bloklnd RIVM Ntionl Institute for Pulic Helth nd Environment, P.O. Box, 3720 BA Bilthoven, The Netherlnds I. Riedmier : C. Becker : H. H. D. Meyer Physiology Weihenstephn, Technische Universitet Muenchen, Weihenstephner Berg 3, Freising, Germny M. W. F. Nielen Lortory of Orgnic Chemistry, Wgeningen University, Dreijenplein 8, 6703 HB Wgeningen, The Netherlnds were either treted using injection nd pour-on ppliction once or three times hving week etween tretments using injection nd pour-on ppliction. Animls were slughtered from 2 weeks fter the lst tretment. Both hir nd lood plsm smples were collected nd nlysed y LC/MS/MS. From the results, it is concluded tht fter single tretment the levels of steroid esters in hir drop to CCβ levels (5 20 µg/kg) fter 5 7 weeks. When tretment is repeted two times, the CCβ levels re reched fter 9 weeks. Furthermore, in plsm, no steroid esters were detected; not even t the low microgrmme per litre level ut in contrst with the pour-on ppliction fter i.m. injection, significnt increse of 7β-testosterone nd 7βestrdiol were oserved. These oservtions suggest tht trnsport of steroid esters fter pour-on ppliction is not only performed y lood ut lso y lterntive fluids in the niml so proly the steroid esters re lredy hydrolysed nd epimerized efore entering the lood. Keywords Liquid chromtogrphy. Mss spectrometry. Hir. Testosterone ester. Estrdiol enzote. Pour-on tretment Introduction Anolic steroids re nned sustnces in the Europen Union ut might still e illeglly pplied s growth promoters in cttle fttening [, 2]. In the serch for suitle smple mtrices, ovine hir hs shown to e n ttrctive smple mtrix for prolonged detectility of residues of nolic steroids nd et-gonists. Such sustnces cn e incorported into hir from lood vi the hir follicle, incorported from swet vi the hir shft, sored from swet on the outside of the hir shft, nd/or sored from

2 76 A.A.M. Stolker et l. exogenic sources such s (environmentl) contmintion or intentionl illegl pour-on tretment [3 6]. The potentil of hir nlysis for the determintion of steroids nd etgonists hs een demonstrted in severl ppers [5, 7 8]. Nturl steroids re usully dministered s synthetic steroid esters ut these re rpidly hydrolysed in vivo into nturl steroids: following orl intke of testosterone undecnote, the unchnged ester could e found in plsm from thletes for 6 h only [9]. In urine, it is hrd to differentite etween the (metolites of) endogenous nturl steroids lwys present nd the identicl (metolites of) nturl steroids from the hydrolysed esters. In doping control, this prolem is usully ddressed y estlishing the 7β-testosterone/ 7α-testosterone urinry rtio (so-clled T/E rtio) [20] nd/or the ppliction of 3 C/ 2 C isotope rtio mss spectrometry [2]. In food surveillnce, reltively high or low findings of 7β-testosterone nd 7α-testosterone in urine re often ignored ecuse of the lck of sttisticlly vlid reference dt of nturlly occurring ckground levels. An interesting lterntive for inconclusive urine nlyses in veterinry control cn e provided y the nlysis of the dministered synthetic steroids themselves, i.e. the nlysis of intct esters of nturl steroids in hir. Gleixner nd Meyer digested hir smples using dithiothreitol to rek up the longitudinl chins of the kertin protein of the hir y reducing the disulfide onds nd pplied tht method to the determintion of the nturl steroids estrdiol nd testosterone []. Hooijerink et l. developed n LC/MS/MS method for the determintion of intct estrdiol enzote in hir using the reducing gent tris(2-croxyethyl)phosphine hydrochloride (TCEP) [22]. Menwhile, tht method hs een further developed towrds true multiresidue screening method for ll kinds of intct esters of testosterone, oldenone nd estrdiol [23] nd dopted y other countries s well [24]. Thnks to this method, severl cses of illegl use of synthetic esters of nturl steroids were discovered in recent yers. Following non-complint finding of synthetic ester of nturl steroid in hir, questions rise regrding retrospective estimtion of the dte of illegl ppliction might rise. In generl, there will e no simple nswers to tht. When no preprtions hve een found, the route of dministrtion remins unknown nd might comprise injection, orl, sulingul, pour-on or other mens. Moreover, severl ppers studying hir segments reported the lck of correltion etween incorportion time nd growth rte of hir [25 27]. This discrepncy is elieved to e due to the fct tht steroid esters re minly incorported vi swet nd seum excretion t the surfce of the skin followed y diffusion into the hir fire [25]. Aprt from tht, interindividul differences in metolistion rtes do occur of course. Dt out detectility in hir following pour-on ppliction re rre. Anielski et l. [28] investigted NorAndrosteDerm ppliction contining prohormones of nortestosterone ut intct esters were not studied. Hooijerink et l. [22] reported estrdiol enzote concentrtions up to 8500 ng g in ovine hir following pour-on ppliction. Thieme et l. [6] compred trnsderml nd orl ppliction