Determination of clenbuterol in bovine liver by enzyme immunoassay

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Analy8ca Chumca Acta, 275 (1593) 227-230 Elsewer Science Pubhshers B V , Amsterdam

Determination of clenbuterol in bovine liver by enzyme immunoassay S D Bucknall, AL MacKenzie, M J Sauer, D J Everest, R Newman and R Jackman Bwchemutry Dzscq~he, Central Vetennary Laboratory, New Haw, Weybrtdge,Surrey KT15 3NB (UK) (Received 20th May 1992)

Abstract Procedures are described for optmusmg and vabdatmg an ELISA method for measurmg clenbuterol residues m bovme bver wlthout pnor sample ennchment Optunal assay conddlons are obtamed by premcubatmg bver supematant wllth an unmoblhsed antibody rmed to clenbuterol-ovalbumen After further mcubatlon wrth a salbutamol-peroxldase coqlugate, &our IS developed usmg a tetramethylbenzldene substrate The assay wll pemut measurement of clenbuterol residues m hver at the maxmmm residue level of 05 ng g-’ Hnth a confidence of > 99% I(eywords Enzymatx methods, Immunoassay, &Agomsts, Bovme bver, Clenbuterol, ELISA method

Clenbuterol 1s a @agonist which 1s licensed only for the treatment of respiratory disorders m cattle and horses and as a tocolytlc At high dose It can be used as a repartltlonmg agent promotmg the conversion of fat to muscle and therefore mcreasmg carcase value and m this respect its use has been banned As clenbuterol is toxic it is necessary to monitor levels of the residue m ammal tissue destmed for human consumption to ensure the maxnnum residue lumt (MRL) for this compound 1s not exceeded To support this laboratory’s vetermaly drug residue momtormg programme an ELISA method was developed for clenbuterol m urme [l] It was necessary to optlmuse and validate this assay for rapidly and rehably measuring residues of clenbuterol m hver

Correspondence to SD Buck&l, Bmchermstry Dwlplme, Central Vetermary Laboratory, New Haw, Weybndge, Surrey KT15 3NB (UK) Elsevler Science Publishers B V

EXPERIMENTAL

An ELISA method for clenbuterol m urme usmg an alkalme phosphatase enzyme system was performed m the presence of either urme or 10% bovme liver homogenate to assess the matrvr effect of the hver (Fig 1) In an attempt to decrease assay tune a horseradish peroxldase (HRP) enzyme label was substituted for the alkalme phosphatase label (Fig 2) The antisera obtamed from an ammal before (248/5) and after (248/6) rechallengmg mth clenbuterol-ovalbumen unmunogen were compared (Fig 3) Utihsmg the 4% cross-reactlvrty of the clenbuterol antibody wth salbutamol, salbutamol-HRP was prepared (by the conjugation of salbutamol henuscuccmate 121to ammated HRF’ usmg the rmxed anhydnde method based on [3]) and substituted for the clenbuterol-HRP conjugate (Fig 4) Sample volume was then mcreased from 10 to 200 ~1 and sample concentration doubled from 10 to 20%

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SD Bucknall et aL /Anal Chun Acta 275 (1993) 227-230 7

100

60

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20

0

Fig 1 Alkaline phosphatase clenbuterol ELBA vs 10% hver (- - - - - -1 homogenate ( -_)

m urme

(w/v) (Fig 5) Figure 6 shows the effect of premcubatlon of the test samples and standards m the wells at approxunately lo-mm mtervals up to 1 h, pnor to addition of salbutamol-HRP

Fig 3 Anttsera bleed 24S/5 (- - - - - -1 vs 248/6 (-_)

The followmg ELISA procedure evolved from these mvestlgatlons Mlcrotltre plates were coated vvlth clenbuterol-spectilc antisera usmg partial denaturatlon of the antibody with glycme to m-

loo, 90 60

F

loo,

t

t 20 10

1

phosphatase Fig 2 Clenbuterol peroxldase ( -_)/alkahne (- - - - - -1 conlugates Assay tmes are 0 5 and 4 5 h, respectlve1y

Ftg 4 Salbutamol peroxtdase (- - - - - -1 vs clenbuterol peroxlcoqugate dase ( -_)

SD B&t&l

et aL/AnaL Ch

Acta 275 (1993) 227-230

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(5%), bovme serum albumm (BSA, l%), and dned pnor to storage at 4°C Bovme hver test samples and negative control hver were prepared by homogenlzatlon 111PBS (005 M, pH 7 0) to @ve 20% (w/v> suspensions which were centrfiged (2500 g, 4”C, 15 mm) The supematant was retamed for assay Standards were prepared as above usmg control negative bovme liver fortlfied with clenbuterol HCl to provide a range between 0 25-8 ng g-l

