Detection, quantification and confirmation of anabolic steroids in

13 Pages·2005·0.67 MB·English

JournalofChromatographyB,829(2005)56–68
Detection, quantification and confirmation of anabolic steroids
in equine plasma by liquid chromatography
and tandem mass spectrometry
Fuyu Guana, Cornelius E. Uboha,b,∗, Lawrence R. Somaa, Yi Luoa,
Jeffery Rudyb, Thomas Tobinc
aUniversityofPennsylvania,SchoolofVeterinaryMedicine,DepartmentofClinicalStudies,NewBoltonCenterCampus,
382WestStreetRoad,KennettSquare,PA19348,USA
bPAEquineToxicologyandResearchCenter,DepartmentofChemistry,WestChesterUniversity,
220EastRosedaleAvenue,WestChester,PA19382,USA
cMaxwellH.GluckEquineResearchCenterandDepartmentofVeterinaryScience,
UniversityofKentucky,Lexington,KY40546,USA
Received5August2005;accepted23September2005
Availableonline10November2005
Abstract
Anabolicandrogenicsteroidsarerelatedtothemalesexhormonesandareabusedinequinesports.Inanefforttodetertheabuseofanabolic
steroids,asensitiveLC–MS/MSmethodwasdevelopedfordetection,quantificationandconfirmationofeightmajoranabolicsteroids(testosterone,
normethandrolone,nandrolone,boldenone,methandrostenolone,tetrahydrogestrinone(THG),trenbolone,andstanozolol)inequineplasma.For-
mationofsolventadductionsoftheanalyteswasobservedunderelectrosprayionization(ESI)conditions,anddesolvationofthesolventadductions
bysourcecollision-induceddecomposition(CID)increasedtheabundanceofthe[M+H]+ionsaswellasthemultiple-reactionmonitoring(MRM)
signals.ESI(+)andAPCI(+)werecomparedwithrespecttosensitivityfortheanalytesandtheformerprovidedbettersensitivity.Thematrix
effectonionsuppressionorenhancementwasevaluated,andwasnegligible.Confirmationoftheanalyteswasperformedusingcriteriaofthreeion
transitionsandLCretentiontimeofeachanalyte.Thelimitofdetection(LOD)andquantification(LOQ)was25pg/mL.Thelimitofconfirmation
(LOC)was25pg/mLforboldenone;50pg/mLfornormethandrolone,nandrolone,andmethandrostenolone;and100pg/mLfortestosterone,THG,
trenbolone,andstanozolol.Theanalyteswereevaluatedforstabilityandfoundtobestableinplasmafor24hatroomtemperature,13daysat
4◦C,and34daysat−20and−70◦C.Themethodwassuccessfullyappliedtoanalysesofequineplasmasamplesforpharmacokineticsstudy.
Thismethodissensitiveandusefulfordetection,quantificationandconfirmationoftheseanabolicsteroidsinequineplasma.
©2005ElsevierB.V.Allrightsreserved.
Keywords: Anabolicsteroid;LC–MS;Plasma;Horse;Dopingcontrol;THG;Testosterone;Boldenone
1. Introduction bythelatestreportsonsteroidabuse[5,6]andtherecentdiscov-
eryof“designer”steroids,tetrahydrogestrinone(THG)[7]and
Anabolicandrogenicsteroidsaresyntheticsubstancesrelated norbolethone [8]. The use of these agents in sports is prohib-
to the male sex hormones (androgens). These agents promote itedbytheInternationalOlympicCommitteeviaitsanti-doping
growthofskeletalmuscle(anaboliceffects)anddevelopmentof arm,theWorldAnti-DopingAgency[9].Theyarealsoprohib-
malesexualcharacteristics(androgeniceffects).Theyareused ited in the horseracing industry by the Association of Racing
intreatingdelayedpuberty,selectimpotence,andwastingofthe Commissioners International [10]. To enforce the prohibition
bodycausedbysomediseases[1].However,anabolicsteroids onanabolicsteroidabuse,effectivemonitoring,detection,iden-
canbeabusedinhumanandanimalsports[2–4],asevidenced tificationandconfirmationmethodsarerequired.Forscreening
andconfirmationofanabolicsteroids,GC–MSisthetechnique
commonlyusedfortheanalysisofurinesamplesbyanti-doping
∗ Correspondingauthor.Tel.:[phone];fax:[phone].
oranalyticaltoxicologylaboratories[8,11–15].
E-mailaddress:[email protected](C.E.Uboh).
1570-0232/$–seefrontmatter©2005ElsevierB.V.Allrightsreserved.
doi:10.1016/j.jchromb.2005.09.045
F.Guanetal./J.Chromatogr.B829(2005)56–68 57
Fig.1. Chemicalstructuresofnineanabolicsteroidsusedinthestudy.
