Urinary Concentrations of Steroids in Bulls under Anabolic Treatment by Revalor-XS® Implant

17 Pages·2016·2.07 MB·English

Hindawi Publishing Corporation
Journal of Analytical Methods in Chemistry
Volume 2016, Article ID 8013175, 16 pages
http://dx.doi.org/10.1155/2016/8013175
Research Article
Urinary Concentrations of Steroids in Bulls under Anabolic
„
Treatment by Revalor-XS Implant
GiancarloBiancotto,1RobertoStella,1FedericaBarrucci,2
FrancescaLega,1andRobertoAngeletti1
1DepartmentofChemistry,IstitutoZooprofilatticoSperimentaledelleVenezie,Vialedell’Universita`10,35020Legnaro,Italy
2DepartmentofPublicHealthandRiskAnalysis,IstitutoZooprofilatticoSperimentaledelleVenezie,Vialedell’Universita`10,
35020Legnaro,Italy
CorrespondenceshouldbeaddressedtoGiancarloBiancotto;[email protected]
Received14July2016;Revised16September2016;Accepted18September2016
AcademicEditor:JoseM.M.Lopez
Copyright©2016GiancarloBiancottoetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttribution
License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperly
cited.
DespitetheEuropeanbanofusinganabolicsinfood-producinganimals,growthpromotersmightstillbeillegallyusedinthe
EuropeanUnion.Tocontrolthefoodchainandguaranteeconsumers’health,thereisaneedofhighlysensitiveanalyticalmethods
fortheidentificationofmarkerresiduesofsuchtreatments.Inthepresentstudy,agroupofbulls(𝑛 = 16)receivedtrenbolone
acetate(200mg)andestradiol(40mg)byacommercialearimplantduringatimerangeof71days,andasecondgroup(𝑛 = 16)
waskeptforcontrol.Theaimoftheresearchwastomeasuretheresidualurinaryconcentrationsoftheadministereddrugs(𝛽-
trenboloneand𝛽-estradiol),theirmainmetabolites(𝛼-trenboloneand𝛼-estradiol),andpossiblealterationsoftheurinaryprofile
ofotherendogenoushormonesmetabolicallyrelated.Theanalyticalmethodwasbasedonliquidchromatography-tandemmass
spectrometry.Resultsshowedaverageurinaryconcentrationsof𝛼-trenboloneand𝛼-estradiolduringtreatmentintherangeof
(0.81÷2.1)ngmL−1and(0.96÷4.4)ngmL−1,respectively,whereas𝛽-trenboloneand𝛽-estradiolexhibiturinaryconcentrations
−1
lowerthan0.22ngmL inbothcases.Dataobtainedfromtheurinaryprofilesofendogenoussteroidsindicatethattheycouldbe
usefultoindirectlydetecttheongoingtreatment.
1.Introduction most challenging tasks in the national monitoring plans of
EU members, involving a wide number of substances with
Anabolic steroids, both natural and synthetic, have been different chemical structures and concentration levels in a
used over the last past decades in animal breeding to varietyofbiologicalmatrices[8–11].Forthisreason,several
improve animal growth and feed conversion efficiency [1– analyticalmethodshavebeendevelopedforthedetermina-
4]. Steroid hormones are molecules included in the classes tionofhormoneresiduesortheirmetabolitesinsamplesof
of estrogens, gestagens, and androgens. In some countries, animal origin [12–14]. Efficient control of the illegal use of
suchastheUnitedStates,theuseofsuchhormonalgrowth anabolic steroids must rely on the direct measurement of
promoters is still allowed. In contrast, they are banned steroidresidues,butalsoontheirdirectmetabolitestotake
in food-producing animals in Europe [5]. To enforce the into account kinetics of elimination and metabolic patterns
prohibitionontheillegaluseofanabolicsteroids,European [15–20]. Gas chromatography-mass spectrometry (GC-MS)
Union(EU)memberstatesarerequiredtomonitoranimals has played the dominant role for the detection of anabolic
for possible abuse [6]. However, the possibility of abuse of steroids in biological samples for many years [21–23]. In
hormonalsubstancesinEuropestillexists,mainlyduetothe recent years, thanks to the rapid advancement of liquid
economic benefits that these substances provide in animal chromatography-tandem mass spectrometry (LC-MS/MS)
breeding [7]. The analysis of the drug residues in animals thathasledtoanincreasedsensitivityimprovingtheoverall
intended for human consumption is probably one of the performance, there has been a shift towards LC-MS/MS
2 JournalofAnalyticalMethodsinChemistry
for the determination of hormones in different matrices The methods were validated following the criteria of Com-
[21,22,24,25]. mission Decision 657/2002/EC, properly adapted for our
Beside the residue analyses, new approaches have been purposes[37].
proposed in recent years to prove illicit treatment with
growthpromoters[26]:somefocusedontargetedmetabolite 2.Experimental
analyses[17,18,27]andothersonuntargetedproteomics[28–
30] or metabolomics approaches [31–33]. Despite all being 2.1.ReagentsandChemicals. Waterwaspurifiedanddeion-
promising, none of them is definitive to establish a unique izedbyanultrapurewatersystem(Sartorius,StedimBiotech,
and robust set of biomarkers of treatment independently AubagneCedex,France);methanolandacetonitrile(HPLC
from drug administration, breed, gender, age, and physi- grade) were from BDH (VWR, West Chester, PA, USA).