of nolic steroids nd concluded tht effective nd durle concentrtions of ctive steroids in lood could e otined following the former tretment. This study ws designed to test the hypothesis whether the incorportion of intct steroid esters into ovine hir following pour-on tretment is detectle. The findings were criticlly compred with results from intrmusculr tretment. To this end, nimls were pour-on treted with typicl hormone cocktil contining testosterone cypionte, testosterone decnote nd estrdiol enzote in different crriers, or y intrmusculr injection. Hir nd lood smples were collected during severl weeks nd nlysed for intct steroid esters y stte of the rt LC/MS/MS. Hir smples were tken y using rzor. The possiility of crry-over y using one rzor to shve different nimls ws investigted s well. Experimentl Chemicls, regents nd solutions LC-C/MS-grde cetonitrile, wter nd methnol were otined from Biosolve (Vlkenswrd, the Netherlnds). Formic cid, cetone, cetonitrile, ethnol, n-pentne, isooctne, dithiothreitol nd Tris(hydroxymethyl)-minomethne were otined from Merck (Drmstdt, Germny). The reduction gent tris(2-croxyethyl) phosphine hydrochloride (TCEP) ws otined from Sigm (St. Louis, MO, USA). Estrdiol enzote (EB), testosterone cypionte (TC) nd testosterone decnote (TD) were otined from Sigm-Aldrich Chemie.v (Zwijndrecht, the Netherlnds). N-methyl-N-trimethylsilyl-trifluoro(o)cetmide (MSTFA) ws otined from Alltech (Ancond, Montn, USA), mmonium iodide ws otined from Fluk (Zwijndrecht, the Netherlnds). The steroids 7α-testosterone nd 7βtestosterone were otined from Sterloids (Newport, Rhode Islnd, US). 7α-Estrdiol ws otined from Diosynth (Morrisville, North Crolin, US), 7β-estrdiol ws otined from Orgnon (Oss, the Netherlnds) nd the internl stndrds 7β-testosterone-d2 nd 7β-estrdiold3 were otined from RIVM (Bilthoven, the Netherlnds). The deuterium-lelled internl stndrds d3-estrdiol enzote, d3-testosterone cypionte nd d3-testosterone decnote were in-house-synthesised s descried in [23]. The crrier solvents for pour-on ppliction Ivomec (mixture of isopropnol nd Ivermectin B, B), DMSO (dimethyl sulfoxide Ph Eur), Mygliol 840 (Triglycerid

3 Testosterone esters nd estrdiol enzote 77 sturte medi Ph Eur), DEGMBE (diethyleneglycol monoutyl ether) nd the crrier for injection Archide oil were otined from Spruyt Hillen.v. (IJsselstein, the Netherlnds). All other chemicls used were of nlyticlregent grde. The derivtiztion regent MSTFA++ for the free steroid nlysis consisted of N-methyl-N-trimethylsilyl-trifluoro(o) cetmide/mmonium jodide/dithiothreitol (,000/2/4; v/w/w). Individul stock solutions of the steroid esters were prepred ydissolvingmgofechcompoundinmlofmethnol. Individul stock solutions of the steroids were prepred y dissolving mg of ech compound in ml of ethnol. Solid-phse extrction columns type Bond Elute LRC-C8 Phse 0 mg nd 500 mg columns were otined from Vrin (Hror City, CA, USA). Equipment The seprtion of the steroid esters ws crried out using n ultr performnce liquid chromtogrphic (UPLC) system, consisting of vcuum degsser, utosmpler nd inry pump (Acquity UPLC system; Wters, Milford, MA) equipped with reversed phse Wters cquity UPLC BEH C8 nlyticl column of 0 2. mm nd.7 µm prticle size. The grdient (solvent A, wter cetonitrile methnol formic cid (300:350:350:20, v/v/v/v); solvent B, cetonitrile methnol formic cid (500:500:20, v/v/v)) ws: 0 min, 0% B; min, liner increse to 0% B; min 0% B. Injection volume ws 40 µl. The UPLC system ws connected to triple-qudrupole mss spectrometer model Quttro Premier (Wters, Mnchester, UK) equipped with n electrospry interfce operting in the positive ion mode. The MRM trnsitions cquired for the different steroid esters of interest re summrised in Tle. The free steroids 7α-, 7β-estrdiol nd 7α-, 7βtestosterone were nlysed y using GC-MS/MS. A Vrin (Plo Alto, Cliforni, US),200 L triple-qudrupole MS/ MS spectrometer equipped with CP8400 utosmpler nd CP-3800 GC ws used. The GC column ws VF-7MS (L=30 m, id=0.25 mm, df=0.25 µm) otined from Vrin. The temperture progrmme used strted t C (hold min); incresed t 20 C/min to 240 C (hold.5 min); incresed with C /min to 243 C followed y n increse of 25 C/min to finl temperture of 340 C (hold 2 min). The GC-MS/MS ws operted in the EI ionistion mode. The MRM trnsitions cquired for the different steroids of interest re summrised in Tle 2. Smples An niml tril hs een conducted t the Physiology Weihenstephn, Technicl University of Münich (Germny). The protocol hs een pproved y the ethicl committee of the Regierung von Oeryern. Twenty vel clves (Holstein Friesin) were otined from identified sources t n ge of 2 weeks. The nimls were housed on strw nd the different tretment groups were housed in seprte pens. The nimls were fed ccording to vel clf prctise, using milk replcer nd roughge, drinking wter ws d li. Pour-on experiments were crried out using hormone cocktils contining three steroid esters. Prior to ppliction the nimls were shven on the ck from neck to til. There, they were