Detection of antibiotics in muscle tissue with microbiologicalinhibition tests: effects of the matrix

Lieve Okerman,* Katia De Wasch and Jan Van Hoof

Department of Veterinary Food Inspection, Faculty of Veterinary Medicine, University ofGhent, Salisburylaan 133, B-9820 Merelbeke, Belgium

Received 26th June 1998, Accepted 26th August 1998

The effects of the tissue matrix on detection limits of antibiotics with microbiological inhibition tests, intended formuscle tissue, were measured. Pieces of frozen meat were laid directly on top of paper disks impregnated withaqueous antibiotic solutions. Inhibition zones were compared with those obtained by the same standard solutionwithout tissue. Only tetracyclines were detected as efficiently with as without muscle tissue. Inhibition zones ofthe beta-lactam antibiotics ampicillin and penicillin G, and the fluoroquinolone antibiotics enrofloxacin andciprofloxacin were smaller when muscle tissue was added to low levels of standard solution. At higher levels thedifferences were not substantial. Inhibition zones of tylosin were smaller and irregular or had disappearedcompletely, while ceftiofur, sulfadimidine, erythromycin, lincomycin, and streptomycin were not detected inspiked muscle tissue at concentrations fivefold higher than the detection limits without tissue. These resultsindicate that ceftiofur, sulfonamides, streptomycin and some macrolide antibiotics cannot be detected in intactmeat with the plates and bacterial strains prescribed in the European Four Plate Test, a test which was initiallyintended as a multi-residue method for muscle tissue. Two plates of this system are not suitable for screeningpurposes; a third one detects tetracyclines and beta-lactam antibiotics in spiked tissue; the fourth one is sensitivefor beta-lactam antibiotics and for some but not all macrolides. Samples spiked with the fluoroquinolonesenrofloxacin and ciprofloxacin can be detected with an additional plate, not included in the Four Plate Test.

Introduction

Microbiological inhibition tests are considered as multi-residuescreening tests for antibiotics in milk, meat or other animaltissues. Several methods have been described to investigatedisks of frozen tissue, which are laid directly on one or moreagar media, each seeded with a susceptible bacterial strain.1–5

An inhibition test method is useful for detection of anantibiotic or a group of antibiotics, if the detection limits ofthese antibiotics are below safe levels or maximal residue limits(MRL). With agar diffusion methods used for animal tissues,such as the Four Plate Test (FPT), detection limits of antibioticshave most often been determined using aqueous solutions ofanalytical standards.1,4 However, residues are detected directlyin undiluted meat with the FPT and other comparable tests, andeffects of this matrix on detection limits never have beendetermined.

Residues of substances other than antibacterials are nowa-days most often detected with immunological or chemicalmethods. To determine the recovery of the analyte or analytes,a spiked sample is prepared by adding a known amount ofanalytical standard to the sample before it is mixed withextraction fluid. The extraction is then followed by a clean-upprocedure and a detection step. Inhibition tests such as the FPTdetect residues in intact meat, without any extraction or clean-up procedures; determination of recoveries is not necessary.During routine testing, spiked samples are not prepared and thisis another reason why matrix effects have never been meas-ured.

The composition and the properties of the medium, used in amicrobiological inhibition test, influence the detection limits ofantibiotics.3,5–7 For example, the pH of the test medium is animportant factor influencing the detection limits of mostantibiotics,3,5 and inhibitory zones produced by 500 ng sulfadi-midin may vary from 0 to more than 10 mm, depending on theorigin of the peptone in the medium.7 It is therefore possible that

tissue components, such as proteins, change the composition ofthe medium and influence the inhibitory zone produced by anantibiotic residue present in the sample.

In the present paper, we describe a method to investigatepossible influences of animal tissues on the detection limits ofcommonly used antibiotics. A piece of blank chicken, pork orbeef meat was applied directly on top of a paper diskimpregnated with an antibiotic standard solution. Inhibitionzones of standards with meat and standards without meat werecompared. The significance of our findings to routine antibioticresidue screening is discussed.