With the advance of LC–MS technology, the emphasis has Stock solution (1.0mg/mL) of all the steroids, and
shifted in favor of this new technology. There are reports of methenolone(internalstandard)wasindividuallypreparedfrom
LC–MS methods for quantitative determination of anabolic drychemicalpowderofeachanalytestandardbydissolvingin
◦
steroids such as testosterone, nandrolone, and trenbolone in methanol,andstoredat4 C.Anintermediatesolutionofmix-
humanorbovineplasmaorserum[16–19],inbovineorequineor ture of the eight analytes each at 10(cid:1)g/mL was prepared by
humanurine[20–28]andinhumansaliva[29–31].Toourknowl- adding 50(cid:1)L of each stock solution (1.0mg/mL) to 4600(cid:1)L
edge,thereisnoreportonLC–MSmethodsforqualitativeand ofmethanolina7mLvial.Workingsolutionsoftheeightana-
quantitativedeterminationofanabolicsteroidsinequineplasma. lytes mixed at 100, 10 and 1.0ng/mL were prepared from the
ThepurposeofthispaperwastodevelopasensitiveLC–MS/MS intermediate mixture solution of the analytes by dilution with
◦
methodforscreening,quantificationandconfirmationofeight methanol,andstoredat4 C.
major anabolic steroids (testosterone, normethandrolone, nan- Stockformatebuffercomprising1.0mol/mLofammonium
drolone,boldenone,methandrostenolone,tetrahydrogestrinone formateand1.0mol/mLofformicacidwaspreparedfromthe
(THG), trenbolone, and stanozolol, the chemical structures of drychemicalpowderandconcentratedformicacid,andthepHof
whichareshowninFig.1)inequineplasma.Theseagentswere thebufferwas3.4.Workingformatebuffersolutioncontaining
chosenforthisstudybasedontheirpotentialforabuseinrace- 2mmol/L of ammonium formate and formic acid, used as LC
horses. mobilephase,waspreparedbydilutingthestockformatebuffer
withHPLCgradewater.
2. Experimental
2.2. Drugadministrationandsamplecollection
2.1. Chemicalsandreagents
Female and castrated male horses ranging in age from 4 to
All the steroids in this study, except THG, were purchased 10 years with an average weight of 550±49kg were used in
fromSteraloids(Newport,RhodeIsland,USA).THGwassyn- thestudy.TheUniversityofPennsylvaniaInstitutionalAnimal
thesized at the Maxwell H. Gluck Equine Research Center, CareandUseCommitteeapprovedthestudyprotocol.Asingle
UniversityofKentucky,Lexington,USA[32].Ammoniumfor- dose(1.1mg/kg)ofboldenoneundecylenate(FortDodge,Madi-
mate (Certified), HPLC grade methanol and water and methyl son,NJ,USA)wasintramuscularlyadministeredtoeachofsix
tert-butyl ether (MTBE) were obtained from Fisher Scientific horses.Bloodsampleswerecollectedbeforedrugadministration
(Pittsburgh, Pennsylvania, USA). Formic acid was purchased andatvariousintervalspost-drugadministration.Thesamples
fromEMScience(Gibbstown,NewJersey,USA). werecentrifuged(2500–3000rpmor776–1318×g)at4◦Cfor
58 F.Guanetal./J.Chromatogr.B829(2005)56–68
15mintoobtainplasma.Theplasmasamplesweretransferred tures (25, 4, −20 and −70◦C) for evaluation of stability of
intoseparatetubes,frozenandstoredat−70◦Cpendinganal- theanalytes.Thesetemperatureswerechosenbasedonsample
ysis. A single dose (1.1mg/kg) of nandrolone decanoate (Fort collection,handling,shipmentandstorageconditionstowhich
Dodge, Madison, NJ, USA) was intramuscularly administered the samples could be exposed before and after receipt at the
to a horse, and plasma samples were collected and similarly laboratory.Toassessstabilityoftheanalytesatambienttemper-
◦
treated. THG (15mg) was intramuscularly administered to a ature (25 C), the plasma samples in test tubes were allowed
horse(539kg),andplasmasampleswerecollectedandtreated to remain on benchtop for 2, 4, 6 and 24h, spiked with IS
asdescribedabove. (1000pg), extracted, and analyzed. For assessment of the sta-
bilityatothertemperatures,theplasmasamplescontainingthe
2.3. Preparationofplasmasamples analyteswerestoredat4◦Cfor13days,andat−20and−70◦C
for34days,thawed,broughttoambienttemperature,spikedwith
Control equine plasma used in this study was collected IS(1000pg),extracted,andanalyzedatdifferenttimeintervals.
from drug-free mares, and was determined to be free of the Themeanconcentrationofeachanalytefromtriplicatesamples
analytes, using the same LC–MS/MRM method described in ateachtemperatureandtimepointwasdetermined.
this paper. Plasma calibrators for internal calibration quan-
tification were prepared by spiking plasma aliquots of 1mL 2.7. Instrumentationandoperatingparameters
each with the working solutions of the mixed eight analytes
and the internal standard, containing each analyte at 25, 50, SampleanalyseswereperformedbyLC–MS(ThermoElec-
100, 250, 500, 1000, 2500, 5000 and 10,000pg/mL, and IS at tron Corp., San Jose, CA, USA) consisting of a Surveyor LC
1000pg/mL. Plasma calibrators were freshly prepared shortly pump with an on-line degasser, a Surveyor autosampler, and
beforeuse.Qualitycontrol(QC)plasmasamples(1.0mL)con- a TSQ Quantum triple stage quadrupole mass spectrometer
tainingISat1000pg/mLandtheeightanalytes,eachat50,500 equippedwithanelectrosprayionization(ESI)sourceoperated
and5000pg/mL,weresimilarlyprepared.Testplasmasamples inpositiveionmode.