treted with ml pour-on cocktil. In totl, set of four pour-on hormone cocktils were prepred nd for ech cocktil, different crrier solvent ws used. The hormone cocktils contined 25 mg of EB, 60 mg of TD nd 60 mg of TC per ml of crrier solvent. The crrier solvents used were Ivomec, DMSO, Mygliol 840 nd DEGMBE. Tle 3 presents the ppliction scheme. Five nimls were not treted t ll nd were used s reference group. Four nimls were treted t dy, 7 nd 4 (repeted tretment) with ml of crrier solvent. Four nimls were treted t dy with ml of hormone cocktil; ech niml ws treted with different crrier solvent. Four nimls were treted on dy, 7 nd 4 (repeted tretment) with ml of hormone cocktil. One niml ws injected intrmusculr in the neck on dy with cocktil of 35 mg EB, 60 mg TD nd 60 mg TC nd one Tle Steroid esters; specific ions monitored for LC-ESI(+)-MS/MS Compound MRM trnsition (m/z) Collision energy MRM trnsition (ev) MRM trnsition 2 (m/z) Collision energy MRM trnsition 2 Rt in min Estrdiol enzote 377> > Estrdiol enzote-d3 380> Testosterone cypionte 43> > Testosterone cypionte-d3 46> Testosterone decnote 443> > Testosterone decnote-d3 446>

4 78 A.A.M. Stolker et l. Tle 2 Free steroids; specific ions monitored for GC-EI-MS/MS Compound MRM trnsition (m/z) Collision energy MRM trnsition (ev) MRM trnsition 2 (m/z) Collision energy MRM trnsition 2 7α-estrdiol 46> > β-estrdiol 46> > β-estrdiol-d3 49> α-testosterone 432> > β-testosterone 432> > β-testosterone-d2 434>2 2.5 niml ws injected on dy, 7 nd 4 with the hormone cocktil. For the injection, rchide oil ws used s the crrier. One niml ws injected three times (dy, 7 nd 4) with rchide oil. For pour-on ppliction, the liquid-contining steroid esters (hormone cocktil) were spred out over the smll (shved) strip t the ck of the niml from neck to til. Hir smples were tken t lest 20 cm off from the ppliction zone. The first hir smples were tken efore tretment. After week (7 dys) the second hir smple ws tken t the next spot (see Fig. ). The smples tken 35 dys fter tretment re tken t the sme spot s the smple which ws tken t dy. Tle 4 nd Fig. descrie the hir smpling scheme including the spots where the smples re tken, the numer of dys fter first tretment nd the weeks the hir hd een growing t the specific spot. Ech smple contined pproximtely 2 5 g of hir. Hir smples were tken y using one rzor ut with one shver hed for ech tretment group. To check for crryover from one hir smple to the other due to the use of only one rzor system, contmintion experiment ws performed. The (worst cse) contmintion experiment ws s follows: piece of skin ws treted (pour-on) using the steroid cocktil. The hir directly on the pour-on spot ws collected y using rzor. The rzor ws clened dry y crefully removing the hir nd lowing some ir through the rzor. No visile pieces of hir were left. The sme rzor ws used to shve n untreted piece of skin. This hir smple ws tested for contining residues of steroids esters. Smples of lood were tken t dys, 2, 3, 4, 5, 6, 7, 4, 2, 28, 42, 56, 70, 84 nd 98. The smples were tken Tle 3 Appliction scheme Animl group Dy of tretment Animl id no. Type of tretment Reference group (n=5 nimls) 75, 82, 85, 9, 93 No tretment Reference group treted (3 times) Dy, 7 nd 4 72 Injection with Archide oil with crrier solvents only 73 Pour-on with crrier solvent DEGMBE 77 Pour-on with crrier solvent DMSO 8 Pour-on with crrier solvent IVOMEC 84 Pour-on with crrier solvent Miglyol Treted once Dy 74 i.m. injection with hormone cocktil 78 Pour-on hormone cocktil in DEGMBE 80 Pour-on hormone cocktil in DMSO 83 Pour-on hormone cocktil in IVOMEC 88 Pour-on hormone cocktil in Miglyol Treted three times Dy, 7 nd 4 7 i.m. injection with hormone cocktil 79 Pour-on hormone cocktil in DEGMBE 87 Pour-on hormone cocktil in DMSO 90 Pour-on hormone cocktil in IVOMEC 92 Pour-on hormone cocktil in Miglyol Hormone cocktil contents 25 mg of estrdiol enzote, 60 mg of testosterone decnote nd 60 mg of testosterone cypionte for ech injection or for ech ml of crrier solvent

5 Testosterone esters nd estrdiol enzote 79 efore ech niml tretment. Blood smples were collected in EDTA Vcutiner, centrifuged nd the plsm ws used for the nlysis. Hir nd lood smples were stored t 20 C. Methods Spot 3 (=8=3) Appliction side Spot (=6=) Appliction side Spot 4 (=9=4) Anlysis of steroid esters in hir Spot 2(=7=2) Spot 5 (==5) Fig. Spots were the hir smples re tken (see lso Tle 4) Hir steroid esters The smples were treted s descried in detils y Nielen et l. [23] with some minor chnges. The hir smples were nlysed without using wshing step. Normlly, the wshing step with wter is to wsh wy mud, etc. The smples were very clen so the wshing step ws omitted. In short, the procedure is s follows: the hir smples were cut into 0.5 cm pieces using pir of scissors. Then, 500 mg ws pulverised using Mikro-dismemrtor S ll mill. Two hundred milligrmmes of the pulverised hir ws weighed into plstic tue nd deuterium-lelled internl stndrds were dded ( ng/g hir). Digestion of the hir ws performed