ological condition. Still, these studies represent a valuable FormicacidandglacialaceticacidwerefromFluka(Buchs,
contribution in the area of research aimed at improving Switzerland);ethylacetateandsulfuricacidwerefromCarlo
the efficacy of controls against illegal practices with growth Erba(Milan,Italy).Activatedcharcoal,dextran,ammonium
promoters. hydroxide,and𝛽-glucuronidase(98,000UmL−1)werefrom
Moreover,formulationsadoptedforillegalpurposesare Helix pomatia and sulfatase type H2 (2,000UmL−1) was
notrevealedbyproducersorfarmers.Consequently,several from Helix pomatia; 𝛽-glucuronidase type IX-A was from
research studies have been implemented through the years −1
E. coli (12,500UmL ); filter paper (Whatman, black rib-
totesttheeffectofdifferentgrowthpromotersadministered
bon) was obtained from Sigma Aldrich (St. Louis, MO,
via different routes [18, 28, 34, 35] and to evaluate residual
USA). Oasis-HLB reversed-phase SPE cartridges (200mg,
levels and target metabolites. In this work a commercially
6mL) were from Waters (Milford, MA, USA); Strata-NH2
available ear implant containing trenbolone acetate and
SPE cartridges (100mg, 1mL) and C18 SPE cartridges
estradiol(Revalor-XS)wastested;thisformulationisusedin
(500mg, 6mL) were obtained from Phenomenex (Tor-
the United States in bovine breeding and is able to release rance, CA, USA). D2-17𝛽-testosterone (D2-17𝛽-T), D4-
a constant amount of hormones over a time period up to 17𝛽-estradiol (D4-17𝛽-E2), and D4-estrone (D4-E1) were
200 days eliminating the need to reimplant the device. The
from CDN ISOTOPES (Pointe-Claire, Quebec, Canada),
pharmacologicaltriallastedfor71days;urinesampleswere
D3-dihydrotestosterone (D3-DHT), D2-dehydroepiandro-
collected before and during the treatment from 16 animals
sterone (D2-DHEA), and D7-4-androstene-3,17-dione (D7-
withimplantandfrom16untreatedanimals(control).Deple-
AED) were from Steraloids (Newport, RI, USA), and D3-
tionstudiesaftertreatmentsuspensionwerenotperformed. 17𝛽-trenbolone (D3-17𝛽-TBOH) was purchased from RIK-
Theresultsofthisworkrepresentthethirdpillarofamul- ILT (Wageningen, Netherlands). 17𝛽-Testosterone (17𝛽-T),
titarget research project aimed at verifying the potentiality
dihydrotestosterone(DHT),4-androstene-3,17-dione(AED),
ofthecombinationofresidueanalysis,proteomics[29],and
dehydroepiandrosterone (DHEA), etiocholanolone (ETIO),
metabolomics [33, 36] to control and prevent illicit steroid estrone(E1),17𝛼-estradiol(17𝛼-E2),and17𝛽-estradiol(17𝛽-
treatments. E2) were from Sigma Aldrich (St. Louis, MO, USA), 17𝛼-
Therefore,thepresentstudyprovidesananalyticalevalu- trenbolone(17𝛼-TBOH)wasfromTorontoResearchChem-
ationof icals (Toronto, Ontario, Canada), and 17𝛽-trenbolone (17𝛽-
TBOH) and 17𝛼-testosterone (17𝛼-T) were purchased from
(a)theurinaryexcretionoftheadministereddrugsand
Steraloids (Newport, RI, USA). Primary stock standard
their main metabolites (alpha epimers) after the
solutions of each analyte were prepared in methanol at a
exogenous administration of trenbolone and estra- concentrationof1000𝜇gmL−1.Intermediatesinglestandard
diolinbulls,introducingusefuldataregardingtheir
solutions for each analyte were prepared in methanol at a
residualconcentrationsinurineduringthetreatment
concentration of 10𝜇gmL−1. An internal standard solution
period;
whichincludedeachdeuteratedcompoundwaspreparedin
(b)the average urinary concentration of other related methanol at a concentration of 0.3𝜇gmL−1. A mixed stan-
steroidsphysiologicallypresentintheanimals,thatis, dard solution was prepared in methanol, with the endoge-
estrone(E1),17𝛼-testosterone(17𝛼-T),17𝛽-testostero- nous compounds at a concentrationof 0.05𝜇gmL−1 for E1,
ne (17𝛽-T), androstenedione (AED), dihydrotesto- 0.125𝜇gmL−1 for AED and 17𝛼-E2, 1𝜇gmL−1 for 17𝛽-T,
sterone (DHT), dehydroepiandrosterone (DHEA), 1.25𝜇gmL−1forDHTandDHEA,2.5𝜇gmL−1for17𝛼-T,and
and etiocholanolone (ETIO), before, in the middle, 12.5𝜇gmL−1 for ETIO. Finally a mixed standard solution
andattheendofthetrialtoverifyapossiblealteration
waspreparedinmethanolfortheexogenoussubstances(i.e.,
intheprofileoftheselectedendogenoussteroids;in
17𝛼-TBOH, 17𝛽-TBOH, and 17𝛽-E2) at a concentration of
addition,theurinaryprofilesofendogenoussteroids
0.125𝜇gmL−1each.Allstandardswerekeptat−20∘C.
oftreatedsampleswerecomparedtothoseofcontrol
animalsusingalogisticregressionmodel.
2.2. Sampling and Animal Treatment. 32 Charolais bulls at
Urine samples were processed using a purification 10–14 months of age were housed in stables and fed for
method based on a solid phase extraction (SPE) cleanup two weeks (acclimatization) after which they were divided
commontoalltargetanalytes,butwithdifferentLC-MS/MS intotwogroupsconsistingof16animalseach, andthetrial
conditions that were optimized for clusters of compounds. began. Animals owing to the treatment group (T) were
JournalofAnalyticalMethodsinChemistry 3
implanted using a commercially available device, Revalor- with5mLofmethanolandconditionedwith5mLofwater.
