determination of sulphamethazine in animal tissues by enzyme immunoassay

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  • Analytzca Chtmtca Acta, 275 (1993) 323-328 Elsener Science Publishers B V , Amsterdam


    Determination of sulphamethazine in animal tissues by enzyme immunoassay

    C Renson, G Degand and G Maghum-Roglster Laboratoue dhtyse des Den&es Ahnentaues dOngme Ammale, Faculti de M&iecme Vthnaue, Unwers~te de L&e,

    B& 842 Sart-Tdman, B-4ooo LGge (Be&m)

    Ph Delahaut

    Centre dEconomte Rurale, B-6900 Ma&e (Belguun) (Received 30th June 1992, revised manuscnpt received 29th September 1992)


    An enzyme mmmnoassay for sulphametbazme was developed usmg an antiserum rzused m rabtuts by unmumaa- tlon agamst a sulphamethazme dlazo derwatrve coupled to bovme serum albumm Horseradish perox~dase coupled to sulphametbazme by a carbodurmde method was selected as a tracer The dose of sulphametbazme that caused 50% bmclmg mhiilfion was 3 8 ng per well wth a 2-h mcubation tune at 37oC and 12 ng per well wrth a 16-h mcubatlon tune at 4C The assay was evaluated by usmg control and sulphamethazme-fortdied mu&e and bver samples These samples were treated by the matnx sohd-phase dqerslon method for the subsequent lsolaaon of the drug The method allowed a dete.&on lmut well below the tolerance tit (0 1 mg kg-) generally appbed for sulphamethazme

    +r& Enzymatic methods, Immunoassay; Ammal &sues, Sulphamethazme, Trues

    Sulphonanudes have a broad spectrum of an- tlbactenal atiwty and are frequently mcorpo- rated 111 feeds as a practical method for the prevention or treatment of a variety of so-called confinement diseases 111 ammals, mamly pigs However, there are concerns for human health because of the possible presence of residues of the drug 111 meat [l] The US Food and Drug Admmlstratlon (FDA) requires a 19day mth- drawal period of feed contammg sulphametha- zme before slaughter and has estabhshed Its tol- erance hmlt m tissues for human consumption at 0 1 mg kg- [2-51 Although dtierent sulphon-

    Correspondence to G Maghum-Roglster, Laboratolre dAnalyse des De&es Abmentares dOngme Ammale, Fa- cult6 de Midecme WtQmlure, Umversk de L&ge, Bit B42 Sart-Tdman, B-4000 Loge (Be&urn)

    armde-antlblotx combmatlons have been ap- proved for use m swme feed, sulphamethazme has been xdenttied as the major problem m ca 95% of all sulphonanxde tissue vlolatlons [6] This paper reports a competltlve enzyme munu- noassay (EIA) for routme screening of sulpha- methazme m tissues


    Reagents and eqtupment Sulphamethazme was obtamed from Janssen

    Chmuca (Geel/Belgmm), sulphaguamdme, sul- phachloropyrldazme, sulphaqumoxahne, sulpha- merazme and sulphadnnethoxme from Sigma (St LOUIS, MO), sulphamlamtde, sulphadlazme and sulphathlazole from Aldrich Wlwaukee, WI),

    ooO3-2670/93/$06 00 Q 1993 - Elsevler Science Pubhshers B V All nghts reserved

  • 324 C Rmson et aL /Anal Chzm Acta 275 (1993) 323-328

    bovme serum albumm (BSA) from Serva (Heldel- berg, Germany), horseradish peroxldase (HRP) from Boehrmger (Mannhenn, Germany), 3,3, 5,5-tetramethylbenzldme (TMB) mlcrowell per- oxldase system from firkegaard & Perry (Galthersburg, MD), Tween 20, cytochrome c and thlmerosal (merthlolate) from Merck (Darmstadt, Germany), aprotmme (trasylol) from Bayer (Brussels), Freunds complete adjuvant from DIFCO (Brussels) and bulk C,, (40 pm, 18% load, end-capped) octadecylsdyl-denvatlzed silica from AnalytIchem International (Harbor City, CA) All other chemicals were of analytlcal- reagent grade or better and were used as re- ceived Delomzed water was purified mth a Mllh- Q system (Waters)

    Buffers were prepared as follows (A) carbon- ate-hydrogencarbonate buffer solution (coatmg buffer) 50 mM pH 9 6 Na,CO, (159 g), NaHCO, (2 99 g) and dlstllled water (to 1 0, (B) phos- phate-buffered salme (PBS)-gelatme solution (0 01 M PBS-O 2% gelatme) (EIA buffer) NaCl (8 6 g), Na,HPO, 2H,O (16 8), NaH,PO, H,O (0 14 g), thunerosal (0 1 g), gelatme (2 g) and distilled water (to 10, (C) washmg solution NaCl (8 76 g), Tween 20 (0 5 ml), dlstdled water (to 1 0, (D) stoppmg solution 6 M H$O,

    All stock and workmg standard solutions were stored at 4C

    ELISA plates (Nunc-Immuno Plate Maxlsorp F96) were obtamed from Nunc (Roslulde, Den- mark) Absorbance of plate wells was measured with a MultIscan MCC/340 nucroplate reader from ICN Flow Laboratorles