y ddition of 2 ml of 25 mm TCEP nd incution t room temperture for h. The tue ws shken y using hedover-hed shker. After ddition of 4 ml of methnol, the tue ws centrifuged for 5 min t,700 g. Next, 4 ml of wter ws dded nd the mixture ws pplied to n ctivted C8 solid-phse extrction (SPE) column. The SPE column ws conditioned y the ddition of, respectively, cetonitrile, methnol nd wter. The column ws wshed with 2 ml of methnol/wter (60/40, v/v) nd eluted with 2 ml cetonitrile followed y 2 ml of ethyl cette. The SPE elute ws evported to dryness under gentle stem of nitrogen gs t 40 C nd redissolved in 200 µl of LC solvent A, wter cetonitrile methnol formic cid (300:350:350:20, v/v/v/v). Finlly, 40 µl ws nlysed y UPLC-MS/MS. For ech steroid ester, two trnsitions (see Tle ) were monitored nd for ech deuterium-lelled nlogue one trnsition ws mesured. The response fctor ws defined s the rtio etween the sum of pek res of the steroid ester trnsitions nd the pek re of the trnsitions of the deuterium-lelled nlogue. For quntifiction, response fctors versus concentrtion plots were constructed. To this end, seven lnk hir smples were fortified with different concentrtions of the steroid esters. The concentrtion of TC, TD rnged from to,000 µg/kg nd the concentrtion of EB from 2.5 to 250 µg/kg. The concentrtions of the deuterium-lelled nlogues were 50, 0 nd 0 µg/kg for ech smple of EB, TC nd TD, respectively. The hir smples were nlysed together with the clirtion smples (Mtrix Tle 4 Smpling scheme for hir smples (see lso Fig. ) Spot Weeks of grown hir 0 (efore tretment) (=) (=2) (=3) (=4) 56 5 (=5) 63 5 (==6) (=2=7) (=3=8) (=4=9) (=5=) 98 5

6 80 A.A.M. Stolker et l. Mtch Stndrds; MMS); concentrtions were clculted using the liner regression method. Anlysis of steroid esters in plsm The nlysis of steroid esters in plsm ws set up for this study nd ws sed on the procedure descried y Shckleton et l. [29]. To ml of plsm, 5 ng of ech internl stndrds nd 4 ml of cetone/ethnol (/; v/v) were dded. After intensive shking nd fter centrifugtion, the superntnt ws trnsferred to clen tue nd the solvent ws evported t 40 C under gentle strem of nitrogen. The residue ws redissolved in 4 ml of methnol nd 6 ml of wter. The complete extrct of ml ws pplied to preconditioned SPE column. The SPE procedure nd the LC-MS/MS procedure were s descried for the nlysis of steroid esters in hir section. Becuse of the expected low concentrtions of steroid esters in plsm the concentrtion rnge used for the clirtion curve ws 0 µg/l. Anlysis of the free steroids 7α-, 7β-estrdiol nd 7α-, 7β-testosterone in plsm The smples were treted s descried y vn Tricht et l. [30]. In short, the procedure is s follows: to ml of plsm, 0.5 ng of ech internl stndrd nd ml methnol were dded. The proteins were denturted nd fter ddition of ml of wter, the mixture ws pplied to C8 SPE column. The SPE ws preconditioned with 3 ml of methnol nd 3 ml of wter. After wshing step with wter nd cetonitrile/wter (35/65; v/v) the nlytes of interest were eluted with 3 ml of cetone. The elute ws collected nd evported t 50 C under gentle strem of nitrogen nd redissolved in 0 µl of methnol nd 2 ml of Tris-uffer ph 9.5. Liquid liquid extrction (LLE) ws performed with 7 ml of n-pentne. This extrction step ws repeted nd the orgnic lyers were collected nd evported (see ove). The residue ws redissolved in 0.5 ml of ethnol nd trnsferred into derivtistion vil nd the ethnol ws evported. The dry residue ws derivtised y dding 25 µl of MSFTFA++ followed y incution of hour t 60 C. The derivtised mixture ws evported nd the residue ws reconstituted in 25 µl of iso-octne; 4 µl ws injected into the GC-MS/MS (pulse pressure 30 psi). The ion rtio ws defined s the rtio etween the re of the MS-MS trnsition (see Tle 2) of the steroid nd the deuterium-lelled nlogue. For 7αestrdiol nd 7α-testosterone, the 7β- deuterium-lelled nlogues were used. For quntifiction, ion rtios versus concentrtion plots were constructed. For qulity control, five lnk plsm smples were fortified with different concentrtions of the steroid. The concentrtion of steroids rnged from 0 to 0.4 µg/l. The concentrtions of the deuterium-lelled nlogues were 0.2 µg/l for ech smple. The plsm smples nd qulity control smples were nlysed together with the clirtion stndrds; concentrtions were clculted using the liner regression method. Results nd discussion There is not much informtion ville regrding the phrmcokinetics of steroid esters dministered to n niml y pour-on ppliction [26]. Therefore, it is of interest to know if the steroid esters finlly rech the loodstrem. To collect informtion out the distriution of the steroid esters it ws decided to nlyse selection of the plsm smples for contining steroid esters (EB, TC nd TC). From literture, it is known [29] tht fter injection of steroid esters, the esters re hydrolysed nd the free steroids re detected in the lood. To find out if the free steroid (7α-, 7β-estrdiol nd 7α-, 7βtestosterone) concentrtion in plsm lso increses fter pour-on