XS(Merck AnimalHealth),containing200mgofTBAand The cartridge was washed with 5mL of water and 5mL of
40mg of E2. Animals of the control group (C) were not methanol/water(50/50,v/v),vacuumwasapplied,andthen
implanted. The trial lasted for 71 days after which animals analytes were eluted with 4mL of ethyl acetate/methanol
wereslaughtered.Urinesampleswerecollectedthedaybefore (50/50,v/v).TheelutedsolutionwasfilteredthroughaNH2
the beginning of the trial (𝑇0), 8 times during the trial (𝑇7, SPE cartridge (100mg, 1mL) previously conditioned with
𝑇21, 𝑇28, 𝑇35, 𝑇42, 𝑇49, 𝑇56, and 𝑇63) and at 𝑇68. Samples 1mL of methanol and the purified extracts were collected
werefrozenandstoredat−20∘Cuntilanalysis.Theanimals directly during loading. The collected solution was evap-
∘
were managed in agreement with the European Directive orated at 45 C, and steroid extracts were reconstituted in
86/609/EEC regarding the protection of animals used for 0.5mLofwater/acetonitrile(70/30,v/v)andanalyzedbyLC-
experimental or other scientific purposes, enforced by the MS/MS.
Italian D.Lgs 116 (January 27, 1992), and with the Directive
2010/63/UE [38–40]. During the experimentation, hay and 2.4. Blank Urine Preparation for Quantification of Endoge-
waterwereavailabletoanimalsadlibitum. nous Steroids. To remove endogenous steroid compounds
fromurineusedformatrix-matchedcalibrationandquality
2.3. Sample Preparation. Urine samples were clarified by control samples, a pool of urine samples (250mL) from
centrifugation(5000×gfor5minutes).Foreachsamplingday vealcalves(naturallycontainingloweramountsofthetarget
2 aliquots (5mL each) from each animal were analyzed to analytes) were stirred in presence of charcoal (1.25g) and
achieveanalytequantificationinduplicate.50𝜇Lofinternal dextran (0.125g) overnight at room temperature, adjust-
standards containing D3-17𝛽-TBOH, D2-17𝛽-T, D4-17𝛽-E2, ing the method proposed by AbuRuz et al. [41]. Urine
D4-E1,D3-DHT,D7-AED,andD2-DHEAataconcentration samples were finally centrifuged at 5000×g for 5 minutes
of0.3𝜇gmL−1wasaddedtoeachvialtoobtainafinalconcen- to remove the charcoal in suspension. Afterwards, the
trationof3𝜇gL−1.Dependingonthegroupofanalytes,the charcoal-depleted urine samples were filtered with filter
preparedurinesampleswereprocessedusingoneofthetwo paper,and5mLaliquotswerestoredat−20∘Cuntilanalysis.
alternative deconjugation approaches described in the next Absence of interfering compounds was checked by analyz-
sections,followedbyacommonpurificationprotocol. ing an aliquot of blank urine samples during each sample
batch.
2.3.1. 17𝛼-TBOH,17𝛽-TBOH,17𝛼-E2,and17𝛽-E2Deconjuga-
tionbyEnzymesfromHelixpomatia. ThepHwasadjustedto 2.5. Analysis by LC-MS/MS. Analysis of steroid residues
5.0 by adding 15mL of acetate buffer 0.2M, pH 5.0, and, if wasachievedusingaProminenceHPLCsystem(Shimadzu,
needed,fewdropletsofHCl6N.Thehydrolysiswascarried Kyoto, Japan) interfaced to an API 4000 triple quadrupole
outfor2hat55∘Cusing50𝜇Lof𝛽-glucuronidaseand50𝜇L massspectrometerwithaturboionspraysource(ABSCIEX,
ofsulfatasefromHelixpomatia. Framingham, MA, USA). Sample extracts were analyzed
using 3 different ionization and gradient elution condi-
2.3.2.E1,DHT,DHEA,AED,17𝛽-T,17𝛼-T,andETIOSolvolysis tions: atmospheric pressure positive chemical ionization
andDeconjugationby𝛽-GlucuronidasefromE.coli. ThepH (APCI+),positiveelectrospray(ESI+),andnegativeelectro-
was adjusted to 7.0 by adding 15mL of phosphate buffer spray (ESI−). Air was used as nebulization and desolvation
0.2M,pH7.0.Thehydrolysisofglucuronideconjugateswas gas,whilenitrogenwasusedascurtaingas.Sourcetemper-
carried out for 2h at 55∘C using 50𝜇L of 𝛽-glucuronidase ature was set to 550∘C with an ion spray voltage of 5500
from E. coli. After deconjugation, samples were cooled at and −4500 for ESI+ and ESI−, respectively, and 375∘C with
roomtemperatureandthesolutionwasappliedtoHLBSPE nebulizer current set at 5 for APCI+. MS/MS experiments
cartridges (200mg, 6mL) already activated using 5mL of were performed using nitrogen as the collision gas at a
methanol and conditioned with 5mL of water. The column pressure of 3.5 ∗ 10−5torr. Ion acquisition was performed
waswashedwith5mLofwater,vacuumwasappliedtodry inselectedreactionmonitoring(SRM)usingthetransitions
cartridges,andsteroidswereelutedusing2mLofmethanol from the precursor ion to the two most abundant product
followed by 2mL of ethyl acetate. The final volume was ions optimizing the declustering potential and the collision
increased adding ethyl acetate and sulfuric acid (50% in energyforeachproduction(Table1).
water) to a final volume of 10mL to obtain a solution Purifiedextractswereseparatedbyreversed-phaseliquid
containingethylacetate/methanol/sulfuricacid(80/20/0.06, chromatography using an Accucore C18 column (100mm
v/v/v).Thesolvolysisofsulfateswascarriedoutfor2hat55∘C × 2.1mm, 2.6𝜇m, 100A˚) from Superchrom (Milan, Italy).