(1.0mL) from drug administration or racehorses were spiked LC separations were performed on an Ace C column
8
withIS(1000pg). (2.1mm×50mm,5(cid:1)m)(Mac-ModAnalytical,ChaddsFord,
PA, USA) with an Ace C guard column (2.1mm×12.5mm)
8
2.4. Extractionoftheanalytesfromplasmasamples (Mac-Mod Analytical) that was at ambient temperature. LC
mobile phase gradient in both composition and flow rate
Analyteswereextractedfromplasmabyliquid–liquidextrac- (Table1)wasusedforresolutionoftheanalytes.
tion (LLE). To each of the prepared plasma samples, MTBE TSQQuantummassspectrometerwascalibratedwithasolu-
(5mL)wasaddedandgentlymixedbyrotorack(Thermolyne, tion of polytyrosine-1,3,6 according to the manufacturer. The
Dubuque, Iowa) for 5min and centrifuged at ∼3000rpm ESIsourceparameterswereoptimizedfornandroloneatLCflow
(∼1409×g) for 5min. The organic layer (top) was decanted of300(cid:1)L/minbasedonpreliminaryresultsindicatingthatnan-
into a fresh glass tube (16mm×100mm) and dried at 50◦C drolonewastheanalyteforwhichthemethodhadthelowestsen-
(Techne Dri-Block DB·3, Duxford, Cambridge, UK) under a sitivity.Theoptimizedsourceparameterswereasfollows:sheath
steady stream of nitrogen. The dried extract was reconstituted gaspressure,45psi;auxiliarygasflow,23(arbitraryunits);spray
in100(cid:1)Lof60%methanolinformatebuffer(2mmol/mL,pH voltage,4500V;capillarytemperature,300◦C;tubelensoffset,
3.4)anda20(cid:1)LaliquotwasanalyzedbyLC–MS. 178V;andsourcecollision-induceddecomposition(CID),20V.
For quantification, the mass spectrometer was set to the data
2.5. Intra-andinter-dayprecisionandaccuracy acquisitionmodeofmultiple-reactionmonitoring(MRM),and
thespecifiedacquisitionparametersareshowninTable2.The
Forevaluationofintra-dayprecisionandaccuracy,threesets acquisitionparameterscommontoallanalyteswere:scanwidth
ofsixduplicateplasmasamplescontainingIS(1000pg/mL)and (m/z)0.70,scantime0.4s,peakwidth(FWHM)0.7forbothQ1
theeightanalytesatthelow(50pg/mL),medium(500pg/mL) andQ3,andcollisiongaspressure1.5mTorr.Dataacquisition
andhigh(5000pg/mL)concentrationswereprepared,extracted,
and analyzed on the same day. As for inter-day precision and
Table1
accuracy,threesetsoftwoduplicateplasmasamplescontaining LCmobilephasegradientincompositionandflowrate
IS(1000pg/mL)andtheeightanalytesatthesamelow,medium
LCruntime Formate Methanol Flow rate
andhighconcentrationswereprepared,extracted,andanalyzed (min) buffera(%) (%) ((cid:1)L/min)
perdayfor3days(sixsamplesintotalforeachconcentration).
0 40 60 300
4.0 40 60 300
2.6. Evaluationofstabilityoftheanabolicsteroidsin
8.0 10 90 300
equineplasma 8.1 40 60 300
8.2 40 60 400
The eight anabolic steroids in equine plasma (1.0mL) at 10.4 40 60 400
10.5 40 60 300
threedifferentconcentrationsof50,500and5000pg/mLwere
prepared as described above, and stored at different tempera- a Ammoniumformate,2mM,pH3.4.
F.Guanetal./J.Chromatogr.B829(2005)56–68 59
5) 21, and analysis were accomplished with Xcalibur software v.1.3
ol 29(3 29,1 (ThermoElectronCorp.,SanJose,CA,USA).
Stanozol 7.89→3293 →3293107(40) 3. Resultsanddiscussion
3.1. ESI(+)MSspectraandsolventadductions
5) 41,
1 2
1( 6, Intheearlyphaseofthemethoddevelopment,sevenanabolic
4 6
→2 →215) steroids were studied with flow injection into the LC with a
THG 6.93313 313239( flow of 300(cid:1)L/min of methanol/HCOONH4 (2mmol/L, pH
3.4) (60/40, v/v). Testosterone (Fig. 2a), normethandrolone
7 (Fig. 2b), and nandrolone (Fig. 2c) formed [M+H]+ ions at
8
1 m/z 289, 289 and 275, respectively, as well as solvent adduct
→
IS 6.00303(15) Iionncso[nMtra+sHt,b+oCldHe3nOonHe]+(Fwigit.h2md)e,thmaentohlaantdmro/szt3e2n1o,lo3n2e1(aFnidg.320e7).