ppliction, the free steroid concentrtion ws mesured in su selection of the plsm smples. The results of the nlysis of hir smples nd plsm smples re descried elow. Determintion of steroid esters in hir smples y LC-MS/MS LC-MS/MS method performnce The nlyticl method for the determintion of steroid esters in hir is descried y Nielen [23]. The method ws in-house-vlidted ccording to EU legisltion 2002/657/ EC [3] for 3 steroid esters including EB, TC nd TD. The vlidtion ws performed ccording to the guidelines for confirmtory method. The CCα nd CCβ were determined y the nlysis of 20 different smples of hir with nd without ddition of steroid esters. The CCα of EB, TC nd TD were respectively 2, 6 nd 6 µg/kg. The CCβ of EB, TC nd TD were respectively 5, 20 nd 20 µg/kg. Recent pulictions [24, 32] demonstrte tht the method is suitle for the nlysis of steroid esters in ovine hir. To use the method s quntittive method, some dditionl vlidtion ws performed. The linerity of the clirtion curve ws checked within the rnge of µg/kg for EB nd of,000 µg/kg for TC nd TD. The coefficient of correltion (R 2 ) ws etter thn 0.98 for ll tested esters. The within-l reproduciility ws determined t the CCβ concentrtion level. In different smple series (nlysed on different dys), (lnk) smple fortified t CCβ level ws

7 Testosterone esters nd estrdiol enzote 8 nlysed. The %RSD ws 20, 5 nd 20% (n=9) for EB, TC nd TD, respectively. For this study determintion of the kinetics of the elimintion the otined results from method vlidtion re dequte. The CCβ-s re t or elow 20 µg/kg, linerity is etter thn Furthermore, deuterted internl stndrds re ville for ech steroid-ester to correct for recovery losses nd the influence of the mtrix resulting in within-l reproduciility results of %RSD <25%. Steroid esters in hir Animl no. 78 (steroid-esters in DEGMBE x) Figures 2 6 present the concentrtion of the steroid esters mesured in the smples of hir. The verticl lines t dy 35 (fter 5 weeks) divided the plot into smples of hir grown Animl no. 74 (injection steroid-esters x) Animl no. 79 (steroid-esters in DEGMBE 3x) Animl no. 7 (injection steroid-esters 3x) Fig. 2 Concentrtion of the steroid esters found in hir smples fter intrmusculr injection Fig. 3 Concentrtion of the steroid esters found in hir smples fter pour-on tretment for 8 2 weeks nd smples grown for only 5 weeks (see lso Fig. nd Tle 4). The horizontl lines in the figures represent the CCβ-s of the steroid esters mesured. Figure 2 represents the results of the niml injected once with the hormone cocktil nd Fig. 2 represents the results of the repeted i.m. injection tretment. The tretment took plce on dys, 7 nd 4. Note tht the concentrtion scle is logrithmic scle. The results otined fter pour-on tretment re presented in the Figs The tretments presented in Figs. 3 6 differ only in the crrier solvent used DEGMBE, DMSO, IVOMEC or Miglyol. Figures 3 6 present the single-tretment results nd the Figs. 3, 6 the repeted pour-on tretment results. For monitoring the illegl use of steroid esters the CCβ concentrtion is most relevnt ecuse t nd ove tht concentrtion, identifiction ccording to the EU criteri is

8 82 A.A.M. Stolker et l Animl no. 80 (steroid-esters in DMSO x) elow the CCβ levels re, for most smples, due to oserved interfering peks most proly due to residues of some ftty compounds like seum. From the results presented in Figs. 2 6 some generl conclusions cn e drwn. The mximum concentrtions mesured for ll pplictions (pour-on nd injection) re for TC with mximum of 9,600 µg/kg y pour-on ppliction in DMSO. In generl, the TC concentrtions re slightly higher thn the TD concentrtion. Due to the concentrtions pplied EB shows the lowest concentrtions in hir. Tle 5 presents how mny dys fter tretment the CCβ concentrtion (lowest concentrtion resulting in noncomplint results) levels re reched. In generl, for single tretment, the CCβ levels re reched fter dys with the only exception of the pour-on ppliction in Animl no. 87 (steroid-esters in DMSO 3x) Animl no. 83 (steroid-esters in IVOMEC x) estosteronedecnote Fig. 4 Concentrtion of the steroid esters found in hir smples fter pour-on tretment Animl no. 90 (steroid-esters in IVOMEC 3x) possile in t lest 95% of ll non-complint cses. For this study, it is importnt to estlish how long fter tretment the CCβ concentrtion level is reched; in other words, how long fter tretment the hir smple is non-complint. All results re sed on single-hir nlysis. When no recovery of the internl stndrd ws mesured nd/or when too mny interfering peks were mesured, the quntifiction ws not possile for the specific nlyte/ smple comintion. These dt re the missing points in the figures. The missing points t the higher concentrtion levels re mostly due to low recovery of the internl stndrds. The used method fits the purpose; however, due to the different smple pre-tretment steps necessry (pulverising, digestion, SPE), the solute recovery is sometimes low. When the internl stndrd is not detected, the quntifiction is not possile. The missing points t or Fig. 5 Concentrtion of the steroid esters found in hir smples fter pour-on tretment