mixing by vortex every 30min. Samples were neutralized Mobile phases were water (A1) and acetonitrile (B1) for
with 60𝜇L of 2M NaOH and the volume was decreased to APCI+,0.02%(v/v)ammoniumhydroxideinwater(A2)and
∘
2mLunderastreamofnitrogenat45 C.Thevolumewasthen acetonitrile/methanol(70/30,v/v)(B2)forESI+,and0.02%
increasedto20mLaddingwatertoreducetheconcentration (v/v) ammonium hydroxide in water (A3) and 0.02% (v/v)
oforganicsolventforthesubsequentpurificationstep. ammoniumhydroxideinacetonitrile(B3)forESI−.Thedif-
ferentgradientsoptimizedforchromatographicseparations
2.3.3.PurificationofSteroids. Afterhydrolysis,sampleswere arereportedinTable2.Theflowrateforthedifferentgradient
−1
cooled at room temperature and the solution was loaded elutionconditionswassetto0.250mLmin andtheinjected
ontoHLBSPEcartridges(200mg,6mL)previouslyactivated volumewas10𝜇L.
4 JournalofAnalyticalMethodsinChemistry
Table1:Instrumentalsettingsoptimizedfortheanalyzedcompounds.
Steroid Ionizationmode Retentiontime(min) SRMtransition(Q1/Q3) Declusteringpotential Collisionenergy
271.1/213.3 60 25
DHEA APCI+ 4.9
271.1/81.2 60 43
D2-DHEA APCI+ 4.9 273.1/213.1 60 24
273.1/123.1 70 28
DHT APCI+ 5.9
291.1/255.1 70 19
D3-DHT APCI+ 5.8 276.1/123.1 70 33
273.1/215.1 70 24
ETIO APCI+ 6.7
273.1/159.0 70 31
17𝛼-TBOH ESI+ 6.5 271.1/253.4 70 31
271.1/199.3 70 36
17𝛽-TBOH ESI+ 5.7 271.1/199.3 70 36
271.1/227.3 70 31
D3-17𝛽-TBOH ESI+ 5.6 274.1/199.1 70 35
17𝛼-T ESI+ 9.7 289.2/109.2 60 35
289.2/97.1 60 35
17𝛽-T ESI+ 8.6 289.2/97.1 60 35
289.2/109.2 60 35
D2-17𝛽-T ESI+ 8.5 291.2/99.0 60 35
287.2/97.4 60 34
AED ESI+ 9.5
287.2/109.1 60 36
D7-AED ESI+ 9.4 294.1/100.1 60 35
17𝛼-E2 ESI− 4.9 271.0/145.0 −100 −54
271.0/239.0 −100 −53
17𝛽-E2 ESI− 4.4 271.0/183.0 −100 −54
271.0/145.0 −100 −54
D4-17𝛽-E2 ESI− 4.4 275.0/145.0 −100 −75
E1 ESI− 5.4 269.0/144.9 −100 −55
269.0/142.9 −100 −75
D4-E1 ESI− 5.3 273.0/144.9 −100 −75
2.6. Data Processing and Statistical Analysis. Steroids quan- 𝑝 value < 0.05 considered as statistically significant. In
tificationwasperformedusingMultiQuantsoftwareversion addition a statistical analysis based on a logistic regression
2.1 (AB SCIEX, Framingham, MA, USA) integrating the model was performed using the statistical software R 2.13.2
area corresponding to the chromatographic peak of each (theRFoundationforStatisticalComputing,2011).Dataon
hormoneandthecorrespondinginternalstandard.Theuri- endogenoussteroidsprofilesofthetwogroupsmeasuredat
nary concentration of exogenous steroids (17𝛼-TBOH, 17𝛽- times 𝑇0, 𝑇35, and 𝑇68 (before, in the middle, and at the
TBOH,and17𝛽-E2)wascalculatedusingamatrix-matched end of the treatment) using solvolysis and deconjugation
calibrationcurveconstructedinurine,whileforendogenous with 𝛽-glucuronidase from E. coli were first standardized
steroids (E1, 17𝛼-E2, DHT, DHEA, AED, 17𝛽-T, 17𝛼-T, and by transforming data to have zero mean and unit variance.
ETIO)thecurvewasconstructedinurinepreviouslytreated Thenstandardizedendogenoussteroidprofileswereusedas
with charcoal to remove endogenous compounds physio- predictorstoinfertheprobabilityofbelongingtothetreated
logically present. In each batch of analysis the curve was group,usingalogittransformoftheprobability.Finally,the
injected at the beginning of the sequence and two fortified predictive ability of the model was assessed computing the
samplesattheendforqualitycontrolpurposes.Samplesthat ROCcurve.
were fortified with exogenous substances were prepared in
urine, while the fortified samples for endogenous steroids 3.ResultsandDiscussion
werepreparedinurinedepletedwithcharcoal.