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TABLE 1

20-

Assays of control her forttied wth clenbuterol HCI Standard concentration (ng g-‘1 0 Ftg 5 Clenbuterol standard curves III10% (- - - - - -1and 20% her homogenate Sample volume, 200 pl ( -_)

crease bmdmg [41 The plates were then washed, 6 x with phosphate buffered salme (PBS, 0 1 M, pH 7 0) contammg Tween 20 (0 05%), sucrose

Inter Mean (%I?/&)

SD cv n

05

10

20

96 125 13 87

18 21 32

47 19 42 32

34 16 46 32

37 38 8

21 33 8

24 51 8

15 44 8

65

Intra

SD CV n

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90 80

I

120

100

so 40 20 10 0 05

1

~'twa

8

16

OLD’ 001

Fig 6 SIX clenbuterol standard curves w~tb premcubatlon tlmcs -, 60, ------, 45, , 30; ---1 20, -,lO, - - ,Omm

01

’

10

I

100

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500

I

900

I

995

999

PROBABImn

F@ 7 Clenbuterol probabhty curves 0, Negative samples, q,05 ngg-’ standard, A, 10 ng g-’ standard

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S D 3ucknall et aL /Am!

100

RESULTS AND DISCUSSION

90

30 70 60

30

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Chm Acta 275 (1993) 227-230

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0.3

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05

1

2

3

5

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Fug 8 Composite clenbuterol standard curve f 2s D -, Intra-, - - - - - -, Inter-assay rz = 0 98

200 ~1 ahquots of samples, controls and standards were added to each well and the plate premcubated (40 mm, room temperature), 50 ~1 of salbutamol-HRP was added and the plate was shaken for 15 mm After mcubatlon the plate was washed with Tween 20 (0 05%) and 200 ~1 of TMB-peroxldase substrate added to each well After shakmg the plate for 15 mm, colour development was stopped by the addition of 50 ~1 of 10% H,SO, Absorbance was read at 450 nm on a Dynatech MR700 ELISA plate reader Usmg this procedure a number of assays were performed using negative control hver fortified with clenbuterol HCl at 0,O 5, 10 and 2 0 ng g-l, to assess both inter- and mtra-assay variation (Table 1) A probability curve was constructed by plottmg percentage cumulative frequency of B/B, (probablhty) agamst B/B, for 176 negative tissue samples and homogenates sp&ed at 0 5 and 10 ng g- ’ (Fig 7)

The followmg conclusions were drawn from the optumsatlon procedures described Although liver matnx had a negligible effect on linear response the assay was not sensitive enough to detect the MRL of 0 5 ng g-’ liver The mtroductlon of clenbuterol-HRP conjugate gave a marginal increase m sensltlvlty and reduced assay time from 4 5 to 0 5 h Rechallengmg the nnmumsed sheep with clenbuterol-ovalbumen resulted m a doubled sensltlvlty Assay sensltnrlty was further improved by the substltutlon of a salbutamol-HRP conjugate owing to the antibodies lower affinity for salbutamol than for the clenbuterol m the test sample By mcreasmg sample volume and sample concentration, sensltlvlty was doubled without srgnlficantly increasing background colour After premcubatlon of the sample with unmobllrsed antibody the B/B, at 0 5 ng g -I was lowered from 62 to 44% At the MRL of 0 5 ng g-’ the mter-assay coefficient of variation (CV > was 2 7% when the number of samples tested was 32 and mtra-assay C V was 3 3% when the number of batches tested was 8 At a B/B, value of approxunately 65% there 1s a 1% probablhty of producmg a false positive result and less than 0 5% probablhty of producing of false negative result at the 0 5 ng g-’ detection level Cross-reactlvlty with other @agonists was found to be salbutamol 4%, terbutahne 2%, lsoproterenol, ractopamme, DL-4-hydroxy-3-methoxy mandehc acid, dlhydroxymadalelc acid and noradrenaline less than 1% The optmused assay provides a robust, sensltlve and specific analytical procedure wrth a lmear range between 0 25 and 8 ng g-’ (Fig 8) suitable for the screenmg of liver samples

REFERENCES 1 R J H Plckett and M J Sauer, Anal Glum Acta, 275 (1993) 269 2 N Beauheu, C Charette, J C K Loo, N Jordan and I J McGdveray, J Pharm Blamed Anal, 3 (1985) 575 3 B F Erlanger, Methods Enzymol, 20 (1980) 85 4 J F Conradle, M Govender and L VLsser J Immunol Methods, 59 (1983) 285