DETERMINATION OF TRENBOLONE IN CATTLE MEAT WITH ELISA METHOD

I

UNIVERSITY,,SS. CYRII, AND METHODIUS,, Ih[ SKOPJEFACULTY OF VETERINARY MEDICINE _ SKOPJE

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BOOKOFPROCE,EDINGS

DAYS OF VETERINARY VIEDICII{E 2OI2

3'd International Scientific Meeting

Republic of Macedonia

2-4 September 2012

Original Article Days of Veterinary Medic ine 20123''d International Scientific Meeting

UDC: 637 .s' 62"07 (497 .7)

DETERMIN.{TIOI\ OF TREI\8OI-,ONE Ii\ CATTLE ME.{T WITI{ELISA MtrTEIOD

uzunov Ristol, Hajrulai-Muslu ?ehrar, Dimitrieska-Stojkovik Elizabetal, stojanovska-Dimzoska Biljanal, sekulovski puur.l sio;r.o"rki veiimiri, Todorovic AleksandrallFood Institute' 2lnstitutefor Biomedicine and Reproduction Faculty of veterinary Medicine,Ss. "Cyril and Methodius, Skopje, Uo"l"jinf,

x Corresponding author: [email protected]. edu. mk

ABSTRACT

[ffi*t'"11}.fit"ffi1:?ffiJe like substances have been recentlv used in livestock production to obtain a high yieldThese anabolic agents are used to increase the weight gain, to improve the food efficiency, storing protein and to decrease fatness"But' depending on the use of anabolic in animal r""a,"rrouolic iesidues tirat may occur in meat and meat products present risksto human health. The present study was undertaken io deteci and quanti$z the [veh of trenbolon. ."riar", (a potent syntheticanalog of testosterone) in meat in Republic of Macedonia. cattle meat samples were collected within period of 12 months as theywere delivered by the authorised veterinary inspectors' a totui or 82 samples or.uttr. meat were anaryzed.for revel of trenboloneby Enzyme-Linked Immunosorbant Assay method. The detection limit was 91.5 ppi ro, the assay. The overal recoveries and thecoefficientsofvariation(cvs)wereintherange

of83.7%-99.6Yoand,4.!o/-o-.g.2%,respectivery,aworkingrangebetwe en25to4o0ppt, and the regression equation of the nnar iirutit;;r;;;; s: y: _0.2451x + 1.[221,I,,2:0.992e. ite average experimentalvalue of trenbolone in meat was 152'2ppt'-This uutu. gou" oo evidence.for trr. il"g"i use of hormon"s i, Republic of Macedonia,but these results do not exclude tte possiuitity of misuJe ortrr.r" potentiaily trarmdt chemicals in future. The."for. it is necessaryto conduct permanent control of these hormones. as a food quality and health safety measure.Key words: Trenbolone, ELISA, validation, residues, cattle meat.

TI\TR.ODUCTION

, Xn recent years, hormones and hormone likesubstances have been recently usea in livestockproduction to obtain a high yilld performance in ashorter period of time.These anabolic agents are used to increase the weightgai3, to. improve thi food efficiency, storing proteinand to decrease fatness. But, depending on th'.;;-;ianabolic in animal feed, anaboi. ...IAres tfrai may

:ccy in meat and meat products present risks to humanhealth. (1-4). Trenbolon.' u..tut. il-u po*.rfuI syntheticllerold.al a_ndrogen, which is used as a growth promotertn caftle. Ir is rapidty hydroly zed, to its metailo li" l;F- trenbolone after administraiio, to .uttle. It is thought11

act on skeleral muscle, ;ird in ougt, .rJG;receptors to increase protein synthesis";r--il;;;;glucocorticoid receptors to reduce the catabolic effects_of

glucocorticoids. Trenbolone u..iut" decreases therate of both Brotein synthest; il- degradation, andynr;l the rate of degradatio; L l.r, than rhe rare ofsynthesis, muscle protein rate increases (5). In manycountries outsicle the EU trenborone acetate is ricensedas growth promoter for steers und-irir.rs. In addition,ltjJ:.ff_rt:us effecriv*.* ;;,y