9 Testosterone esters nd estrdiol enzote Animl no. 88 (steroid-esters in Miglyol x) Animl no. 92 (steroid-esters in Miglyol 3x) Fig. 6 Concentrtion of the steroid esters found in hir smples fter pour-on tretment DEGMBE for which the CCβ levels re reched fter >56 dys (8 weeks) fter tretment. For the pour-on ppliction in Miglyol, some relevnt dt points re missing ut y extrpolting the elimintion plot for TC it is expected tht the CCβ is reched etween 56 nd 63 dys fter tretment. For repeted tretment, the following conclusions cn e drwn: it is ovious tht the highest concentrtion levels of steroid esters re reched fter the first tretment nd tht there is hrdly ny concentrtion differences etween injection nd pour-on ppliction. The mximum elimintion rte of steroid esters into the hir is proly reched lredy fter the first tretment nd so, dditionl tretments do not result in higher concentrtions levels in the hir smples. The numer of tretments do not hve effect on the mximum concentrtion level in hir ut on the numer of dys the concentrtions re t high level (>,000 µg/kg). Consequently, fter repeted tretment, it tkes more time efore the CCβ levels re reched. By repeted tretment, the high level is (in comprison with the single tretment) continued for 2 28 dys then the concentrtions strted to decrese. For TC, the CCβ level is reched for ll ppliction fter >9 dys. The only exception is the pouron ppliction in IVOMEC for which TC reched the CCβ level fter 43 dys ut these results re not very relile ecuse rther unexpected the TC concentrtion strted to increse fter 63 dys. For the steroid esters EB nd TD, the CCβ levels re reched t dys (9 weeks) fter injection or pour-on tretment with the crrier solvent DMSO or IVOMEC. For the pour-on ppliction in DEGMBE nd Miglyol, slightly longer elimintion times for EB nd TD viz dys ( 2 weeks) re oserved. It is difficult to drw conclusions out the comprison of injection nd pour-on ppliction. Only two nimls were injected with the hormone cocktil. This experiment in which one niml ws injected once nd one niml ws injected three times shows tht the steroid ester concentrtions in the hir smples collected fter injection were roughly t the sme level s fter pour-on tretment. It seems tht the ppliction procedure does not ffect the steroid ester elimintion rte into the hir. Note tht the smples of hir collected efore tretment (t=0 smples) did not contin ny of the steroid esters t detectle concentrtions. Bsed on the results otined, it ws decided not to nlyse ll the hir smples collected from the nimls treted with only crrier solvent or untreted (reference group) nimls. From these tretments, only the smples tken t week 0, 5,, nd 4 were nlysed. The smples did not contin ny of the steroid esters t detectle concentrtion. Importntly, the smple of hir collected during the contmintion (crry-over) experiment did not contin ny of the steroid esters t detectle concentrtion. Determintion of steroid esters in plsm y LC-MS/MS A method ws set up to monitor steroid esters in plsm. From literture, it is known tht fter ppliction of 35 mg testosterone esters to humn ody, the lood concentrtions fter 96 h is out µg/l [29]. For tht reson, the method ws set up in the concentrtion rnge of 0 µg/l. The method ws vlidted for qulittive nlyses only ut to ech smple, the internl stndrds were dded nd the recovery of the internl stndrds (criteri S/N of internl stndrd hs to e >3) ws used s sensitivity nd recovery check. The lowest clirtion point ws t µg/l nd therefore the LOD ws set for ll steroid esters t this specific concentrtion level. All smples of plsm collected from the nimls treted once nd three times (injection nd pour-on) with the hormone cocktil were nlysed for contining

10 84 A.A.M. Stolker et l. Tle 5 Numer of dys fter tretment the CCβ concentrtion levels re reched Injection Pour-on ppliction DEGMBE DMSO IVOMEC Miglyol Unrelile results; concentrtion increses significntly fter 63 dys Single tretment Estrdiol enzote Testosterone cypionte >56 Testosterone decnote >49 Repeted tretment Estrdiol enzote Testosterone cypionte >9 >9 > >9 Testosterone decnote steroid esters. In none of the smples detectle concentrtions (> LOD) of steroid esters were monitored As mentioned ove, the method ws prtilly vlidted nd for tht reson selection of the plsm smples ws send to the EU Community Reference Lortory for hormones the RIVM (Bilthoven, the Netherlnds) nd were nlysed under ISO/NEN 7025 ccredited conditions for contining steroid esters. The su selection consists of the following smples: plsm collected from Dys 0 to 42 of the niml injected (3 ), Dys 0 to 4 of the niml injected nd the first five smples collected fter pouron ppliction ( nd 3 ) y using DEGMBE s the crrier solvent. Agin, no steroid esters were detected. The LOD levels for EB, TC nd TD for the LC-MS/MS method used were 0.2, 0.3 nd µg/l, respectively. Tht no steroid esters were detected in the plsm