Materials and methods

The FPT described in the Manual of Reference Materials andMethods to Detect Veterinary Drug Residues, is intended todetect residues of beta-lactam antibiotics, tetracyclines, sulfo-namides, aminoglycosides and macrolides in muscle tissue ofslaughter animals.1 A fifth plate, with low detection limits ofquinolones and fluoroquinolones,2,5 was tested with threeantibiotics belonging to this group.

1. Media used for maintainance of Escherichia coli andMicrococcus luteus strains and for preparation of inocula

Tryptone soya agar (TSA) (CM131, Oxoid, Basingstoke,England) and Tryptone soya broth (TSB) (Oxoid CM 129) wereprepared and autoclaved as indicated by the manufacturers.

Mist. desiccans (MD) was used as a medium intended forconservation of bacterial strains at 220 °C and was prepared asfollows: 6 g of dextrose was added to 20 ml Brain HeartInfusion Broth (Difco 0037-17-08; Detroit, USA), preparedaccording to manufacturer’s instructions but not autoclaved.

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This solution was sterilized by filtration and added to 60 mlinactivated horse serum. The medium was kept frozen formaximum 12 months before use.

2. Bacterial suspensions

Bacillus subtilis spore suspension: Merck (Darmstadt, Ger-many) No. 10649 is a ready-to-use suspension.

M. luteus bacterial suspension: the ATCC9341 strain wasprepared as described in the Manual of Reference Materials andMethods to Detect Veterinary Drug Residues,1 but MD wasused instead of culture broth to maintain the stock inoculum. Afew colonies were suspended in 0.5 ml MD in sterile Eppendorftubes; these tubes were kept frozen until needed.

E. coli suspension: A freeze-dried strain of E. coliATCC11303 was reconstituted and inoculated onto TSA in aPetri dish. The plate was incubated for 24 h at 37 °C andinspected for purity. Sterile Eppendorf tubes with 0.5 ml MDwere inoculated with several colonies of the E. coli strain. Thisstock inoculum was kept at 220 °C for maximally two years.When needed, the stock inoculum was thawed and inoculatedonto a TSA plate. After overnight incubation at 37 °C the platewas inspected for purity. Ten ml of TSB were inoculated withseveral colonies obtained on the TSA plate and incubatedovernight. The TSB culture, which contained at least 5 3 108

colony forming units of the E. coli strain per ml, was diluted 1+ 9 in sterile TSB and added to the prepared medium cooled to45-50 °C.

3. Media for residue testing

Test agar pH 6 (Merck; dehydrated medium 10 663), test agarpH 7.2 (Merck; dehydrated medium 15 787) and test agar pH 8(Merck; dehydrated medium 10 664) were prepared andautoclaved. After cooling to 45–50 °C, the bacterial suspensionand the supplement (if necessary) were added.

Five different inoculated media were used for antibioticdetection: medium I, test agar pH 6, seeded with B. subtilis;medium II, test agar pH 7.2, with trimethoprim (Sigma, St.Louis, MO, USA; no. T-7883) added to a final concentration of0.05 mg l21, and seeded with B. subtilis; medium III, test agarpH 8, seeded with B. subtilis; medium IV, test agar pH 8, seededwith M. luteus; medium V, test agar pH 6, seeded with E. coli(0.1 ml of the diluted suspension).

Sterile Petri dishes (diameter 90 mm) were filled with 5 ml ofthe prepared and seeded media.