3 and stanozolol (Fig. 2g) formed abundant [M+H]+ ions at
handrolone 213(18) 253,231,21 (imoF/nizgs.2[8M27f),+3fHo0r1+mCaendHd3dO3o2mH9]i,n+raeansttpme[Mc/zti+v3e1Hl9y],,+3b3iuo3tnwanaetdakm36/sz1o.l2vT7er1netnwbaiodthldoounucett
met → → noticeable solvent adduct ion [M+H+CH OH]+ at m/z 303.
Nor 4.57289 289(18) The tendency of the analytes to form [M+3H]+ ion and sol-
ventadductionsisrelatedtotheirchemicalstructures(Fig.1).
6) 09, Forexample,trenbolonehasfourconjugateddoublebondsand
1 1
sterone →109( →253,6) bthoelsdeendoonuebalendbomnedtshasntadbroilsizteenothloen[eMha+vHe]t+hrieoenssufcohrmboenddsu,nadnedr
Testo 4.46289 28997(1 tbhaeseE)SthIatcosntadbitiiloizne.sSthtaeno[Mzo+loHl ]h+asioan.pHyorawzeovleer,ritnegsto(satewroenaek,
ne 21 normethandrolone and nandrolone have only two conjugated
o 1
ostenol 1(15) 3,149, idoonusb,leanbdonthdusst,hsaotlmveanyt naodtduscutffiicoinesnt[lMy s+taHbi+lizCeHt3hOeH[M]++wHe]r+e
dr 12 17 formed because solvent molecules can stabilize positive ions
an → →
Meth 3.92301 301(15) (eaxiwsteilnlaknhoywdrnateexdafmorpmle).iIsntshhaotrmt,ethtaelmioinnosridnifafqeureenocuesisnocluhteimon-
icalstructuresofthesevenanabolicsteroidsmentionedabove,
17) 145, specifically the number of conjugated double bonds, accounts
Nandrolone 3.39→275109( →275239,109(17) afodrdItunhceatddiodifniftseior[enMntc+oeHcinh+efomCrHimc3aaOltipoHrn]o+opfedrtethsieecsr[iMboefd+thHaeb]o+avnieoa.bnoslaicndstseorloviednst,
the organic solvent used in LC mobile phase also had an
5) 35, effect on formation of solvent adduct ions and, thus, affected
e 1(1 3,1 intensity of the [M+H]+ ions. Compared with methanol, ace-
n 2 7
Boldeno 2.99→2871 →2871121(15) titoorannisotrfwilteihtheshtaohnweaelaydntaeabsosaltcircqounsigtreeerrdoitdwesni,dthaesnaccryeevttoeonailfteordirlmebyinsothltvheeeMnLtSCadsfldpueoccw-t
uisitionoftheanalytes Trenbolone 2.98→271199(20) →271227,199,183(20) m(vnadFteoa/innzrgmmtt.a3/esz3d3ot)hd0l.2vau,8neUc3n9dtn3t,irloo0ian2klods8eand9[neumMdcaet(+nt3Fh[d1HiMag6n.2+,o+7lCr35,HebH.sa)+pc3,AeCeCctcaNoHtenint]v3do+ieCtnrlwNyiinlt,iaer]tin+hblfeduotiretorosamnltlnoosseonosdwtee[fdirMoto(ohrFnmm+iebgieHo(n.dFla]din+3geatc.nb)i3soouoannnal)es--t,
Table2ParametersforMRMacq Retentiontime(min)mzIontransition(/)forquantification(collisionenergy,V)mzIontransitions(/)forconfirmation(collisionenergy,V) (ataastieFtndcvrneiddoegdtulno.esycgntn.3taeTicdntriyrh)ooi,eplnzteomrost,oelfeniotaosotdnlhrnedmr(aeFnnal7cisadfgy.otfir9.eloonvd3esfieVgttateyn)ocntfaeotoattthhldoroamednnnmiui/retzcermpitt3(lhrFee2ioaoti8athgnno,na.osnd3nl3t4omhae[2l3af),e,fi,n3t3na]thmr)n1iae:td2nyenatobahc(tlonhPeatlAdutnooosonf3n=loe7i.hrt80Tarm(.i,sF2hlersiiehegosVhsi.lrgpvae3heesflfoncae)a--rrt,
60 F.Guanetal./J.Chromatogr.B829(2005)56–68
Fig.2. ESI(+)MSspectraoftestosterone(a),normethandrolone(b),nandrolone(c),boldenone(d),methandrostenolone(e),trenbolone(f),andstanozolol(g)showing
formationof[M+H]+ionsaswellassolventadduct[M+H+CH3OH]+ionswithmethanol.Spectraa–garefromtoptobottom.[M+H]+and[M+H+CH3OH]+
ionsarem/z289and321fortestosterone(a)andnormethandrolone(b),m/z275and307fornandrolone(c),m/z287and319forboldenone(d),m/z301and333
formethandrostenolone(e),m/z271fortrenbolone(f),andm/z329and361forstanozolol(g),respectively.Eachanabolicsteroid(10(cid:1)g/mL)wasintroducedby
syringeinfusionat20(cid:1)L/minintoLCmobilephase(CH3OH/HCOONH4,60/40,v/v)flow(300(cid:1)L/min)tothemassspectrometer.Thetemperatureoftheion
transfercapillarywas275◦C.