Steroidconcentrationsarereportedasmean±standard 3.1. Selected Treatment. The majority of the studies dealing
error of the mean (SEM). The methods used for hormone with sexual steroids administration for growth promotion
quantification were validated according to 2002/657/EC were performed using a combination of androgens and
CommissionDecision. estrogens administered by single or multiple injections [28,
The difference in measured variables between C and T 42–44].Giventhelackofofficialinformationregardingthe
groups was assessed using Student’s unpaired 𝑡-test, with illicit practices employed in livestock production, different
JournalofAnalyticalMethodsinChemistry 5
Table2:ChromatographicconditionsadoptedforthedifferentLC- days, and, around that time, the remaining coated pellets
MS/MSanalyticalmethods. begin to deliver their content into the bloodstream for
up to 200 days. In this study, for practical and economic
(a) reasons, the treatment could last for 71 days, after which
APCI+ A1(%) B1(%) animals were sacrificed, preventing us from performing a
drugexcretionprofilingovertheremainingca.130days,and
Time(min) H2O ACN
a following desirable depletion study. Still, achieved results
0.00 70.0 30.0
offeracleardatasetofurinaryconcentrationsofprecursors
1.00 70.0 30.0
and metabolites during a treatment mimicking an illicit
1.01 62.5 37.5
administration for anabolic purposes, typically lasting few
6.00 50.0 50.0
weeks. The aim was to provide data regarding the urinary
6.50 10.0 90.0 concentration of the administered drugs (17𝛽-TBOH and
7.50 10.0 90.0 17𝛽-E2) and their main metabolites (17𝛼-TBOH and 17𝛼-
8.00 70.0 30.0 E2) to help understanding the expected residual levels in
10.50 70.0 30.0 case of illicit employment of such formulation. Therefore,
theextraction/purificationprotocolwasadjustedspecifically
(b)
forthese4molecules.Moreover,theresultingprocedurewas
ESI+ A2(%) B2(%) slightlymodifiedinthehydrolysisstep,forthedetermination
Time(min) H2O+0.02%NH4OH ACN/MeOH(70/30) ofother7endogenoussteroidsthatwereanalyzedbefore,in
0.00 70.0 30.0 the middle, and at the end of the trial to evaluate possible
1.00 70.0 30.0 alterations of the pattern of endogenous steroids due to
the anabolic treatment. The efficacy of the selected implant
1.01 65.0 35.0
wasverifiedcalculatingthefeedconversionindexofcontrol
7.00 62.5 37.5
(9.54) and treated animals (8.51), attesting for increased
7.01 25.0 75.0
performancesaftertreatment.
8.50 25.0 75.0
8.75 10.0 90.0
3.2. Analytical Methods Development. Preliminary experi-
9.75 10.0 90.0 mentswereperformedtoselecttheconditionsforthetriple
10.25 70.0 30.0 quadrupole analyzer (API 4000, AB SCIEX, Framingham,
12.75 70.0 30.0 MA,USA)andthemobilephasecomposition,byindividually
infusing the steroid standards. Acetonitrile with water and
(c)
methanolwithwater,inpresenceofdifferentmodifierssuch
ESI− A3(%) B3(%) as0.1%formicacid,or0.02%ammoniumhydroxide,or5mM
Time(min) H2O+0.02%NH4OH ACN+0.02%NH4OH ammoniumacetate,bothinESI+andinESI−werecompared.
0.00 70.0 30.0 Acetonitrile with water in presence of 0.02% ammonium
1.00 70.0 30.0 hydroxide gave the best result in terms of signal intensity,
1.01 62.5 37.5 but,toachievethedesiredperformances,differentionization
techniqueswerecombined.ESI−waschosenfortheanalysis
6.00 50.0 50.0
ofestrogens;positiveionizationmodewasselectedtodetect
6.50 10.0 90.0
androgens, most of which were analyzed in ESI+, whereas
7.50 10.0 90.0
APCI+ source was chosen in the case of DHEA, DHT,
8.00 70.0 30.0
and ETIO. The latter molecules are characterized by the
10.50 70.0 30.0
presence of a keto-group in position 17 and the lack of
proximal electron-donor moieties. Thus, to achieve higher
signalintensityandlowerinterference,theAPCIsourcewas
drug administration schedules have been tested [19, 35]. used as previously reported in the literature [25, 45]. Using
Nonetheless,avarietyofvariablessuch asthechoiceofthe suchionizationmode,thepresenceofammoniumhydroxide
compounds, dosage, medium for drug resuspension, route, negatively affected the signal intensity; thus three separate
duration,andtimingofadministrationcannegativelyaffect chromatographic and ionization conditions combined with
a well-designed trial. For the mentioned reasons, in our acommonpurificationprocedurewereused.
study, animals were treated using a device named Revalor- During the method development, the best purification
XS, because it is a commercial formulation regularly used conditions were determined comparing C18 and HLB SPE
in the USA and could therefore be easily available on the cartridgescombinedwithNH2 cartridges,toreduceatmost
market; furthermore the usage and the effectiveness on the the matrix effect and ensure a satisfactory recovery for the
feed conversion index are known. This ear implant device analyzed molecules. C18 (500mg, 6mL) and HLB (200mg,
is made of coated and uncoated pellets containing 200mg 6mL)SPEweretestedinparallel:SPEactivationconditions
of TBA and 40mg of E2 that are continuously released. and loading volumes of sample extracts were the same
Accordingtothemanufacturers’instructions,fouruncoated for both of them. Different percentages of methanol/water
pellets can immediately release both steroids for about 70 (25/75, 50/50, and 75/25, v/v) solutions were tested for
6 JournalofAnalyticalMethodsinChemistry
17𝛼-Trenbolone
1.5e5 9.0e4 17𝛼-Trenbolone
271.1>253.1
Interfering
Intensity (cps) 01..50ee55 peak Intensity (cps) 36..00ee44 271.1>253.1 Inteprefaekring
0.0e0 0.0e0
4.0 5.0 6.0 7.0 4.0 5.0 6.0 7.0
Time (min) Time (min)
(a) (b)
Figure 1: Representative SRM chromatograms for 17𝛼-TBOH. (a) shows the most intense transition (271.1 > 253.1) monitored using
organicmobilephasenotcontainingmethanol,while(b)showsthesametransitionacquiredinpresenceofmethanolintheorganicphase
(acetonitrile/methanol70/30,v/v).InbothSRMchromatogramsthepeakof17𝛼-TBOHisintegratedandtheinterferingpeakphysiologically
presentinbovineurineisindicated.