-and lactating cuil

fo** !::,^: rncreased weight gain accompanied bv,_

..". rar oeposrtron) was reported (6). The^European.".?*:l.,Communiry (EEC)banned the use of anabolic

with nafural origins (such as estradiol and testosterone)and some synthetic hormones such as tr."uil"".^'*animal

_husbandry 0-g). The permitted limit ;;i"";for trenbolone is I ppb in musile (CRt GUIDANCEPAPER (7 Decemb er 2OOl11. tn nepubti.

"f M;;;;;r;;

the use of hormones as growth promoters has beenpud-r illegal too. The aim of this study was to detect thelevels of trenbolone residues in the meat in Republic oiMacedonia with ELISA methods.

MATERIAI,S AND METHODS_ - Y.{ samples were collected from March 20lI toMarch 2012. The samples were kept frozen until use. Weanalyzed a total of 82 cattle meat samples.

Reagents. Most of the reagents that we used werecontained in the RIDASCREEN Trenbolone test kiifrom. R-Biopharm AG, Darmstadt, C.*ur-" fliicontain: Microtiter plate with 96 wells tf Z strlps?itn gremovable wells each) coated with capture antibodies, 6standard solutions, 0 ppt (zero standard), 25 ppt,sn;i100.ppt, 200 ppt and 400 ppt trenbol on in' 40 ,i'^r;;;i',conjugate (peroxidase_ ionjugated trenbolone), anti_trenbolone antibody, substratl (iontaining rr.u p.ro*ia",chromogen (containing, t.1y-"tfrffUJrriair'.1, ,Csolution (containing 1 N sulfuri. u.id;, .or;ujut. uriantibody dilution buffer.

i,'*Xll1i:o;,. ?: Sl;rth accere*i",; l; H"T ffifji:It.iSpni', ::1ltt9 states Food and Drug Administration

Tlo, o "**ffi : ;#,T.1T flir,ffi.IfjHX',?:Methanol and tertiary butyl methyl ether wereof analytical grade and purchased from Merck. pBS

(Fho sphate bu,ffer s o luti oO, d 7 rnL4; pLI J,.2, w a* pr.p u[ A

2-4..Septemher.ZO.@ti27

Days of Veterinary Medicine20123'd International Scientific Meeting

Origircat Article

E rylfg 1.79-9 pdiu.ry dihydrogen phosphate hydrate

$iIaP9. ,I IJro) with__e.61 g- aisooium hydr&;;phosphate dihydrate (NarHpOo x 2 HrO) andg.7 g sodi"umchloride gtacl) and fiiling up io 1006 d disstiflId water-20 mM PBS buffe1 pH 7.2, was prepared by mixing0.55_ g^sodium dihydrogen phosphaie hvdrate (irtaH^po]

{.. HzO) w_i!h _J S,S g disodium hydiogen pfr"rpiriJ

{ihv*Iate H1,IPO. x 2 HrO) and 9 g .oaiu- chtoride1na!U and hflmg up to 1000 ml disstilled water. Fromfortifled samples and calculation of recovery we usedexternal standard trenbolone (Sigma_ Aldrichi.

The standard and samples were analyzed- in Ouplicatl

3"ii:;1'T:*::::"l': -1 9t! o| the diluted ;;;;

. Apparatus. Microtiter, plate spectrophotometer(tsIO RAD model 680) (450 nm), evaporator, mixer,shaker, vortex, centrifuge, balance,'fr.IDe* CtS ;"il;;micropipette (20 pI,50 pl, 100 pl,200_1000p1).

