Francesca Diana

Maurizio Paleologo

& Lidija Persic

Validation of two enzyme immunoassays for aminoglycoside residues according to European Decision 657/2002 2 4 6

Keywords: Immunoassays, Screening -ELISA Additives/Contaminants: Veterinary drug residues -antibiotics Food Types: Meat, Milk gentamicin, neomycin, EIA, drug residues 42

Aminoglycoside antibiotics are commonly used in the treatment of bacterial infections 28 in human and veterinary practice. Because of their toxicity, the European Community has established Maximum Residue Limits (MRL) in foodstuffs of animal origin (EEC 30 No 2377/90). In the present work the performance of two new enzyme immunoassays (EIA), I'screen Gentamicin and I'screen Neomycin, for the quantitative detection of the 32 aminoglycosides gentamicin and neomycin in milk and tissue are described. The validation of these EIAs has been performed in accordance to criteria of the European 34 Decision 657/2002. Assays sensitivity at the MRLs was 95% for milk samples and 100% for tissue samples, while specificity was 100% at 33% and 25% of the MRLs for 36 milk and tissues, respectively. The performance of these EIAs indicates that they can be used as easy screening methods in the analysis of aminoglycosides in milk and tissue 38 samples.

Test samples

Twenty raw milk samples, derived from untreated cows, were kind gifts from AAFVG 110 (Associazione Allevatori del Friuli Venezia Giulia). Twenty tissue samples (ten bovine and ten swine muscles) were purchased in a supermarket, guaranteed as residue free 112 ("Prodotti con Amore", COOP Italia).

114

Gentamicin EIA

The anti-gentamicin antibody was raised in rabbit by three cycles of immunization with 116 gentamicin-BSA conjugates, obtained using EDC and Sulpho-NHS [START_REF] Hermanson | Zero-lenght cross-linkers[END_REF]. Microtiter plates were coated with anti-gentamicin antibodies purified on a Protein A 118 Sepharose CL-4B column. The assay design is a direct competitive enzyme immunoassay, because during the first incubation, standards/samples and gentamicin-120 HRP-conjugate (obtained by EDC and Sulpho-NHS mediated conjugation procedures, [START_REF] Hermanson | Zero-lenght cross-linkers[END_REF]) compete for the antibodies binding sites on the microtiter plate (30 122 minutes, room temperature). The amount of enzyme conjugate which remains bound after washings is inversely proportional to the amount of the analyte in 124 standards/samples and is determined by measuring the absorbance after a developing reaction step of 30 minutes using tetramethyl benzydine (TMB) as chromogenic HRP-126 substrate. To stop the reaction, a sulphuric acid solution is then added, and the absorbance is measured at 450 nm by a microplate reader (Sunrise, Tecan). The anti-neomycin antibody was raised in rabbit by three cycles of immunization with neomycin-BSA conjugates, obtained using EDC and Sulpho-NHS [START_REF] Hermanson | Zero-lenght cross-linkers[END_REF].

144

Microtiter plates were coated with anti-neomycin antibodies purified on a Protein A Sepharose CL-4B column. As the gentamicin assay, the I'screen Neomycin test kit is a 146 competitive enzyme immunoassay. The assay is carried out in the same way, except that the first incubation is for 20 min and the second for 10 min. Data handling was also 148 performed with the same instrument, the same software, the same curve fitting. Also in this case two different calibration curves for the analysis of milk or meat samples were 150 used, but both curves were freshly prepared at each analysis.

Sample preparation

Sample preparation was the same for both EIAs. The milk samples were refrigerated 162 and centrifuged at 4°C for 10 minutes at 3000g. The fat was discarded and the skimmed milk was diluted 10 times with the dilution buffer provided by the kit. The tissues 164 samples were extracted according to protocols previously reported [START_REF] Haasnoot | 452 Immunochemical detection of aminoglycosides in milk and kidney[END_REF][START_REF] Brown | Improved sodium hydroxide 426 digestion method without homogeneization for extraction of gentamicin from renal tissue[END_REF][START_REF] Fox | Total extraction of aminoglycosides from guinea pig and bullfrog tissues with sodium hydroxide or triochloroacetic acid[END_REF], with some modifications. Briefly, to 1 g of tissue 4 166 ml of a 3% trichloroacetic acid solution (TCA) were added. After homogenization for 1