tionship is in agreement with Field’s rule [34]. Understanding CID was used for desolvation to improve sensitivity for the
oftherelationshipbetweenprotonaffinityofanorganicsolvent analytes.
anditstendencytoformsolventadductionsishelpfulinselect-
ingLCsolventforLC–MSstudiesofanalytesthatmaynotform 3.2. ESI(+)MS/MSspectra
stable[M+H]+ions.
Abundant [M+H]+ ions are always desired for sensitive, Product ion spectra of the anabolic steroids are shown in
qualitativeandquantitativemethods.Conversely,solventadduct Fig. 4. Testosterone and normethandrolone are different com-
ionsareundesirablesincetheydecreaseabundanceof[M+H]+ poundswiththesamemolecularformulaand[M+H]+ion(m/z
ions.Tominimizeformationofsolventadductionsinthisstudy, 289). However, their product ion spectra were different from
methanol was chosen over acetonitrile as solvent for the LC eachother(Fig.4aandb).Testosteronehadanabundantproduct
mobile phase. To desolvate the undesired solvent adduct ions ionatm/z97thatwasveryweakintheproductionspectrumof
[M+H+CH3OH]+, temperature of the ion transfer capillary, normethandrolone; normethandrolone had two abundant prod-
thedesolvationdeviceofthemassspectrometer,wasincreased uctionsatm/z231andm/z213thatwereveryweakintheprod-
◦
from 275 to 350 C. The increase in temperature reduced the uctionspectrumoftestosterone.Testosterone,nandrolone,and
intensitybutdidnotcompletelyabolishformationofthesolvent boldenonearesimilarinchemicalstructure,butweredifferent
adduct ions. This result indicated that the increase in temper- inproductionspectra(Fig.4a,candd).Normethandroloneand
ature alone was not sufficient for desolvation of the solvent nandrolone,ahomologouspair,resultedinspectrawithdiffer-
adductions.However,carefulselectionofthesourceCIDvolt- entproductionprofile(Fig.4bandc).However,boldenoneand
agewassuccessfulindesolvationofthesolventadductionsbut methandrostenolone,anotherhomologouspair,showedspectra
didnotfragmentthe[M+H]+ ions.Forthisreason,thesource withsimilarproductionprofile(Fig.4dande).Thoughsimilar
F.Guanetal./J.Chromatogr.B829(2005)56–68 61
Fig.3. ESI(+)MSspectraoftestosterone(a),normethandrolone(b),nandrolone(c),boldenone(d),methandrostenolone(e),trenbolone(f),andstanozolol(g)
showingformationofsolventadduct[M+H+CH3CN]+ionswithacetonitrileatm/z330,330,316,328,342,312and370,respectively.Spectraa–garefromtop
tobottom.Eachanabolicsteroid(10(cid:1)g/mL)wasinfusedbysyringeat20(cid:1)L/minintoLCmobilephase(CH3CN/HCOONH4,60/40,v/v)flow(200(cid:1)L/min)tothe
massspectrometer.Thetemperatureoftheiontransfercapillarywas275◦C.
instructure,THGandtrenboloneproduceddifferentproduction rather unexpected since APCI is considered suitable for neu-
profiles(Fig.4fandg).Inshort,thedifferencesinproduction tralandsmallmoleculeswithoutanyaminoorcarboxylgroup
spectraofstructurallysimilaranabolicsteroidsorinm/zvalue and is commonly used for determination of anabolic steroids
of[M+H]+ionsofthehomologouspairsprovidesolidbasisfor [19,25,35].However,ourresultsagreewiththoseofotherinves-
differentialdetectionandconfirmationoftheanabolicsteroids. tigators [22,36] showing that ESI is equally suitable for deter-
BasedontheMS/MSspectra,specificproductionsoriontransi- minationofanabolicsteroidsandthus,itwaschosenforusein
tionswerechosenfordetection,quantificationandconfirmation thisstudy.
of the anabolic steroids by MRM. The selected ion transitions
forMRMoftheanabolicsteroidsaresummarizedinTable2. 3.4. Extraction
Theanabolicsteroidswereextractedfromequineplasmaby
3.3. ESIversusAPCI
liquid–liquid extraction. These agents are neutral compounds
andwereextractedfromequineplasmabyMTBEwithoutany
The two commonly used atmospheric-pressure ionization
adjustmentinpH.MTBEwasused,basedonitsusefulnessin
(API) modes, electrospray ionization (ESI) and atmospheric-
theextractionofbeclomethasone,asyntheticcorticosteroid,ina
pressurechemicalionization(APCI),wereevaluatedforsensi-
previousstudybyourlaboratory[37].Theextractionefficiency
tivity in determining the analytes. With ESI, the lowest quan-
ofMTBEfortheanabolicsteroidswasabove74%(Table3).