Extraction procedure. Fat and connective tissuewere removed from the muscle and 10g of the groundmuscle was homogenized, with 10mL of OZ*IZ pgSbuf[er by mixer for_ 5min. 29 of homogenized samplewere mixed with 5mL of tertiary Uutyt methyl ether(TBME) in a centrifugal screw Lup iiut and shakenvigorously by vortex for 30-60min. rhe contents werecentrifuged at 3000rpm for 10min. The supernatant waskept and the extraction with TBME was iepeated. The

conjugate (peroxidase conjugated t.rtort.rJ,r;;'r#:added. Then 20pL of standardsadded. After that 50pL of the a,uteoolntiiffiJ:,:,-was added and after mixing gently Uv ..ljrg',il,Jplate manually, the contents were mcubateci zt rnn*temperature for 2h. The riquid poured out of th. ,^iJ#and after removal of liquid completely, .il *.il ;;;':filled with distilled water (250pL). Afteruir;ils;';;water was also discarded; washing was repear.j'r,romore times. Then, 50pL of substrate (urea p.roniajand 50pL of chromogen (tetramethylbenriO*.1- *].Jadded. After mixing thoroughly unO incubating" ili30min at room temperature and dark" 100uL of srn-solution (lM sulphuric acid) was added" after;;;;the absorbance was read at 450nm. Colour *rr;;i;for 60min. Data were analyzed,using a special ,"t*"..RIDAWIN ELISA (R-B iopharm, Oarmstadt, GentranirtlThe mean absorbance varues obtained for the ,t"rJ"ii,and the samples divided by the absorbance value of thefirst standard (zerc standard) and multiplied by 100 ;;;the o/o absorbance. The zero standu.d *u, ,:hr, *uJ;equal to 100 0h and the absorbance values were qr"i.Jin percentages.

Table 1- cross reactivity of trenbolone antibody with various compounds

Compound Cross reactivify (%)l7B-Trenbolone

Trendione

17s-trenbolone

19 nortestosterone

Testosterone

Estradiol

Zeranol

DES

100

100

82

0,06

<0,01

<0,01

<0,01

<0,01

<0,01Chloramphenicol

supernatants were combined and evaporated then thedried extract was dissorved in lml- oi g0% methanol.The methanolic solution was diluted with 2mL of 20mMPBS-buffer and applied to a RIDA Clg column-(."1t;phase extraction column with C1g end_capped ;";il;;of an ayerage particle size of 50pm) in tirf f"il;;*;manner:

- Column was rinsed by flowing of 3mL methanol(100%); Column was equilibrated b"y injectio" oiZ_flBf - Buffer; 3q! of iample *u, ioud.d "; ;r;;Column was rinsed by injection of 2mL methanol fq|%i;Column was dried by pressing nitrogen through ft f;;3min; Sarnple was elufed slo,i,ty by-injection"of frnl,methanol (80 %). An ariquot of the eluate *u, Jit,r1.awith.water (1+1, v/v), then 20SL per well of r.ruttirjsolution was used in the test.

^ Test procedure. Ridascreen ELISAkit was obtainedfrom- ry Biopharm GmbH, Germany. Trenbolonestandard solution used for the caribration curve *.r. ,t1.":.^ll of 0, 25,50, 100, 200, and 400 ppt trenbolone in40 % methanol,, whereas the antibody^used fraO .ros,reactivity with other related .o.pounds, as indicatJby the manufacturer's literature and shown in Tabre 1.

Method validation. The limit of detection (LOD)was obtained by spiking with 0, 5 times nom Mnpiwhich 1 ppb is according the guidance letter fromCommunity Reference Laboratories, (7 December2007). The method recovery was determined at threelevel by_spiking meat samples with 0,5; I and 1,5 timesfrom MRPL level. For determination of repeatabifity, tfr.same steps were repeated on two occasions in the iameanalytical conditions. Detection capabilities (ccB) wasevaluated by analyzing 20 spiked samples at b,5 ffan-Pl,

level. A fypical ELISA standard ,u*i is presented inFigure 1' Final trenbolone concentrations in meat werecalculated by taking the average recoveries into account.