Antibiotic standards were all purchased from Sigma, with theexception of enrofloxacin, ciprofloxacin and ceftiofur, whichwere kindly provided by the pharmaceutical companies Bayer(Leverkusen, Germany) and Pharmacia and Upjohn (Puurs,Belgium). Detection limits of antibiotics frequently used inveterinary medicine were determined as follows: two-folddilutions were prepared and 0.01 ml of these dilutions wereapplied to 6 mm diameter paper disks. Each dilution was testedfour times on the medium considered to be most sensitive for theantibiotic tested. Concentrations producing a 12 mm diameterinhibition zone were calculated. Detection limits were asfollows: on medium I: penicillin G (sodium salt), 0.4 ng;ceftiofur (sodium salt), 9 ng; Ampicillin trihydrate, 3 ng;oxytetracycline, 8 ng; tetracycline, 5 ng; chlortetracycline,0.5 ng; doxycycline, 1 ng; on medium II: sulfadimidin, approx.50 ng; on medium III: streptomycin sulfate, 20 ng; on mediumIV: erythromycin, 1 ng; tylosin, 10 ng; lincomycin, 20 ng;penicillin G, 0.5 ng; ampicillin, 0.8 ng; ceftiofur, 2 ng; onmedium V: enrofloxacin, 2 ng; ciprofloxacin, 1 ng; flumequin,10 ng.

4. Influence of the matrix

Three plates, from one batch of medium, were used for eachconcentration tested. Four paper disks (diameter 6 mm) werelaid on each plate, at a distance of 10 mm from the edge of theplate, and 0.01 ml of an appropriate standard solution wasadded. As soon as possible, and always within 15 min, pieces offrozen chicken, pork and beef muscle tissue were each laid upontwo paper disks, on opposite sides of the plate. Each meatspecies was tested twice on one plate while the other two paperdisks on the plate served as controls. The muscle tissue had beenbored with a cork borer of 8 mm diameter and cut into disksapproximately 2 mm thick, as prescribed for the FPT. Thevolume of such tissue disks is 0.4 3 0.4 3 3.14 3 0.2 = 0.1mm3 and the weight can be estimated about 0.1 g. Platescontaining media I, II, III and V were incubated overnight at30 °C, while plates with medium IV were incubated for 24 h at37 °C. Diameters of inhibition zones of standard with muscletissue were measured and compared with zones observedaround standard disks without tissue.

Results

All results are summarized in Table 1. Six observations wereobtained with each concentration of antibiotic without meat,and six with meat. No obvious differences were seen betweenthe different meat species (data not shown in the table). Theranges of zones obtained with aqueous solutions of antibioticsand the ranges of zones obtained with meat spiked with the samelevel can be found in Table 1.

1. Beta-lactam antibiotics

Beta-lactam antibiotics were detected on two plates, butdetection limits on plate IV were lower than on plate I. Meatspiked with levels slightly higher than the respective detectionlimits did not always produce inhibition zones. Meat spikedwith 3 ng penicillin G (0.03 mg kg21) was detected on bothplates. Plate I was less sensitive for ampicillin: meat spiked with4 ng per disk was not always detected on plate I, but zones werealways wider than 12 mm on plate IV.

2. Cephalosporins

Ceftiofur was detected on plate I and on plate IV. Addition ofmeat affected the detection of ceftiofur substantially on bothplates.

Results on plate I were as follows: at 20 ng no zones occurredaround the disks with meat. At 50 ng, which is five times higherthan the detection limit, inhibition zones larger than 12 mmwere only observed in two of six cases, while the zonessurrounding the six control disks were larger than 23 mm. Thehighest level, 100 ng per disk, was only tested on plate I;inhibition zones were observed around the six meat disks, butusually they were small and in one case less than 12 mm.

Detection limits of ceftiofur were lower on plate IV. Two ofsix meat samples spiked with 20 ng ceftiofur and all six meatsamples spiked with 50 ng ceftiofur were detected on this plate.Large variations of inhibition zones of samples spiked with thesame level were seen.

3. Tetracyclines

Low quantities, approximately equal to the detection limits,were not always detected in spiked meat. With higherquantities, the zones were almost equal around paper disks with

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or without meat. Chlortetracycline and doxycycline weredetected at concentrations far below 0.1 mg kg21; the detectionlimits of oxytetracycline and tetracycline in spiked meat wereapproximately 0.1 mg kg21.

