determination of sulphamethazine in animal tissues by enzyme immunoassay

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

323

Determination of sulphamethazine in animal tissues by enzyme immunoassay

C Renson, G Degand and G Maghum-Roglster

Laboratoue d’htyse des Den&es Ahnentaues d’Ongme Ammale, Faculti de M&iecme V’thnaue, Unwers~te de L&e, B& 842 Sart-Tdman, B-4ooo LGge (Be&m)

Ph Delahaut

Centre d’Economte Rurale, B-6900 Ma&e (Belguun)

(Received 30th June 1992, revised manuscnpt received 29th September 1992)

Abaa-act

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 4’C 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 sulphamethazme before slaughter and has estabhshed Its tolerance hmlt m tissues for human consumption at 0 1 mg kg-’ [2-51 Although dtierent sulphon-

Correspondence to G Maghum-Roglster, Laboratolre d’Analyse des De&es Abmentares d’Ongme 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 mununoassay (EIA) for routme screening of sulphamethazme m tissues

EXPERIMENTAL

Reagents and eqtupment Sulphamethazme was obtamed from Janssen

Chmuca (Geel/Belgmm), sulphaguamdme, sul- phachloropyrldazme, sulphaqumoxahne, sulphamerazme 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 peroxldase system from firkegaard & Perry (Galthersburg, MD), Tween 20, cytochrome c and thlmerosal (merthlolate) from Merck (Darmstadt, Germany), aprotmme (trasylol) from Bayer (Brussels), Freund’s 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 received Delomzed water was purified mth a Mllh- Q system (Waters)

Buffers were prepared as follows (A) carbonate-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 4°C

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 4°C 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 4°C 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) contammg 100 mg of BSA The reactlon mixture was left for 18 h at 4°C Unreacted material was sepa- rated from the conjugate by chromatography on Sephadex G-50 Hrlth 0 05 M ammomum carbonate 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 Freund’s 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 - 20°C 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) drssolved 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 4°C for 18 h Excess of sulphamethazme was ehmmated by chromatography on Sephadex G-25 column m PBS buffer, the column was first washed wrth PBS buffer, the solutron was then added to the column and the enzyme 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 sulphamethazme 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 4°C for 16 h. The coatmg solutron was asprrated, then 50 ~1 of standard solutions of sulphamethazme (range 0 2-100 ngl or trssue extracts 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 sulphamethazme-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 4°C or for 2 h at 37°C The wells were washed three times wrth washing solutron C to remove unbound material TMB solution (150 ~1) was then added to each well and the plate was mcubated at 37°C m the dark After 30 mm the reacuon was stopped by addmg 50 ~1 of 6 M H,SO., to each well, m the same order and at the same rate as the substrate solutron was added so as to keep the reaction ume constant for all of the samples The plate was gently shaken before absorbances were measured at 450 MI wrth the nucrotrtre plate reader

The cross-reactrvrtres (CR) of the antrserum were determmed by preparmg dose-response curves for a range of sulphonanndes and were calculated vvlth the equatron

CR= Mm1 sulphametllaPne requucd to reduce colour production by 50%

nmol other drug rrxtured to reduce colour pmductmn by 50%

Catiratwn The percentage bmdmg was calculated from the absorbance obtamed m the absence (I?,) and the presence U?) of sulphamethazme 111 standards and samples as follows bmdmg (o/o) = (B - NS/B, - NS) X 100, where NS rep- resents the non-specrfrc bmdmg, I e., the absorbance measured m wells uncoated wrth the antrserum Cahbratron graphs were prepared by plotting log(sulphamethazme concentratron) against percentage bmdmg or agamst logltiper- centage bmdmg) (logrt-log plot)

Calculatwn Results were calculated accordmg to the method of Rodbard and Frazrer [14] by mterpolatron from a cahbratron graph where the bound actrvrty, expressed as the logrt of the ratio (m percent) between absorbance mcrease per 30