tity of the injected analyte standards detectable by MRM
was 5pg each of normethandrolone, nandrolone, boldenone, 3.5. Matrixeffect—ionsuppressionorenhancement
methandrostenolone, trenbolone and stanozolol; whereas with
APCI, that quantity was 20pg each, which was four times as Matrix effect is a special phenomenon associated with
highasthatbyESI.ThisresultshowedthatESIprovidedhigher LC–MS/MSdeterminationofdrugsfrombiologicalfluidssuch
sensitivity for the analyte standards than APCI. The result is asplasmaandurine[38–40].Endogenouscomponentsextracted
62 F.Guanetal./J.Chromatogr.B829(2005)56–68
Table3
Extraction efficiency, accuracy and reproducibility (coefficient of variation, CV) for quantification of testosterone, normethandrolone, nandrolone, boldenone,
methandrostenolone,THG,trenboloneandstanozololinequineplasma
Added(pg/mL) Testosterone Normethandrolone Nandrolone Boldenone Methandrostenolone THG Trenbolone Stanozolol
Extractionefficiency(%)a
50 95 105 96 92 92 81 94 82
500 89 86 88 89 86 85 83 74
5000 83 84 84 84 84 80 86 77
Intra-dayaccuracy(%)b
50 112 114 106 106 108 114 104 100
500 105 106 105 104 104 105 101 114
5000 93 97 94 93 93 93 95 111
Inter-dayaccuracy(%)c
50 96 84 108 90 56 100 92 90
500 101 106 105 102 103 102 104 103
5000 98 100 99 98 98 100 97 99
Intra-dayCV(%)b
50 21 24 19 14 20 19 22 5.8
500 3.3 5.4 5.1 5.0 4.4 7.8 6.3 8.6
5000 4.7 4.3 4.3 4.6 4.4 5.4 4.5 4.4
Inter-dayCV(%)c
50 20 18 38 11 10 25 10 8.0
500 8.0 5.0 9.0 7.0 7.0 13 8.0 12
5000 4.2 5.6 3.8 4.8 4.7 5.8 4.6 8.4
a Extractionefficiency=peakareaofananalyteextractedfromplasma/peakareaoftheanalytestandardaddedtotheextractofblankplasma.Thevaluewasthe
resultaveragedfromsixduplicatesamplesanalyzedonthesameday.
b Intra-dayaccuracyandprecisionresultswereobtainedfromsixduplicatesamples(n=6)foreachconcentrationoftheanalyteanalyzedonasingleday.
c Inter-dayaccuracyandprecisionresultswereobtainedbyanalyzingsixduplicatesamplesforeachconcentrationoftheanalyteonthreeseparatedays(two
sampleseachday).
along with analytes from plasma or urine may suppress or arepresentedinFig.5.Alltheanalyteswerewellresolvedeither
enhance ionization of the analytes in electrospray source if chromatographically(Fig.5)ormassspectrometrically(Fig.4).
theyco-elutewiththeanalytesfromLCcolumn.Matrixeffect Inthechromatogramsofblankplasmaextract,thoughtherewas
mayimpairaccuracyandreproducibility.Forthisreason,matrix apeak(att =7.34min)intheiontransitionchannelforTHG,it
R
effectwasevaluatedundertheexperimentalconditionsusedin waswellseparatedfromthatofTHGanddidnotinterferewith
this study, and the results are shown in Table 4. Negative val- determination of THG. Quantification of the analytes was not
uesrepresentionenhancement,andpositivevaluesrepresention interferedwithbyendogenouscomponentsintheplasma.
suppression.Ionenhancementorsuppressionwaslessthan13%, Confirmationoftheanabolicsteroidsinequineplasmawas
whichwouldbewithinacceptablerandomexperimentalerrors, performedusingcriteriaofthreeiontransitionsandLCretention
exceptinthecaseofTHGatlowconcentration(50pg/mL).In timeofeachanabolicsteroid(Table2).Specifically,therelative
total, this result indicates that matrix effect caused by plasma intensitiesofthreemajorproductionsofagivenanalytewere
wasnegligible. unique(Table5)andtheydidnotappreciablychangeoverthe
concentrationrangetestedandsowereusedforconfirmationof
3.6. LC–MS/MRMfordetection,quantificationand thepresenceoftheanalytesinplasma.Variationoftherelative
confirmation ionintensitieswithin20%isusuallyacceptableforconfirmation
purposes[28].