smples ws not completely unexpected. Shckleton [29] lredy descried tht fter ppliction of 35 mg of testosterone esters to humn ody the lood concentrtions fter 96 h is out µg/l. In other words, rpid hydrolysis of steroid esters tkes plce s soon s the steroid esters rech the loodstrem. Determintion of free steroids in plsm y GC-MS/MS GC-MS/MS method performnce The nlyticl method for the determintion of free steroids in plsm nd the vlidtion of the method re descried y vn Tricht et l. [30]. The method ws vlidted ccording to EU legisltion 2002/657/EC [3] for 20 nturl hormones including 7α-, 7β-estrdiol nd 7α-, 7βtestosterone. For this study, the linerity of the clirtion curve ws checked for the ng/l rnge. The coefficient of correltion (R 2 ) ws etter thn A summry of the method vlidtion results is presented in Tle 6. The mesurement uncertinties sed on within-lortoryreproduciility (n=2) re for 7α- nd 7β-estrdiol 25 nd 20%, respectively, nd for 7α- nd 7β-testosterone 40 nd 46%, respectively. For this study determintion of the kinetics of the elimintion the otined results for method vlidtion re dequte. The CCβ levels re t or elow 6 ng/l for estrdiol nd 50 ng/l for testosterone, linerity is etter thn The detection limits of this method re low enough to mesure the concentrtion of nturl hormones (endogenous hormones) in plsm. Furthermore, deuterted internl stndrds re ville for ech steroid to correct for recovery losses nd the influence of the mtrix. Although the oserved uncertinties in the mesurements of the free testosterone re reltively high, the method ws useful for the determintion of the elimintion kinetic. The testosterone mesurement showed high vriility during this study, proly due to nturl vrition of testosterone concentrtions. Free steroids in plsm GC-MS/MS results A selection of the plsm smples were nlysed for 7α-, 7β-estrdiol nd 7α-, 7β-testosterone. The smples nlysed were the smples collected from the niml injected with the hormone cocktil ( nd 3 ). Injection is trditionl wy of treting the niml with steroid esters. It is of interest to nlyse the plsm smples collected fter injection of the hormone cocktil nd to compre the results with the results of the pour-on ppliction. Since, only minor differences were oserved in the steroid ester concentrtion of hir smples y using different crrier solvents for pour-on ppliction, it ws Tle 6 Vlidtion prmters for the GC-MS/MS nlysis of steroids in plsm; dpted from [30] Compound CCα (in ng/l) CCβ (in ng/l) 7α-estrdiol 9 6 7β-estrdiol 7 2 7α-testosterone β-testosterone 30 50

11 Testosterone esters nd estrdiol enzote 85 decided to nlyse only one set of the plsm smples fter pour-on ppliction. For the nlysis, the smples collected fter pour-on ppliction of the hormone cocktil with the crrier solvent DEGMBE ( nd 3 ) were selected. These smples were selected ecuse the corresponding hir smples contined the reltively high levels of steroid esters. Possily, these high levels would correspond with high plsm levels resulting in detectle concentrtions (ove CCβ). Figures 7 nd 8 present the results otined for the nlysis of 7α-, 7β-estrdiol nd 7α-, 7β-testosterone in plsm smples. In Fig. 7, the results for injection nd in Fig. 8 the results for pour-on re presented. Figures 7 nd 8 re results otined fter single tretment; Figs. 7 nd 8 re results otined fter repeted tretment. Note conc. (ng/kg) conc. (ng/kg) Animl no 74 ( injection steroid-esters x) Dys fter tretment lf-estrdiol et-estrdiol lf-testosterone et-testosterone Animl no 7 ( injection steroid-esters 3x) Dys fter tretment lf-estrdiol et-estrdiol lf-testosterone et-testosterone Fig. 7 Concentrtion of free steroids found in plsm fter intrmusculr injection conc. (ng/kg) conc. (ng/kg) Animl no 78 (steroid-esters in DEGMBE x) Dys fter tretment lf-estrdiol et-estrdiol lf-testosterone et-testosterone CCβ β-testosterone = 50 ng/l CCβ -testosterone = 40 ng/l CCβ β-estrdiol = 2 ng/l CCβ α-estrdiol = 6 ng/l Animl no 79 (steroid-esters in DEGMBE 3x) Dys fter tretment lf-estrdiol et-estrdiol lf-testosterone et-testosterone CCβ β-testosterone = 50 ng/l CCβ -testosterone = 40 ng/l CCβ β-estrdiol = 2 ng/l CCβ α-estrdiol = 6 ng/l Fig. 8 Concentrtion of free steroids found in plsm smples fter pour-on tretment the non-liner time xis. The first plsm smples were collected just efore the first tretment (Dy 0) followed y, 2, 3, 4, 5 nd 6 dys fter tretment. The next smples were collected t dy 4, 2, 28 (2, 3 nd 4 weeks fter the first tretment) nd t dy 42, 56, 70, nd 98 dys (6, 8, nd 4 weeks fter tretment). Smples tken t dy 4 nd dy 2 were collected efore the second nd third tretments took plce. When no recovery of the internl stndrd ws mesured nd/or when too mny interfering peks were mesured, the quntifiction ws not possile for the specific nlyte/smple comintion. These dt re the missing points in the figures. Especilly round the CCβ levels, dt points re missing due to some interfering peks in these specific smples. Additionl reserch is necessry to improve the roustness of the method.