RESULTS AND DISCUSSIONThe calculation of the gained results was made by

RIDAWIN software. For construction of the calibrationcurve the mean of the absorbance values obtained for thestandards was divided by the absorbance varue of thefirst standard (zero standards) and multiplied by 100. Theabsorption is inversery proportional to ih. .on..ntrationof trenbolone. As can 6e ieen in Fig.I, the trenbolonecalibration curve was found to be vitualy rinear in the20 to 400 ppt.

128 2-4 September 2012, Ohrid, R. of Macedonia

Original Article Puyr of Veterinary Medic ine 20123''d International Sgyntf\, t;;;;

:-:l---i:::L:1:.:ri::: L:: l:::t:'::1:::f--r:f::.j

ri::]

Conqentrailon 'roo.ooo

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l

:

'ii))

1i

il

lllll))

tlI

ll

ijii

l

il

rilrlll

il.l

rilri.l

tl|,).'

Figure'1 Linearity of caribration utrvefor trenborone standards (25_400 ppt)

"-:",,:ff }ff filHi&u;x'fi H::ff ff ilrffi ":#", j,t,lJ*,::f 3-,Tj.1]:*p,*mrnnoa A A(\A --- r tt

__ -vr !rvuuvrvuv Willirange 0 - 400 pft, R2:0.9 g2g: .-"'- vvu /v ue, t,c aDsorDance ratto and trenbolone concentrationias evaluated over the

90,O7o

80,ool"

70,Ao/o

60,o%

50,oo/o

*.*-y = -O,24S1Ln(x) + 1 "6221R2 = O,992a

q --'"'"il.olo\- 4o,oyo

30,o9lo

20,oo/o

1O,Oyo

o,oyo

l

o.ooo200,ooo 250,ooo

STD_Conc

Figure.2. Calibration cttrvefor trenbolone standards e_a00 ppt)

ilI

l-.tI

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IjI

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*il -i-l-; *,lrli-ili1riiilirilli |,-,, J-riiilr

ii i;--,11 lr l

lili,,,Li-- Ii -lllrrrlirittri

11'lt i .--i i- -- +li,ii;iLl-'- | j--.-'*j-ll;,ii:ti

lriillI

ilrlu

jil

iii1 +,llil

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-iiirli:--- ..-: ,..*-l 1t,,irlii,.ll.rii50,ooo 100,ooo 150,ooo

The precision of the method was calculated by measured cy%.The precision dates are shown inTable 2!.

Concentrarion (ppt) cv%

3.0

0.6

1.8

1.5

0.7

v_o^o.-/.

6.5

4.1

Standards

CV for fortified samples

0.0

25.0

50.0

100.0

200.0

400.0

<nn n

1000.0

1500.0

Table 2. Precision of the method (% CV)

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2-4September20@

Days of Veterinary Medicine 20123''d International Scientific Meeting

Origirtat ArtiileThe results of method recovery (n:18) and repeatability (n:54) are presented in table 3"

Tuble 3. Recovery and repeatabiiity

Validationparameter

No. ofreplicates

Spiked concentrationnglkg

Determinatedconcentration nglkg

VIeanrecovery o/o

Coeffrcient ofvaiationoh

RecovervJ

6

6

6

500

1000

1 500

4t8.6

962,1

1494,9

83.7

96.2

99.6

8.2

6.5

4.1

Repeatability18

18

18

500

1000

1500

442.4

9fi.41432.7

88.4

91.I9s"5

r0.2

3.6

4.7

Validation of the method used in on trenbolonedetermination resulted in the mean recovery of g3.7%_9 9 - 6% and repeatabi lity of 88 . 4%-9 5,5yo w rthco effi cientof variation (CV) of 4.1%-8.2yo and3.6%-10.2%.

The analyses of the meat samples showedconcentrations from 66.7 ppt to 25S.g ppt (meanconcentration of trenbolone was 152.2pp0.