TheLC–MS/MRMchromatogramsofblankplasmaextract Internal standard calibration quantifications of the analytes
andtheeightanalytesspikedintoequineplasmaandextracted wereperformed,andaplotoftheratioofthechromatographic
Table4
Ionsuppressionorenhancement(%)showingmatrixeffectonESILC–MS/MSoftheanabolicsteroidsa
Added(pg/mL) Testosterone Normethandrolone Nandrolone Boldenone Methandrostenolone THG Trenbolone Stanozolol
50 −4.0 8.8 12 0.3 −5.9 −27 −7.4 11
500 −5.8 −9.3 −9.2 −4.9 −8.3 −5.3 −12.3 4.9
5000 −6.1 −7.0 −4.6 −6.0 −6.9 0.6 −7.7 1.8
a Ionsuppression(%)=[1−(Aextr/Ast)]×100,whereAstisthechromatographicpeakareaofacertainquantityofadrugstandardandAextristhepeakareaofthe
samequantityofthedrugstandardaddedtotheextractof1.0mLofequineplasma.Eachvalueistheaveragedresultofthreeduplicatesamples.Thenegativesign
indicatesionenhancement,whereasthepositivesignindicatesionsuppression.
F.Guanetal./J.Chromatogr.B829(2005)56–68 63
Fig.4. MS/MSspectraoftestosterone(a),normethandrolone(b),nandrolone
(c), boldenone (d), methandrolone (e), THG (f), trenbolone (g), stanozolol
(h), and methenolone (i). Each of the analytes at concentration of 1(cid:1)g/mL
inCH3OH/HCOONH4 (2mmol/L,pH3.4)(50/50,v/v)wasintroducedinto
theESIsourcebysyringeinfusionat5(cid:1)L/min.Collisionenergywas16Vfor
testosterone;18Vfornormethandrolone;17Vfornandrolone;15Vforbolde-
none,methandrostenolone,THG,andmethenolone;and40Vforstanozolol.
SourceCIDvoltagewas20Vforalltheanalytes.
Fig.5. LC–MS/MRMchromatogramsofblankequineplasmaextractandeight
anabolicsteroidanalytes(250pgeach)spikedintoequineplasma(1.0mL)and
peakareaofeachanalytetothatofISagainsttheanalytecon- extracted.Thepeak(tR=7.34min)intheiontransitionchannelforTHGwas
from an unknown endogenous plasma component but did not interfere with
centrationwaslinearintherangeof25–10,000pg/mLforeach
determinationoftheanalyte(tR=6.97min).
oftheanabolicsteroidsspikedintoequineplasma.Inthispaper,
we define the limit of detection (LOD) as the lowest concen-
trationofananabolicsteroidspikedintoplasmathatresultedin
theselectedreactionmonitoring(SRM)signalfivetimesgreater
64 F.Guanetal./J.Chromatogr.B829(2005)56–68
Table5
Productionintensityratios(%)usedforconfirmationoftheanabolicsteroidsa
Concentration(pg/mL) Mean S.D.
25 50 100 250 500 1000 2500 50000 100000
Testosterone
m/z109/97 100 86 76 89 85 82 87 87 8
m/z253/97 19 24 20 21 23 22 23 22 2
Normethandrolone
m/z231/213 77 76 70 67 77 77 77 74 4
m/z253/213 61 71 68 71 82 75 77 72 7
Nandrolone
m/z145/109 48 59 59 56 53 51 53 55 54 4
m/z239/109 45 43 52 57 53 54 53 54 51 5
Boldenone
m/z173/121 32 31 25 28 28 29 28 28 28 29 2
m/z135/121 56 60 51 54 53 55 54 54 53 54 3
Methandrostenolone
m/z149/121 92 86 89 93 90 86 90 89 89 2
m/z173/121 22 18 19 20 19 20 20 20 20 1
THG
m/z239/241 51 60 49 52 49 54 51 52 4
m/z266/241 35 32 34 37 35 33 34 33 4
Trenbolone
m/z183/199 36 22 33 28 28 27 27 29 5
m/z227/199 49 43 55 48 47 46 46 47 4
Stanozolol
m/z107/329 21 21 22 22 22 22 23 22 3
m/z121/329 27 26 26 27 27 27 28 27 3
a Theanabolicsteroidswerespikedintoequineplasmaandextractedpriortoanalysis.
thannoise,thelimitofquantification(LOQ)asthelowestquan- (25pg/mL) by this method. This method is also more sensi-
tifiableconcentrationofananabolicsteroidspikedintoplasma, tivethantheLC–MSmethodsfordeterminationofsomeofthe
andthelimitofconfirmation(LOC)asthelowestconcentration anabolicsteroidsinhumanorbovineplasmareportedbyother
of an anabolic steroid spiked into plasma that was confirmed investigators[16–19].
by the criteria of LC retention time (Table 2) and product ion
intensityratio(Table5).LODandLOQwere25pg/mLforall 3.7. Stabilityoftheanabolicsteroidsinequineplasma
the analytes. LOC was 25pg/mL for boldenone; 50pg/mL for understorageconditions
normethandrolone, nandrolone, and methandrostenolone; and
100pg/mL for testosterone, THG, trenbolone, and stanozolol. Thestabilityresultsshowedthattheanabolicsteroidsspiked
The accuracy of this method was within 84–114% for all the intoequineplasmawerestablefor24hatambienttemperature,
analytes,andtheprecisionexpressedascoefficientofvariation for13daysat4◦C,andfor34daysat−20and−70◦C(Table6).