the washing step. An acceptable compromise between drug samples and spiked ones. Using acetonitrile and water with
recovery and removal of interfering compounds was found 0.02%ammoniumhydroxideasmobilephase,apeakshow-
whencouplingHLBtoNH2cartridgesusingmethanol/water ing the same transitions of 17𝛼-TBOH (271.1 > 253.1, 271.1
(50/50, v/v) mixture: when higher percentage of methanol > 199.1) was observed around the same retention time in
waschosen,recoveriesofmorepolarcompoundsworsened. blank urine. Different gradient elution and isocratic con-
Similarly, also different solutions of elution were tested in ditions were tested and compared to achieve a separation
parallelonbothSPE:bestrecoverieswereachievedwiththe of the interfering peak from 17𝛼-TBOH. Finally, these two
proposedcombinationofmethanolandethylacetate. different compounds were successfully separated using a
For deconjugation of steroids glucuronides and sulfates slow gradient (Table 2) and a mobile phase composed of
we used two different techniques. Helix pomatia juice con- acetonitrile/methanol (70/30, v/v) without the ammonium
tainingarylsulphataseandHelixpomatiajuicecontaining𝛽- hydroxide that was maintained only in the aqueous solu-
glucuronidase that are commonly used in many screening tion. By means of this organic mobile phase the use of
and confirmatory methods [8, 46] were employed for the immunoaffinitypurificationwasnotrequired(Figure1).
detection of the administered sexual steroids and their
epimers. The choice of using two Helix pomatia juices with 3.3. Validation Study. The reported LC-MS/MS methods
specificactivitytowardssulfatedandglucuronatedconjugates were developed and validated according to Commission
is necessary to quantify the total concentration of steroids, Decision 2002/657/EC, properly adapted for endogenous
withoutneedtoanalyzeeachsampleforsteroidscontentboth steroids.
intheirfreeformandasconjugates. Thespecificityofthemethodswasverifiedanalyzing20
Fortheanalysisofendogenoussteroids,acombinationof blankbovineurinesamplesfortheexogenoussubstancesand
𝛽-glucuronidaseproducedinE.colifollowedbymethanolysis 20 blank charcoal-depleted urine samples for the endoge-
inacidicconditions[47]wasused.Indeed,previousstudies nous compounds. No interfering peaks were found at the
demonstratedthattheuseofHelixpomatiajuicecanproduce expectedretentiontimeofthetargetanalytes.Inparticular,
misleading results due to the presence of other enzymes an endogenous peak showing the same transitions as 17𝛼-
that are responsible for steroids conversion [48, 49] that TBOHbutwithanalteredionratiowasfoundtobepresent.
couldaffectthecorrectdeterminationof17𝛼-T,AED,DHEA, This peak did not interfere with the quantification of 17𝛼-
DHT, and ETIO. This different approach was used for the TBOHresiduesbecausethechromatographicseparationwas
secondary goal of performing multivariate analyses based acceptable (Figure 1). Linearityof the response was verified
on the urinary profile of endogenous steroids to develop with 5 calibration levels, including a blank urine sample,
a probabilistic model able to discriminate treated animals usingalackoffittest.Thecurvewasinjectedatthebeginning
fromnontreatedones. Accordingly, urinesamples collected ofeachbatchofanalysis.Independentcalibrationcurveswere
atthebeginning,inthemiddle,andattheendoftheinvivo built in each of the three separate working sessions, during
trial (i.e., 𝑇0, 𝑇35, and 𝑇68) were also processed using this methodvalidationperformedonfortifiedsamples(Table3).
alternativedeconjugationmethod,tospecificallyquantifyE1, The correlation coefficients (Pearson 𝑅) of the standard
17𝛽-T,17𝛼-T,AED,DHEA,DHT,andETIO. curveswerefoundtobebetterthan0.996.Thestandardcali-
The selection of the chromatographic conditions was brationcurveswerepreparedinurineforthequantificationof
performed comparing the SRM spectra from blank urine exogenoussubstancesandincharcoal-depletedurineforthe
JournalofAnalyticalMethodsinChemistry 7
Table3:Validationparametersinurine.Foreachvalidationlevel,18fortifiedsampleswereanalyzedandusedtocalculateCC𝛼,CC𝛽,mean
recovery,andCV%.