12 86 A.A.M. Stolker et l. Although it is difficult to drw conclusions sed on one dt set for injection nd one dt set for pour-on, some oservtions re worthwhile to mention. It is ovious tht the concentrtion of the free steroids in plsm fter injection is significntly higher thn the concentrtions of free steroids fter pour-on ppliction. It is of interest to see tht fter injection (Fig. 7), the highest levels of free steroids re mesured etween nd 3 dys fter the first tretment nd tht the mximum concentrtions reched for the 7β-forms re etween,000 nd 2,000 ng/l nd for the 7α-forms re much lower, viz. 0 ng/kg. For the pour-on ppliction (Fig. 8), the highest levels of free steroids re lso mesured etween nd 3 dys fter the first tretment, however, for this ppliction, the mximum concentrtions reched re much lower (mximum of 4 ng/l for 7β-testosterone). A second remrkle difference etween injection nd pour-on results is tht for the pour-on ppliction, there is no significnt difference in mximum concentrtions for the four steroids determined. In other words, the mxim reched for 7α-, 7β-estrdiol nd 7α-, 7β-testosterone re within the smll rnge of ng/l in contrst, for injection, 7β-concentrtions re pproximtely ten times higher thn 7α-concentrtions. Regrding the steroid elimintion time, it is cler tht 5 dys fter repeted injection (Fig. 7), the steroid concentrtions re still ove CCβ levels. Due to the second injection t Dy 7 nd the third injection t Dy 4, the concentrtions of the free steroids re higher thn the t=0 concentrtion even fter Dy 6. It hs to e mentioned tht the elimintion time is defined s the time it tkes to rech the concentrtion of less thn or equl to CCβ or to rech the t=0 concentrtion in cse of nturl occurring ckground for exmple for 7α-estrdiol nd/or 7α-testosterone. Regrding the steroid elimintion time fter the pour-on ppliction, it is concluded tht the CCβ or t=0 concentrtions re reched within 5 dys fter the first ppliction. No increse of steroid concentrtion is monitored fter the second nd third pplictions. The 7α-testosterone concentrtions mesured t dy 42 nd dy 98 in Fig. 8 re proly outliers due to some interfering compounds. The results re not confirmed in Fig. 8. It is possile tht there is n increse of free steroid concentrtions fter the second nd third pplictions (Fig. 8) ut due to the smple collection scheme, the possile increses were not monitored. The increse of free steroid concentrtion is expected in the smples collected 3 dys fter ppliction; in this study, in the smples of Dy 8, 9 nd nd Dy 5, 6 nd 7, ut, unfortuntely, no smples were collected t these specific times. From phrmokinetic experiments, it is known tht the steroid esters when entering the loodstrem re hydrolysed to the free steroids viz. 7β-testosterone nd 7β-estrdiol. Furthermore, efore elimintion, free steroids re epimerized to α-testosterone nd α-estrdiol. C7 epimeriztion is mjor pthwy for steroids. This is lso confirmed y the elimintion kinetic descried y Pinel [33]. In [33], it is demonstrted tht the min metolites detected in urine fter i.m. injection of 7β-estrdiol enzote nd 7βnortestosterone lurete re 7α-estrdiol nd 7αnortestosterone. From the results otined for the concentrtion of the free steroids in the present study, it is concluded tht fter i.m. injection, high concentrtions of β-steroid esters enter the lood nd re quickly hydrolysed to their free form 7βtestosterone nd7β-estrdiol nd fter epimeriztion to the free 7α-steroids. However, fter pour-on ppliction, the concentrtions of free β-steroids re much lower proly due to the fct tht not ll steroid esters enter the lood. It is possile tht fter pour-on ppliction, the trnsport of the steroid esters to the hir is y nother route thn y lood trnsport, e.g. other extrvsculr or interstitil fluids [26]. So fter pour-on ppliction, the steroid esters re lredy hydrolysed, epimerized nd eliminted efore entering the lood resulting in much lower concentrtions of the free β- steroids. After i.m. injection, the steroid esters enters the lood very quickly re hydrolyzed to the free β-steroids which re detected in the first 5 dys fter injection. Conclusions From the niml tril performed y i.m injection nd pouron ppliction of steroid esters using different crrier solvents, it is concluded tht: no significnt differences re oserved etween the elimintion rte of steroid esters in hir fter i.m. injection nd pour-on ppliction of steroid esters. For oth wys of ppliction, the concentrtions of steroid esters rech the CCβ levels (5 20 µg/kg) 5 7 weeks fter single tretment nd 9 weeks fter repeted (three times) tretment. The identity of the crrier solvent during pour-on ppliction does not hve significnt influence on the finl steroid ester concentrtion in hir. Even though high concentrtions of steroid esters re mesured in hir, no detectle concentrtion of steroid esters (< µg/l) were mesured in the plsm of the treted niml. This is proly due to the quick hydrolysis of steroid esters to free steroids. The only difference in i.m. injection nd pour-on ppliction mesured in this study is the concentrtion of free β-form steroids in plsm. The concentrtion of free β- form steroids mesured in plsm re much higher (ten times reching 2,000 ng/l) fter injection thn fter pour-on tretment. The concentrtions of the α-form steroids re not significntly different. From these results, it is supposed tht fter the pour-on tretment, the trnsport of steroid esters to the hir is not only y lood ut lso y other

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