4. Aminoglycosides

Only streptomycin was tested. No inhibition zone was observedaround the meat pieces spiked with 100 ng streptomycin (1mg kg21), which is five times higher than the detection limit ofan aqueous solution.

5. Macrolides and lincosamides

Two macrolides and one lincosamide were tested on plate IV.Meat spiked with 80 ng lincomycin, corresponding with 0.8mg kg21 tissue and four times higher than the detection limit,was not detected (Fig. 1).

Erythromycin and tylosin were tested with meat at concentra-tions five-fold higher than the detection limits, 1 ng and 10 ng,respectively. No inhibition was seen with meat spiked with 5 ngerythromycin (0.05 mg kg21), while the zones of meat spikedwith 50 ng tylosin (0.5 mg kg21) were small and irregular. Meatspiked with 40 ng (0.4 mg kg21) erythromycin yielded positiveresults; diameters of zones ranged from 17 to > 30 mm.

Erythromycin and tylosin were also detectable on plate III,but the detection limits were 2 and 20 ng, respectively. Theeffects of samples spiked with 5 ng erythromycin (0.05mg kg21) and 50 ng tylosin (0.5 mg kg21) were analoguous tothose observed on plate IV. Meat spiked with 40 ng (0.4mg kg21) erythromycin was not always detected.

6. Sulfonamides

Only sulfadimidin was tested. No zones were observed roundmeat disks spiked with 200 ng and 400 ng sulfadimidin(corresponding to 2 and 4 mg kg21).

Table 1 Comparison of inhibition zones observed with paper disks impregnated with aqueous antibiotic solutions and identical disks layered with pork,beef or chicken meat

Medium Antibiotic (and detection limit)Paper disktested/ng

Diameters of zoneswithout tissue (range ofsix observations)/mm

Diameters of zones with muscle tissue(range of six observations)/mm

I Penicillin G (0.5 ng) 0.4 8–12 No inhibition1.2 17–22 10–181.5 18–21 13–183 23–26 22–25

Ceftiofur (9 ng) 20 17–20 No inhibition50 24–26 < 8–15

100 28–29 9–16Ampicillin (3 ng) 4 13–15 Absent or small and asymmetric

10 24–26 17–2315 25–26 20–2430 29–31 25–30

Doxycycline (1 ng) 1 10–12 Absent or asymmetric2.5 17–18 15–184 18–22 17–22

Chlortetracycline (1 ng) 1 12–13 9–152.5 17–19 15–204 20–22 20–21

Tetracycline (5 ng) 5 10–11 < 8–1220 18–21 18–21

Oxytetracycline (8 ng) 10 13–15 12–1520 15–16 13–1740 21–25 20–25

II Sulfadimidin (50 ng) 200 16–18 No inhibition400 20–25 No inhibition

III Streptomycin (20 ng) 100 18–22 No inhibitionTylosin (20 ng) 100 19–21 Absent or small and asymmetricErythromycin (2 ng) 10 16–20 No inhibition

40 > 30 9–18IV Penicillin G (0.5 ng) 1.2 23–25 21–25

3 22–26 22–26Ampicillin (0.8 ng) 4 25–29 24–29Ceftiofur (3 ng) 10 17–20 No inhibition

20 28– > 30 Mostly absent50 > 30 13–25

Tylosin (10 ng) 50 22–24 Absent or small and asymmetricErythromycin (1 ng) 5 19–24 No inhibition

40 > 30 17– > 30Lincomycin (20 ng) 80 28–29 No inhibition

V Flumequin (10 ng) 10 13–16 No inhibition20 21–25 13–2250 25–27 18–2280 29–30 25–29

Enrofloxacin (0.5 ng) 1 18–20 No inhibition2 22–25 18–255 25–29 25–298 28–33 27–29

Ciprofloxacin (0.3 ng) 1 21–23 < 8–112 24–27 23–275 28–29 28–308 29–31 26–31

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7. Quinolones and fluoroquinolones

Low concentrations of flumequin, enrofloxacin and cipro-floxacin (10, 1 and 1 ng per disk, respectively), which weredetected in aqueous solutions, did not produce an inhibitoryzone of 12 mm when meat was laid upon the disks. Higherconcentrations of enrofloxacin and ciprofloxacin (5 ng per disk)gave nearly equal zones with and without meat. The zones of50 ng flumequin were smaller with meat than without meat.