(a)
17𝛼-TBOH 17𝛽-TBOH 17𝛼-T 17𝛽-T
−1
Validationlevel(ngmL ) 0.5 0.75 1.0 2.5 0.5 0.75 1.0 2.5 2.5 5.0 20.0 35.0 1.0 2.0 8.0 14.0
−1
Calibrationlevel(ngmL ) 0.3 1.0 2.0 3.0 0.3 1.0 2.0 3.0 3.0 20.0 40.0 60.0 1.2 8.0 16.0 24.0
Meanrecovery(%) 110.0 108.1 105.0 104.2 102.8 102.6 101.2 104.3 97.9 98.0 98.2 99.6 103.7 92.8 99.6 100.6
Intermediateprecision(CV%) 6.8 6.8 8.5 9.4 4.9 4.5 7.2 7.1 7.6 8.0 9.3 8.4 4.6 7.5 3.6 3.7
CC𝛼(ngmL−1) 0.23 0.17 1.42 0.55
CC𝛽(ngmL−1) 0.39 0.29 2.41 0.93
(b)
AED DHEA DHT ETIO
−1
Validationlevel(ngmL ) 0.3 0.5 1.0 1.5 2.0 5.0 10.0 15.0 2.0 5.0 10.0 15.0 20.0 50.0 100.0 150.0
−1
Calibrationlevel(ngmL ) 0.2 1.0 2.0 3.0 3.0 10.0 20.0 30.0 3.0 10.0 20.0 30.0 30.0 100.0 200.0 300.0
Meanrecovery(%) 101.7 88.4 93.6 94.1 109.8 99.9 97.5 106.6 94.1 95.8 97.4 100.4 101.1 81.6 91.2 104.1
Intermediateprecision(CV%) 3.1 7.6 3.4 4.8 12.3 14.1 13.5 11.9 12.4 7.7 6.1 6.6 2.7 6.9 6.5 11.2
CC𝛼(ngmL−1) 0.08 2.29 1.09 18.62
CC𝛽(ngmL−1) 0.14 2.51 1.85 31.68
(c)
17𝛼-E2 17𝛽-E2 E1
−1
Validationlevel(ngmL ) 0.5 1.0 1.5 2.5 0.5 0.75 1.0 2.5 0.1 0.4 0.7 1.0
−1
Calibrationlevel(ngmL ) 0.3 1.0 2.0 3.0 0.3 1.0 2.0 3.0 0.06 0.4 0.8 1.2
Meanrecovery(%) 91.4 96.9 99.1 101.2 98.2 97.7 100.9 103.3 91.0 97.5 98.6 96.4
Intermediateprecision(CV%) 8.0 6.1 4.0 4.8 11.6 7.4 6.5 5.7 9.7 4.4 4.0 4.2
CC𝛼(ngmL−1) 0.13 0.15 0.05
CC𝛽(ngmL−1) 0.22 0.26 0.08
endogenous steroids. Representative SRM chromatograms Decision limit (CC𝛼) and detection capability (CC𝛽)
for target drug residues and their main metabolites at the were calculated according to Commission Decision 2002/
lowest calibration curve level are shown in Figure 2. The 657/EC(forforbiddensubstances)bytheISO11843calibra-
SRM chromatograms of the other analyzed compounds are tion curve procedure following the approach proposed by
reportedinFigure3. Verdon et al. and by Lega et al. [50, 51]. The CC𝛼 values
During method validation for the exogenous molecules together with the CC𝛽 for the investigated molecules are
(17𝛼-TBOH, 17𝛽-TBOH, and 17𝛽-E2), seven blank urine reportedinTable3.
samplesfortifiedwiththesteroidsatfourconcentrationlevels Six blank urine samples were fortified at the calculated
(Table3)werepreparedandanalyzedinthreedifferentwork- CC𝛼with17𝛼-TBOH,17𝛽-TBOH,and17𝛽-E2andanalyzed
ing sessions by two different operators. The same approach to verify the reliability of the estimated decision limits.
was applied for the endogenous steroids (17𝛼-E2, E1, 17𝛽- In all samples each compound was correctly detected and
T, 17𝛼-T, AED, DHEA, DHT, and ETIO), using charcoal- quantified,attestingforacceptablemethodperformanceeven
depleted urine. The validation range for endogenous com- belowextrapolatedCC𝛽.
poundswaspreliminarilydefinedanalyzing10urinesamples Youdentestwasadoptedtoverifymethodruggednessto
collected from bulls under field conditions and taking into minorchanges[52];sevenvariables(i.e.,pH,deconjugation
account instrument sensitivity. For all the molecules under timeandtemperature,washingsolutioncompositionduring
investigation the recovery was corrected by a deuterated SPEpurification,elutionsolventcomposition,mobilephase
internal standard. ETIO was quantified using D2-DHEA composition, and evaporation temperature) were modified
to correct for recovery. The deuterated-𝛽-isomer form of with respect to the prescribed procedure to evaluate meth-
a given steroid was used to correct for both the 𝛼- and ods ruggedness to minor changes. The quantitative results
the 𝛽-epimer. Because 𝛼-isomers and deuterated-𝛽-isomers were not significantly different compared to those obtained
are not eluted at the same retention time, there may be employing the standard conditions, proving that limited
no compensation of the matrix effects. In these cases, our variations (±5%) of the chosen variables do not affect the
results showed an acceptable recovery ranging from 81.6% methodperformance.
for ETIO to 110.0% for 17𝛼-TBOH, displaying an interme-
diate precision (CV%) lower than 15% for all the tested 3.4. Urinary Steroids Profile. To draw the excretion profile
molecules. during the treatment period (Figures 4 and 5), all 32 urine
8 JournalofAnalyticalMethodsinChemistry
17𝛼-Trenbolone 17𝛽-Trenbolone
100 100
%) %)
Blank urine nal intensity ( 50 271.1>253.4 nal intensity ( 50 271.1>199.3
g g 5.7
Si Si
6.5
0 0
4.0 5.5 7.0 4.0 5.5 7.0
Time (min) Time (min)
17𝛼-Estradiol 17𝛽-Estradiol
100 100
%) %)
Blank urine nal intensity ( 50 271.0>145.0 nal intensity ( 50 271.0>183.0
Sig Sig 4.4
4.9
0 0
3.0 4.5 6.0 3.0 4.5 6.0
Time (min) Time (min)
(a)
17𝛼-Trenbolone 17𝛽-Trenbolone
100 100 5.7
%) %)
e y ( y (
Fortified urin Signal intensit 50 271.1>253.4 6.5 Signal intensit 50 271.1>199.3
0 0
4.0 5.5 7.0 4.0 5.5 7.0
Time (min) Time (min)
17𝛼-Estradiol 17𝛽-Estradiol
100 4.9 100 4.4
%) %)
ed urine ntensity ( 50 271.0>145.0 ntensity ( 50 271.0>183.0
Fortifi Signal i Signal i
0 0
3.0 4.5 6.0 3.0 4.5 6.0
Time (min) Time (min)
(b)
Figure2:SRMchromatogramsofthetargetdrugresiduesandtheirmainmetabolites.In(a)ablankurinesampleisreported,andin(b)the
samesamplespikedatthelowestlevelofthecalibrationcurveconstructedinurinefortheexogenousmoleculesandincharcoal-depleted
urinefor17𝛼-E2.