Our test results are of importanie as they giveinformation about the use of tienbolor. pr.puiuti"o^in national animal husbandry and in the foodindustry.The European Economic Community (EEC) bannedthe use of anabolic compounds as gro*ttr acceleratorsin food animals while the United Stites Food and DrugAdministration (USFDA) permitted the rimited ur. oisome hormones with natural origins (such as estradioland testosterone) and some synthitic hormones such astrenbolone in animal husbandry (l-4). The permittedlimit values for trenbolone is i ppb'in muscle (CRLGUIDANCE pApER (7 Decemb& 2007)). A surveycamied out in R. Macedonia from March zo-tt to Marcir2012 demonstrated that the incidence of residues oftrenbolone in tissues of slaughter animals is not aproblem, which means that trenbolone gave no evidenceof illegal use of these hormones in R. Macedonia. TheNational Residues control plan guarantees fulfilmento! lhe requirements which are ofimportance to healthof both humans and animals as well as marketing ofanimals, food and other products of animal origin.

REFER.ENCES1. The Presence of Some Anabolic Residues in Meat and

Meat Products Sold in Ustanbul, B.lent NAZLI**,Hilal .OIAK, Ali AYDIN, Hamparsun HafrapUfcYafriDepartment of Food Hygie-ne and Technology, Facuhyof Veterinary Medicine, Ustanbul University, 34320,

AvcYlar, Ustanbul - TIIRKEY, Received: 27.06.2A032. Cecave, M.J., Hancock, D.L. : Effects of anabolic steroids

on nitrogen metaborism and growth of steers fed cornsilage and cornbased dietssuppremented with urea orcombinations ofsoybean meal and feathermeal. J. Anim.Sci., 1994; 72: 515-522.

3. Moran, C., Ourke, J.F., prendiville, D.J., Bourhe, S.,Roche, J.F.: The effect of estradiol trenbolonee acetate, orzetanol on growth rate, mammary development, carcasstraits, and plasma estradiol concentrations of beef heifers.J. Anim. Sci., 1991; 69:4249- 425g.

4. Sawaya, W., Lone, K.p., Hasain, A., Dashti, B., dl_Zenki,S.: Screening for estrogenic steroids in sheep and chicicenby the application of enzyme-linhed immunosorbent assayand a comparison with analysis by gas chromatography-mass spectromehy. Food Chem., l99g; 63:563_569.

5. Human helt and trenbolone residue in bovine meat, IB.Jannat, 2M. R. Oveisi, *2N. Sadeghi, 2M. Hajimahmoodi,Iran. J. Environ. Health. Sci. Eng., 2007,yol.4. No. 4, pp.203-206

6. Screening of trenbolone-l7Bin milk samples aftelapplieation of trenbolone acetate to a cull cow, IrisG. Lange, Andreas Daxenberger, Heiru-ich I{. D.Meyer Institut fiir physiologie, Forschungszentrumflir Milch und Lebensmittel, Technische UniversitetMiinchen Weihenstephaner Berg 5, D_ 95350 Freising_Weihenstephan, Germany

7. council directive 96l22lEC of 29 aprir 1996 concerningthe prohibition on the use in stock framing of certainsubstances having hormonal or thyrostatic action andof beta-agonists and repealing Directives Bll60ZlEEC,88 / 1 46 E,EC and 88 I 299 IEEC

8. council directive 96l23rEC of 29 april 1996 on measuresto monitor certain substances and residues thereof in riveanimals and animar products and repearing Directives85/358iEECand 861469/EEC and Decisions g g/rgTEECand9l/664lEEC

IJU 2-4 Septentber 2012, Ohrid, R. of Macedonia

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Original Article Days of Veterinary Medicine20123'd International Scientific Me eting

OIXPEAEJIYTAIbE HA TPEHEOJIOH BO MECO OA TOBEA.dco ELrsA MET0A

Ysynon Pmctol, Xajpynau-Mycnlly 3exp al, flwruurpr.recKa-Crojr<onuft Enus aderat,CrojauoBoKa-AuM3ocKa Eunjanar, CerynoBcKr4 flanne,, Crojxon.*, g., krMkrpz,