326 C Renson et aL /AnaL Chm Acta 275 (1993) 323-328

rmn, at each concentration of sulphamethazme (B) tided by the bound acttvrty m the absence of unlabelled sulphamethazme (B,), was plotted agamst IogGulphamethazme concentration) The Rodbard and Frazier procedure of calculation was adapted (Logrvet, Wodecq, Belgmm) to enzyme mmnmoassay usmg EXCEL (Microsoft) on a Macintosh computer

a

40-

20-

RJZSULTS

Typtcal calibration graphs obtamed (a) after a short mcubatron time (2 h at 37°C) and (b) after a long mcubation time (overnight at 4°C) are pre- sented m Fig 1 The day-to-day variability of the loglt-log plot was determmed from ten cahbra- tion graphs for both types of mcubation For 2-h competitions at 37”C, the mean slope f standard deviation (S D ) and relative standard deviation (RSD) was -(062fO02) (3%) and the mrd- point of the curve at 50% bmdmg mhibmon (ED501 was (3 8 f 0 25) ng per well (6 6%) For competrttons overmght at 4”C, these values were respectively -(O 69 f 0 011 (2%) for the slope and (12 f 0 05) ng per well (4%) for the mid-pomt (n = 10)

0 - --..---I - . .-.-“I . . --..-. * - - *I 1 10 100 Ma0

ng/rrll

B/no (*I

b

40-

20-

Nme sulphonanndes were assessed for cross- reactivity with the anti-sulphamethazme antiserum The cross-reactlvmes (CR) of the antibody are given m Table 1 It is clear that other com- monly used sulphonamrdes, except sulphamerazme, had no srgmficant effects Compared with sulphamethazme, sulphamerazme, with the unique dtfference of one methyl group at the pmrdmyl C-4 position, showed 13% cross-reac- t1v1ty

o+ . . . . ..‘.I . . . . . ..‘. . . . ..ml 91 1 10 100

ng/vell

Fig 1 Cahbratlon graphs for the enzyme unmunoassay of sulphamethazme (a) 2-h mcubatlon at 3PC, (b) 16-h mcubatlon at 4°C

Evaluations of the accuracy and reproducibil- rty of the assay m muscle and hver samples are given m Table 2 The accuracy of the method was checked by recovery experunents performed on

TABLE 1

Percentage cross-reactlon of the antwrum antl-dlazotied sulphamethazme coupled to bovme serum albumm

Substance Cross-reaction (%) B Substance Cross-reactlon (%) a

Sulphamethazme loo Sulphamerazme 13 2 Sulphadlmethoxme 01 Sulphaguamdme < 0 01 Sulphaqumoxahne <OOl

Sulphachloropwdazme <OOl Sulphathlazole <OOl Sulphandanude <OOl Sulphadlazme <OOl

’ Cross-reaction (%) = nmol sulphamethazme required to reduce colour production by 50%

nmol other drug required to reduce &our production by 50%

C Renson et aL /AnaL Chun Acta 275 (1993) 323-328 327

TABLE 2

Accuracy and reproduclblhty of the assay m muscle and hver samples a

Muscle samples

ACCUWY

Added (Pg kg-‘)

Measured &gkg-Y

Recovery (%6)

100 &J13*34 81 250 2225*93 89

Reprodunblhty

Added (tig kg-‘) R S D (%I

100 250

Intra-assay (n = 8) Inter-assay (n = 8)

74 113 82 12 2

Lver samples

AccUl?lCy

Added (CLg kg-‘1

100 250

Measured Recovery (CLg kg- ‘) (%)

918+42 92. 2353*112 94

Added (fig kg-l) R SD (%I

100 250

Intra-assay (n = 8) Inter-assay (n = 8)

81 102 78 124

’ Sulpbamethazme concentrations were determmed by enzyme unmunoassay after addltlon of known amounts of sulphamethazme to a muscle or bver sample from an untreated pig Values are means of e&t determmatlons (blank values subtracted) f S D

spked tissue samples Known amounts of sulphamethazme were added to tissue samples at two concentrations and assayed m tnphcate

The recovenes were near to 100% and the difference between the true value and the mean of the determmatlons was well below the limits (-20% to + 10% for real concentrations > 10 pg kg-‘) recommended by the EEC Commlsslon Declslon (87/410) WI The reproductblhtles also fitted the recommendations of the EEC (for real concentrations > 10 pg kg-‘, R S D < 0 15%)