JournalofAnalyticalMethodsinChemistry 9
17𝛼-Testosterone Dehydroepiandrosterone
9.7 4.9
100 100
%) %)
y ( y (
nsit 50 nsit 50
nte 289.2>109.2 nte 271.1>213.3
al i al i
n n
g g
Si Si
0 0
7.0 9.0 11.0 2.25 3.75 5.25
Time (min) Time (min)
Dihydrotestosterone 17𝛽-Testosterone
100 100
%) %)
y ( y (
nsit 50 nsit 50 289.2>97.1
nte 273.1>123.1 nte 8.6
al i al i
n n
Sig 5.9 Sig
0 0
5.5 7.0 8.5 7.0 9.0 11.0
Time (min) Time (min)
D2-Dehydroepiandrosterone D3-Dihydrotestosterone
4.9 5.8
100 100
%) %)
y ( y (
nsit 50 nsit 50
nte 273.1>213.1 nte 276.1>123.1
al i al i
n n
g g
Si Si
0 0
2.25 3.75 5.25 5.5 7.0 8.5
Time (min) Time (min)
D2-17𝛽-Testosterone Estrone
8.5 5.4
100 100
%) %)
Signal intensity ( 50 291.2>99.0 Signal intensity ( 50 269.0>144.9
0 0
7.0 9.0 11.0 4.0 5.5 7.0
Time (min) Time (min)
(a)
Figure3:Continued.
10 JournalofAnalyticalMethodsinChemistry
Androstenedione Etiocholanolone
100 9.5 100 6.7
%) %)
y ( y (
ensit 50 ensit 50
nt nt 273.1>215.1
al i 287.2>97.4 al i
n n
g g
Si Si
0 0
7.0 9.0 11.0 5.5 7.0 8.5
Time (min) Time (min)
D4-Estrone D7-Androstenedione
100 5.3 100 9.4
%) %)
y ( y (
ensit 50 273.0>144.9 ensit 50
nt nt 294.1>100.1
al i al i
n n
g g
Si Si
0 0
4.0 5.5 7.0 7.0 9.0 11.0
Time (min) Time (min)
(b)
Figure3:SRMchromatogramsofthetargetsteroidsspikedatthelowestlevelofthecalibrationcurveconstructedincharcoal-depletedurine.
Theretentiontimeandmonitoredtransitionsarereportedforeachanalyte.
samples at each sampling day were analyzed in duplicate. 17𝛼-TBOH and 17𝛽-TBOH were found to be present in
Mean concentration values and standard errors are sum- urinestartingfromtheinitialstageofthetrial.Theyalready
marized in Table 4. For such purpose, we included in the reachedtheirmaximumconcentrationonthefirstsampling
calculations all the analytical values above CC𝛼: for those point,displayingurinarylevelsthatdecreasedslowlyduring
below the decision limit, a zero value was assigned. When thefollowing63daysoftreatment.At𝑇63,theconcentration
calculatedmeanconcentrationswerebelowCC𝛽,theywere was almost halved compared to 𝑇7. This time point is very
reported in italic for reader information. Data were not close to the moment in which the uncoated part of the ear
normalizedtocreatinineorspecificdensity,suchapproaches implant is supposed to terminate the drug release, while
being not yet fully accepted as reference standards and the coated part should begin its degradation allowing the
consideringthegoodprecisionofcollecteddata. delayedreleaseofitscontent(accordingtothemanufacturer’s
Theurinaryexcretionprofileof17𝛼-TBOH,17𝛽-TBOH, instructions). This fact is confirmed by data collected at
17𝛼-E2, and 17𝛽-E2 was monitored during the entire trial 𝑇68 which showed a slight increase in 17𝛼-TBOH and 17𝛽-
(Figure 4). No drug residues of the exogenous compounds TBOHlevelandbythesimilarbehaviorobservedfor17𝛽-E2
were detectable in urine from control animals. Previous between 𝑇56 and 𝑇63 (Figure 4). On the other hand 17𝛽-E2
research studies conducted to reproduce putative illicit vet- behavesinadifferentwaywithrespecttoTBOH,especially
erinary practices were performed administering anabolic at the beginning of the trial (𝑇7), where all the treated
steroids by intramuscular injection [28, 42–44]. In such samplesdisplayconcentrationswellbelowtheCC𝛼.Indeed
conditionshighconcentrationsoftargetcompoundsortheir its presence can only be indirectly hypothesized thanks to
metabolites were recorded immediately after injection and thecomparisonoftheurinaryconcentrationofitsmetabolite
were rapidly excreted within few days requiring repeated 17𝛼-E2thatissignificantlyhigherinTsampleswithrespect
injections to achieve the anabolic effect. Conversely, the to C samples at 𝑇7 (Figure 4). Conversely, from 𝑇21, 17𝛽-
ear implant used in the present study slowly released its E2concentrationshowedaslowincreaseuntil𝑇49.Itisalso
content, showing low residual urinary concentration of important to note that, at each sampling point throughout
17𝛽-TBOH and 17𝛽-E2 throughout the whole treatment the trial, one-third of the treated samples showed 17𝛽-E2
period. The excretion profile is significantly different from concentrationbelowthecalculatedCC𝛼,confirmingthatthe
the typical excretion kinetic observed after intramuscular tested device may reproduce a putative anabolic treatment
injection. basedonslowreleaseofsteroids.