(CV)waswithin13%(Table3)exceptinthecaseoflowcon- Thenumberofdayschosenforstabilitystudywasbasedonthe
centration (50pg/mL) where it was higher because variations maximumnumberofdaysasamplewaslikelytobestoredprior
werehigherattheconcentrationclosetothelimitofquantifica- to analysis by the laboratory. Stability of the anabolic steroid
tion.ItshouldbenotedthattheLOQ(25pg/mL)fortheanalytes extractsinthesamplesolventonautosamplerduringovernight
spikedintoplasmawasinagreementwiththatfortheauthentic analysis of samples was also observed over a 24h period, and
analytestandards(5pginjectedontocolumn),giventhefactthat theanalyteswerestablefortheperiodofobservation.
onefifthoftheanalytesspikedintoandextractedfromplasma
(20(cid:1)Loutof100(cid:1)Lofsolutionoftheextractedanalytes)was 3.8. Preliminaryapplicationsforspecificityofthemethod
usedforanalysisbyLC–MS.
Radio immunoassay (RIA) is a very sensitive technique The method described above was successfully applied
and is widely used for quantitative determination of testos- to analyses of: (1) plasma samples collected from horses
teroneinplasma.TheLC–MS/MRMmethoddevelopedinthis post administrations of boldenone, nandrolone, and THG
study is more sensitive for testosterone than RIA when the for pharmacokinetic studies; (2) official racehorse samples
LOD(4ng/dL=40pg/mL)andLOQ(20ng/dL=200pg/mL)by for THG screening; and (3) stallion plasma samples for
RIA [41] are compared with the LOD (25pg/mL) and LOQ quantification of endogenous testosterone in research horses.
F.Guanetal./J.Chromatogr.B829(2005)56–68 65
Table6
Stabilityoftheanalytesunderdifferentstorageconditionsa
Added Time Testosterone Normethandrolone Nandrolone Boldenone Methandrostenolone THG Trenbolone Stanozol
(pg/mL) (pg/mL) (pg/mL) (pg/mL) (pg/mL) (pg/mL) (pg/mL) (pg/mL) (pg/mL)
Ambienttemperature
50 2h 57 47 61 54 56 51 49 21
50 4h 50 54 55 54 55 47 57 21
50 6h 52 61 56 53 55 49 49 32
50 24h 42 49 44 44 48 37 47 39
500 2h 530 543 541 538 530 510 504 230
500 4h 562 577 555 569 566 536 518 303
500 6h 579 597 588 573 590 530 510 347
500 24h 532 530 509 535 543 527 505 450
5000 2h 5210 5165 5143 5176 5112 5026 4670 2284
5000 4h 5199 5245 5166 5214 5173 4908 4705 2866
5000 6h 5565 5669 5521 5598 5561 5182 4900 3446
5000 24h 4955 5018 4637 4952 4951 4768 4538 4029
4◦C
50 3days 48 55 52 50 52 45 51 31
50 4days 61 53 60 38 41 38 46 48
50 12days 43 48 53 44 51 51 50 46
50 13days 48 37 51 45 48 42 38 48
500 3days 552 499 547 547 532 514 507 511
500 4days 519 477 488 488 484 471 465 516
500 12days 456 421 450 452 449 461 424 469
500 13days 463 446 453 463 471 448 457 416
5000 3days 5148 5010 5251 5251 5045 5092 4972 4822
5000 4days 4939 4920 5110 5110 4968 4999 4838 5236
5000 12days 4546 4585 4448 4603 4617 4438 4488 4696
5000 13days 4734 4663 4738 4735 4735 4094 4523 3658
−20◦C
50 13days 44 46 46 44 44 35 45 55
50 20days 46 52 50 45 42 46 46 50
50 27days 47 45 50 52 48 54 46 43
50 34days 52 47 43 47 49 50 47 50
500 13days 465 482 475 469 465 451 445 461
500 20days 475 491 492 477 483 458 464 527
500 27days 457 462 461 467 466 435 456 453
500 34days 438 450 425 438 435 418 397 456
5000 13days 4821 4788 4781 4771 4751 4598 4599 4503
5000 20days 4687 4723 4638 4674 4656 4581 4469 4729
5000 27days 4387 4370 4377 4345 4370 4227 4041 4232
5000 34days 4084 4103 4099 4071 4116 4214 3950 4243
−70◦C
50 13days 43 46 37 41 42 43 39 47
50 20days 45 53 69 43 41 45 37 47
50 27days 49 50 48 47 42 47 46 40
50 34days 52 50 55 46 51 52 48 47
500 13days 465 474 453 458 468 467 455 466
500 20days 486 503 494 468 471 479 466 497
500 27days 451 479 460 439 436 464 435 428
500 34days 443 453 452 420 436 444 453 421
5000 13days 4611 4716 4620 4488 4567 4585 4587 4697
5000 20days 4478 4514 4379 4192 4387 4310 4135 4650
5000 27days 4573 4763 4570 4422 4471 4467 4486 4418
5000 34days 4124 4298 4158 4032 4093 4216 4387 4144
a Eachvaluerepresentstheresultaveragedfromtriplicatesamples(n=3).