Discussion

Microbiological inhibition tests are intended for screening offoods for residues of antibiotics. The purpose of such tests is to

select samples which probably contain one or more analytes andwhich should be investigated with more sophisticated im-munochemical and/or chromatographic methods. Screeningtests should be simple, cheap, easy and fast. Multi-residuescreening methods are preferred to methods detecting only oneanalyte. Inhibition tests such as the FPT fit into that profile.3

In routine residue testing, the great majority of samples areevaluated on the basis of a screening test. Nevertheless, mostlaboratories still give too little attention to the reliability of theirscreening methods. Of course, the validation characteristicsdiffer between screening and confirmation methods. Analystsprefer confirmation methods that do not give false positiveresults, while a limited number of false positives is accepted inthe case of a screening test. On the other hand, the number offalse negative screening results should be as low as possible,because negative samples are accepted without further analy-sis.

Our simplified spiking method demonstrated that the FPT isnot suited for detection of many antibiotics in muscle tissue. Afalse negative result can be defined as follows: a negative resultfrom a sample spiked with a specified level of analyte,preferentially corresponding with the MRL or safe level. Thenumber of false negative results was 100% for severalantibiotics when meat was added to the standard compound,although the sensitivity of the plates was optimal (Table 2).

Sulfadimidin will not be detected with the FPT. The detectionlimit of sulfadimidin is approximately 50 ng; this correspondswith 0.5 mg kg21 tissue, which is far above the MRL.Moreover, 400 ng are not detected when combined with 0.1 g offrozen meat; this corresponds with 4 mg kg21 tissue. It is clearthat samples with sulfadimidin levels higher than the MRLcannot be detected with this method. This finding correspondswith unpublished results of routine tests in 375 pork meatsamples, obtained in our laboratory. Not one of 21 samplescontaining sulfadimidin levels above the MRL was detectedwith the FPT. Two samples with more than 1mg kg21

sulfadimidin did not cause inhibition on the plate supplementedwith trimethoprim.

The same applies for lincomycin. Lincomycin is onlydetected on the pH 8 medium seeded with M. luteus, but thedetection limit is not sufficiently low to meet the MRL. Evenlevels much higher than the MRL will not be detected in meat.The FPT should not be used for detection of this antibiotic.

Furthermore, the aminoglycoside streptomycin, which is stilloften used in combination with penicillin via the parenteral wayin large farm animals, is not detected in spiked meat with theFPT. The method is also not suited for detection of tylosin. Onthe other hand, plates III and IV are very sensitive for

Fig. 1 Inhibitory zones of 80 ng lincomycin on pH 8 medium seeded withM. luteus: 0.01 ml of an aqueous solution, containing 0.008 mg ml21

lincomycin, cause a zone of 28–29 mm diameter, but not when a disk offrozen meat is laid upon it. Growth is even more abundant around the spikedtissue, compared to the edges of the plate.

Table 2 Maximal residue limits of antibiotics in muscle tissue, and usefulness of the FPT or a fifth medium intended for detection of quinolones for eachantibiotic or antibiotic group

Group Antibiotic MRL/mg kg21Detection of MRL inaqueous solution?

Detection of MRL in spikedmuscle tissue?