Togopona( Anexcan4pal1I'Iucmumym

3a xpaHa,2l'Irucmumym sa fuLtoaedwtluua w penpodyrct4uja, @arcynmem 3a semepuHapua uedut4urua,Yuueepsumem "CB. Ruputt u Memoduj',, Cxonje, Maxedouuja

*Anrop 3a Kope croAeirquj a : ri steuzunov@fvm. ukim. edu. mk

AIICTPAKTBo uocle'4HHBe ro'qllHrr xpoMoHllTe I{ culcrauul'ITe crrulrlvr Ha xopMoHr{ ce Kopkrcrar Bo oADIeAyBaI.ero Ha 4o6rarorot sago6rnarre Ha BI'IcoKo TIpLIHocHI'r KapaKTepr,rcrrrrr{ 3a rroKparoK BpeMeHcKr{ rrepnoA.osue aHa6orHrIH ce ynotpe6l'naut au iaoraMyBarse Ha Maca'ra, no.qo6pyrirbe ua ex{er<ror Ha r{cKopr,rcryBar*e Ha xpaHara,AenoHl4palbe Ha [poreI4HI'ITe u HaManyBaILe Ha MacHoro rKI'rBo. Ho no.u"raroa, oA EpHMeHara na ana6oar4rlrrre no 4o6r,rrovnaraxpaHa' HIIBHI{ p*fiwrr KoI{ Moxe ga ce jarar Bo Mecoro H IrpoI{3BoAI'ITe oA Meco npercraByBaar p}r3[rrl, sa s4panjero na rqrrero.onaa cryguja 6erue cnpoBeAeHa sa AereKrl'rp a=be vtxnanrrQlrryrar.e Ha HilBoara Ha p*urwog rpen6onoH (cunrerr.rrr*lr aHaJrorHa recrocrepouot) Bo [pklMepoqu oA Meco oA Peny6"rrura Maxe4onuja. flpurraeporlgTe oA roBeAcKo ueco 6ea co6apanu noTe*or Ha 12 nrecequ, a 6ea 4ocraB)rBaH, o,{ oBJlacreHnre Berepr.rHap'r.r r,r'crreKTop,. Brynro g2 npnrraepoKa roBe.qcKo ueco 6eaaHaJIrr3IrpaHH 3a yrBpAyBalf,e Ha HLIBoro ua rpeu6oloH co Engrnucxra BpsaH urvrprocop6eHreu uerog.'Jllru,ror Ha 4er:exqujaIIpH orpeAeJlyBalbero Seue 91,5 ppt' Bxynuuor aHzuIItrrIqKI{ flpr{Hoc ra xoe6uqlaeHT Ha rapujauqa (cv) 6e,re Bo orrceror oA83'7-99'6You4'1-8'2oZ,coo4ner,o,copu6or"ro,rcer o1,25 lo400ppt.PaaenKaHaperpecujanaKoHeqHarauuxu6uqr.lja6eure: y: -0'2451x * 1,6221, R2: 0,9928. Brynuara eKcIIepI,IMeHTarHo o[peAeJreHa BpeAHocr Ha rpen6oroHor Bo veco 6erue 152,2ppt' oraa BpeAHocr yKaxyBa AeKa HeMa HeJIeraJIHa ynorpe6a Ha xopMoHr{ no peny6nuxa MaxeAonrja, no oBr{e p*yJrraru ue jaHcKrr) DBaar MoxHocra 3a 3noylorpe6a ra oBlre fioreHqajamro turerHx cyrrcraHr{r{}r Bo r,rAHr,r'a. 3apagn roa, HeorrxoA'o e Aa cecnpoBeAyBa uoctojaua KoHTpona Ha oBLIe xopMoHI'I KaKo MepKa 3a KoHTpoJra Ha KBiurareror Ha xpauara n 6es6eguocr 3a sgpanjero.K.nyvnu r6oporu: rpen6onon, ELISA, aarigarJuja, p.=rayr, roBeAcKo Meco.

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2-4 September 2012, R. of Macedonia 131