    Zmmwwgen preparatton Sulphamethazme was dlazotlzed and coupled

    to bovme serum albumm [7,8] Prepratwn of the drazo denvatrve Sulpha-

    methazme (57 mg, 0 27 mm00 was dissolved m 4 ml of 0 5 M H,SO, and the solution was left overnight wth stlrrmg at 4C A solution of sodmm mtnte (19 mg, 0 27 mm00 m dlstllled water (1 ml) was added drowse to this nuxture wth stvrmg m the dark at 4C The formation of dlazotlzed sulphamethazme was detected by the appearance of a deep yellow colour after reaction wth N,ZV-dnnethylamlme 191 The formatton of

    the dlazo derlvatlve was also momtored by thm- layer chromatography on silica gel 60 Alu Rol sheets (Merck) after nugratlon m the solvent mnc- ture ethyl acetate-ammoma solution-methanol (30 + 1 + 15, v/v/v) After spraymg of the sheet vvlth a 0 1% solution of 2,7-dlchlorofluoresceme m ethanol (Merck), it appeared that the R, value of sulphamethazme m this system was 0 79, whereas its dlazo derlvatwe did not migrate The reaction was contmued until the disappearance of the sulphamethazme spot The reaction was stopped by addltlon of a solution of ammonium sulphamate (7 mg) m water (140 ~1)

    Couplrng to bovrne serum ahunm The solution of the dlazo derwatlve was added m 50-~1 por- tions to 4 ml of carbonate buffer (pH 10) contam- mg 100 mg of BSA The reactlon mixture was left for 18 h at 4C Unreacted material was sepa- rated from the conjugate by chromatography on Sephadex G-50 Hrlth 0 05 M ammomum carbon- ate solution (pH 8) The conjugate was then lyophlllzed

    Anthdy pregaratwn The mununogen was used to prepare an anti-

    serum Fwe rabbits (New Zealand breed) were munumzed by the protocol of Valtukatls et al [lo] with 1 mg of duuotlzed sulphamethazme- BSA conjugate m complete Freunds adJuvant The ammals recewed five mJectlons vvlth an mter- val between inJectIons of 1 month After the thud mJectlon, blood samples were taken from the ear vem at lo-day intervals to follow the appearance of the antibodies

    The crude antiserum obtamed was dlmded mto ahquots and stored at - 20C after dllutlon m 0 1 M PBS-glycerol nuxture (serum-PBS-glycerol, 1 + 9 + 10) correspondmg to a fmal dilution of 1+ 19

    Enzyme conjugate preparatwn The sulphamethazme horseradlsh peroxldase

    coqugate was prepared by a m&led carbodl- mude method [11,12] Sulphamethazme (10 mg) was dissolved m a nuxture of pyrldme (400 ~1) and dlstdled water (400 ~1) and the solution was added to horseradish peroxldase (20 mg) drs- solved m distilled water (2 ml) A lo-mg amount

  • C Rensm et at! /Anal Chm Acta 275 (1993) 323-328 325

    of l-ethyl3-(3-drmethylammopropyl)carbodr- lrmde hydrochlorrde was added and the solution was strrred at 4C for 18 h Excess of sulphameth- azme was ehmmated by chromatography on Sephadex G-25 column m PBS buffer, the col- umn was first washed wrth PBS buffer, the solu- tron was then added to the column and the en- zyme conjugate preparatron was eluted wrth 3 5 ml of PBS buffer

    Tins conjugate was then diluted (1 + 20, v/v> m a buffer contammg 80 ~01% of a solution of 75 mlofPBS+2gofBSA+12Smlofglycerol+20 mg of thunerosal, 20 ~01% of aprotmme and 0 1 wt % of cytochrome c

    Pretreatment of the samples The muscle and hver samples were treated

    accordmg to the matrrx solid-phase drspersron method [13] The matenal CC,,, octadecylsrlyl-de- rrvatrzed srlrca) was washed usmg a vacuum box successrvely with 30 ml each of hexane, drchloromethane and methanol to remove con- tammants, then 0 5 g of tissue was added to 2 g of the prewashed C,, matenal m a glass mortar The sample was gently blended wrth a glass pestle to produce a semi-dry, homogeneous mater& Tlns was poured mto a syrmge barrel column (10 ml) contammg two filter-paper drscs Whatman No 1) Two other filter-paper discs were placed on the top of the materud and a syrmge plunger was used to compress the sample to a volume of 4 ml A RIO-~1 drsposable pipette up was attached to the end of the column to increase the resr- dence tnne of the elutmg solvent m the column The column was washed wtth 8 ml of hexane by gravrty flow (excess of hexane was removed by applymg a posrtrve pressure) and sulphametha- zme was eluted with 8 ml of drchloromethane The eluted fraction was evaporated to dryness under nitrogen and the residue was drssolved in 250 ~1 of PBS-gelatme (buffer B)

    Assay procedure The sulphamethazme antrserum drluted 1+

    15 000 111 coatmg buffer A (100 ~1) was added to the umer 60 wells of a polystyrene nucrotrtratron plate (the well to well varratrons are greatly re- duced by onnttmg results from the peruneter

    wells) The plate was covered wrth a plastrc fihn (plate sealer from Tech Gen, Zelhk, Belgmm) and stored at 4C for 16 h. The coatmg solutron was asprrated, then 50 ~1 of standard solutions of sulphamethazme (range 0 2-100 ngl or trssue ex- tracts from control or sprked samples were drs- pensed, m tnphcate, mto mdrvrdual wells on the nucrotrtratron plate already coated wrth the spe- crfic anubodres Subsequently, 100 ~1 of the sul- phamethazme-enzyme conjugate solution drluted 1 + 10000 111 PBS-gelatme (buffer B) was added The plate was covered with a plate sealer and, after shakmg, mcubated overmght at 4C or for 2 h at 37C The wells were washed three times wrth washing solutron C to remove unbound ma- terial TMB solution (150 ~1) was then added to each well and the plate was mcubated at 37C m the dark After 30 mm the reacuon was stopped by addmg 50 ~1 of 6 M H,SO., to


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