Beta-lactam Penicillin G 0.05 Yes, on plates I and IV YesAmpicillin 0.05 Yes, on plates I and IV Not always on plate I

Cephalosporins Ceftiofur 0.2 (B)0.5 (P)

Yes, on plates I and IV NoMost often not found

Tetracyclines Oxytetracycline 0.1 Yes, on plate I YesTetracycline 0.1 Yes, on plate I YesChlortetracycline 0.1 Yes, on plate I YesDoxycycline 0.1 Yes, on plate I Yes

Sulfonamides Sulfadimidin 0.1 No NoAminoglycosides Streptomycin 0.5 Yes, on plate III NoMacrolides Erythromycin 0.4 Yes, on plates III and IV Not always on plate III

Tylosin 0.1 Yes, on Plate IV NoLincosamides Lincomycin 0.05 (P) No NoQuinolones Flumequin 0.05 No No

Enrofloxacin 0.03 Yes, on plate V YesCiprofloxacin 0.03 Yes, on plate V Yes

* B = beef; P = pork.

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erythromycin. Although the detection was affected substantiallyby the addition of a meat disk, all samples spiked with 40 ngerythromycin, corresponding with the MRL of this antibiotic,produced a zone wider than 12 mm on plate IV.

Only tetracyclines and quinolones are detected nearly equallywell with as without meat. Probably all samples with levels ofoxytetracycline, doxycycline, tetracycline, chlortetracycline,enrofloxacin or ciprofloxacin equal to or higher than the MRL,will be detected. This applies only to spiked samples; it is stillpossible that residues of one or more antibiotics belonging tothese groups do not diffuse completely into the medium, whenthey are present in naturally contaminated samples.

However, our experience with routine samples indicates thattetracyclines are easily found with the FPT. A large survey onantibiotic residues in poultry, beef, veal and pork meat obtainedfrom retail outlets revealed that 2% of the samples inhibitedB. subtilis. The majority of these positives contained a tetra-cycline.8 Similar observations were made with other routinemeat samples (unpublished data). In 1997, we tested 1768poultry and 173 pork samples for presence of inhibitingsubstances on plate I of the FPT. We found, respectively, 103(5.8%) positive poultry samples, and 20 (8.3%) positive porkmeat samples. A limited number of these positive samples wereconfirmed using an enzyme immunoassay (EIA) and with aliquid chromatography-mass spectrometry (LC-MS) method,and we found a fair correlation between the surface of theinhibition zones and the actual amount of doxycycline measuredwith LC-MS.9 In this series, doxycycline levels higher than theMRL were not found in samples with inhibition rings smallerthan 5 mm (diameter of the zone ≈ 18 mm). This approachesthe zone produced by the same level of doxycycline in anaqueous solution. On the basis of these data, it can be assumedthat the method is suitable for detection of tetracyclines unlessother observations contradict this statement.

Low levels of the beta-lactam antibiotics penicillin G andampicillin are detected more readily in aqueous solutions thanin spiked samples. Detection of MRL levels is, however, stillpossible for both antibiotics. The detection limit of ampicillin islower on the pH 8 medium seeded with M. luteus than on thepH 6 medium seeded with B. subtilis. Penicilline G levels equalto the MRL can be found in spiked meat on both media. MRLlevels of ceftiofur are not detected in spiked meat on the pH 6plate with B. subtilis, and not always on the plate seeded with M.luteus.

In routine practice we have confirmed the presence of beta-lactam with EIA in samples which had reacted positively on oneor more of the plates of the system (unpublished observations).High levels of the fluoroquinolones enrofloxacin and cipro-floxacin were found with LC-MS in two FPT positive samplesof chicken meat.8 Up till now, we do not have sufficient data tocompare the actual levels with inhibition zones, and it cannot beascertained that all naturally contaminated samples with levelsabove the MRL will cause inhibition zones on the appropriateplates. This should preferably be confirmed with incurredsamples.