When used to analyse muscle samples, the hmlt of detection (mean determmed concentratron for 20 blank samples + 3 tnnes the S D > 1161 of thrs assay was established to be 4 8 pg kg-’

and the hnut of determmatlon (mean of 20 blank samples+6tnnestheSD)was54pgkg-’ In liver tissue, the hnut of detection and hmlt of determmatlon were calculated to be 5 1 and 5 7

c1g kg-‘, respectively

DISCUSSION

The enzyme nnmunoassay developed for the determmatlon of sulphamethazme residues m pig tissues extracts 1s based on a competrtion between sulphamethazme present m the samples and sulphamethazme labelled by horseradlsh peroxldase Its prmclple 1s smular to that of assays previously set up for the analysis of artlfiaal anabohcs [17] and clenbuterol [18] Associated with the matlw sohd-phase dlsperslon procedure of extractlon, this assay could be used as a routme method for the screenmg of sulphamethazme m exported and imported hver or muscle of pigs Indeed, the present screenmg control m Belgium IS based on thin-layer chromatography [19], which can occasionally lead to false-positive results The enzyme mununoassay has a hnut of detection of about 5 pg kg-l, which IS well below the tolerance lnmt of 0 1 mg kg-l that has been established for the concentration of sulphamethazme residues m edible tissues Numerous nnmuno- chemical methods have already been pubhshed for the determmatlon of sulphonanudes m blood, annnal tissues and urine Further, several test lots are commeraally avadable However, these lots are expensive for use in screenmg of large series of samples and for this reason the present system was developed Its performance was found to be slmllar to that described by McCaughey et al [ll] for the determmatlon of sulphadlmldme m pig urine

The authors thank the Belgian Instltut d’Ex- pertise V&&man-e for finanaal support They are grateful to Dr M O’Keeffe and his staff (Dunsmea Research Centre, Dubhn) for the de- velopment of the sample pretreatment method An exchange vlslt to their laboratory was made possible with finanaal support from the FLAIR (Food Lmked Agro Industrial Research, con-

328 C Renson et aL /And Chum. Acta 275 (1993) 323-328

cm-ted action No 8) programme The authors are also grateful to Dr W J McCaughey for his gen- erous sift of enzyme unmunoassay reagents

REFERENCES

1 R C Ltvmgston, J Assoc Off Anal Chem ,68 (1985) 966 2 G S Jackson, J Vet Med Assoc, 176 (1980) 1141 3 M K. Perez, J Tox~col Enwon Health, 3 (1977) 837 4 Fed Remst ,42 (1977) 62211 5 Code of Federal Regulations, New Ammal Drugs for Use

m Ammal Feeds, US Government Prmtmg Office, Wash- mgton, DC, 1983, No 21, Part 558 145

6 RB Aahworth, R L Epstein, MH Thomas and LT Froblsh, Am J Vet Res , 47 (1985) 25%

7 I Yamamoto and K. Iwata, J Immunoassay, 3 (1982) 155 8 JR Fleeker and L.J Lovett, J Assoc Off Anal Chem,

68 (1985) 172 9 H Brunn, Fle&nvxtschaft, 68 (1988) 1476

10 J Vatuluutls, J B Robbms, E Nleschlag and G T Ross, J Chn Endocrmol, 33 (1971) 988

11 W J McCaughey, CT Elhott and S R H Crooks, Vet Ret ,126 (1990) 323

12 T Chard, An Introduction to Radlommmnology and Re- lated Techmque, Elsevler Blomedrcal Press, Oxford, 1982, Pm

13 R Long Austin, LC Hsleh, M S Malbrough, CR Short and S A Barker, J Agrx Food Chem ,38 (1990) 423

14 D Rodbard and G R Frazier, RIA Data Processmg, Matenal Techmcal InformatIon Semce, Sprmgfields, VA 1973, PB 217366 and PB 217367

15 DCclslon de la Comrmss~on (87/410) du 14/07/1987, Off J Eur Commun 11/08/87 (1987) No L223/18

16 D&sum de la &nnuss~on (89/610) du 14/11/1989, Off J Eur Commun 2/12/89 (1989) No L 351/39

17 G Degand, P Schrmtz and G Maghum-Romter, J Chro- matogr ,489 (1989) 235

18 G Degand, A Bemes-Duyckaerts and G Maghum-Rogls- ter, J Agnc Food Chem , 40 (1992) 70

19 B W de Bukansk~, J M Degroodt and H Beemaert, L.ebensm -Unters -Forsch , 187 (1988) 242

determination of sulphamethazine in animal tissues by enzyme immunoassay

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