The causes of the differences between detection limits ofantibiotics in aqueous solution and in spiked muscle tissue werenot investigated. With some antibiotics the difference was onlyobserved at low levels. This can be due to variation in diffusioninto the agar layer. The phenomenon was not observed withhigh levels of tetracyclines and the fluoroquinolones cipro-floxacin and enrofloxacin. It was seen, but to a lesser extent,with beta-lactam antibiotics and the quinolone flumequin. Apossible explanation for the nearly total absence of inhibitoryzones with high levels of sulfadimidin, ceftiofur and macrolides

is a change in composition of the medium. The modified FPTprescribes the use of a thin layer of agar medium; only 5 ml arepoured into 90 mm plates, which is just enough to cover thesurface. Thin agar layers cause higher sensitivities for aqueoussolutions of antibiotics. On these thin layers 2 mm thick piecesof frozen meat are laid. Soluble cell contents as well as theantibiotics diffuse into the agar. Normal muscle tissue has a pHlower than 6. It is clear that the pH of the medium is influencedby the matrix. Furthermore, the diffusion of proteins or othernutrients into the medium may influence the detection ofantibiotics too. The definition of matrix effect, as used inanalytical chemistry, is an influence of the matrix on thesensitivity of the sensor.10 Obviously, this is the case with theinhibition test: very often test strains grow more abundantlyaround blank meat disks, and this phenomenon was evenobserved around the spiked tissues, as can be seen in Fig. 1.

When considering the FPT, it was concluded that the plateswith media at pH 7.2 and pH 8, both seeded with B. subtilis andintended for the detection of sulfonamides and aminoglyco-sides, are not of any interest. They do not permit the detectionof muscle tissue spiked with sulfadimidin or streptomycin levelsfar above the MRL. Only tetracyclines and beta-lactamantibiotics can be detected up to the MRL level in spiked muscletissue. One or two plates are sufficient for that purpose. Theplate seeded with M. luteus detects penicillin and ampicillin upto the MRL level, but is not suited to detect all macrolides andlincosamides in spiked tissue. The addition of a plate seededwith E. coli to the system will facilitate the detection ofquinolones and fluoroquinolones. These statements are basedupon the demonstration of an effect of the tissue matrix, addedto the antibiotic standards. Further evidence is needed that themajority of naturally contaminated or incurred samples, withresidue levels equal to the MRL, will also react positive.

Acknowledgements

We thank Martine Boonaert and Ghislaine Vermassen fortechnical help, and Chris Puttevils for the photograph. Weappreciated helpful discussion with Régine Fuselier.

References

1 Veterinary Drug Residues—Residues in Food Producing Animalsand their Products: Reference Materials and Methods, ed. R. J.Heitzman, Blackwell Scientific Publications, Oxford, 1994, pp. Sg3/1–8.

2 L. Ellerbroek, Fleischwirtschaft, 1991, 71, 187.3 G. O. Korsrud, J. O. Boison, J. F. M. Nouws and J. D. MacNeil,

J. AOAC Int., 1998, 81, 21.4 A. M. Ferrini, V. Mannoni and P. Aureli, Arch. Lebensmittelhyg,

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H.-J. Pluta and G. Arndt, Arch. Lebensmittelhyg, 1997, 48, 3.6 J. F. M. Nouws, M. van Schothorst and G. Ziv, Arch. Leben-

smittelhyg., 1979, 30, 4.7 L. Ellerbroek, Arch. Lebensmittelhyg., 1998, 39, 7.8 L. Okerman, J. Van Hoof and W. Debeuckelaere, J. AOAC Int., 1998,

81, 51.9 K. De Wasch, L. Okerman, C. Croubels, H. De Brabander, J. Van

Hoof and P. De Backer, Analyst, 1998, in the press.10 Chemometrics: a Textbook, ed. D. L. Massart, B. G. M. Vandeginste,

S. N. Deming, Y. Michotte and L. Kaufman, Elsevier, Amsterdam,1988, ch. 8, pp. 115–117.

Paper 8/04903C

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