Results and discussion

172 Immunoassays performance I'screen Gentamicin. Figure 1 shows calibration curves obtained (10 runs) for the 174 gentamicin enzyme immunoassay, i.e. the buffer and the milk calibration curves. In the preliminary work carried out during the development of the assay, no matrix effect was 176 observed for tissue samples when analysed in respect to a calibration curve made from gentamicin in buffer solutions (data not shown), obtaining a specificity of 100% for 178 negative tissue samples. On the contrary, the spiking of milk samples using a buffer calibration curve determined an over-estimation of the analyte content because of 180 matrix interference. Therefore, in order to compensate for matrix effects and to achieve higher specificity, it was necessary to provide two different calibration curves. for the buffer calibration curve and 5-250 µg L -1 for the milk calibration curve. Since sample preparation procedures determine a ten folds dilution factor, these calibration 194 curves allow the analysis of samples contaminated with amounts of gentamicin in the range of 25-2500 µg kg -1 for tissue and 50-2500 µg L -1 for milk. Therefore 196 contamination levels around MRLs can be easily detected without the need of excessive sample dilution, providing an accurate measurement.

198

The intra-assay precision of I'screen Gentamicin (calculated as Coefficient of Variation 200 %, CV) was obtained testing three duplicates of buffer and milk standard solutions. CVs of the mean absorbances were always less than 5%. The intra-assay and inter-assay dose 202 CVs, evaluated in three runs, were below 10% in between 5 and 100 µg L -1 , both for milk and buffer standard solutions.

204

The gentamicin EIA is highly specific, since cross reactivities against other 206 aminoglycosides (neomycin, streptomycin, kanamycin) are below 0.1%. It is stable after storage at +4°C for 1 year, since after incubation of the kit at 37°C for 1 week, assay 208 performances (OD values and calibration curve IC 50 ) are not significantly changed (data not shown) [START_REF] Deshpande | Enzyme Immunoassays: from concept to product 444 development[END_REF]. 210 I'screen Neomycin. Figure 2 shows buffer and milk calibration curves obtained for 212 neomycin assay (mean of 10 runs) and Table II summarizes their parameters. As for the gentamicin assay, the milk calibration curve is necessary to compensate matrix effect of 214 milk samples, while tissue samples can be analysed using a buffer calibration curve.

The values obtained for LODs, IC 50 , slope and R 2 show also for this assay a high quality 216 of calibration, as well as a high repeatability of results in case of buffer calibration, MRL of neomycin are 500 µg kg -1 for muscle, 5000 µg kg -1 for kidney and 1500 µg L -1 222 for milk. On the basis of LODs reported in Table II, the calibration ranges chosen were 10-1000 ng/ml for the buffer calibration curve and 50-1000 µg L -1 for the milk The intra-assay precision of I'screen Neomycin was determined testing the standard 232 solutions with three duplicates. CVs of the mean absorbance were always < 10%. The intra-assay dose CV testing buffer standard solutions was below 10% in between 10 and 234 100 ng/ml. The intra-assay dose CV testing milk standard solutions was below 5% in between 100 and 500 µg L -1 . Dose inter-assay CVs, evaluated in three different 236 experiments, were less than 15% for buffer standard solutions and less than 20% for milk standard solutions.

238

The specificity of the assay was studied by testing other aminoglycoside antibiotics 240 (gentamicin, streptomycin, kanamycin): cross-reactivities were in all cases below 0.1%.

The stability of the reagents was evaluated incubating the test kit at 37°C for one week: Milk samples. According to EU regulations, a screening method for the detection of veterinary drug residues must guarantee 5% or less false negative results

254

(2002/657/EC). To be cost effective, this method must guarantee that a food sample containing residues at a concentration lower than the MRL should be classified as The detection capabilities (CCβ) were therefore 100 µg kg -1 for I'screen Gentamicin 278 and 1500 µg kg -1 for I'screen Neomycin. It can be concluded that both assays had a good diagnostic specificity and a high sensitivity at the MRL. shown in Figure 3. The correlation obtained between spiked and measured concentrations was quite good for gentamicin (r 2 = 0.979, S y.x = 32.49 µg L -1 ), while it 286 was weaker for neomycin (r 2 = 0.710, S y.x = 398.9 µg L -1 ). The slope of the linear regression was, on the opposite, close to 1 in case of neomycin (1.10), higher in case of dosing ranges, but the bias was higher in case of neomycin: the mean recoveries at each spiking ranged in fact from 128% to 152% for gentamicin, and from 132% to 201% for 294 neomycin.

296

I'screen Gentamicin precision was calculated as Coefficient of Variation % (CV) of the intra-(n=3 duplicates) and inter-assay measurements (n=3 days) of a blank sample 298 fortified with amounts of analyte corresponding to 0.5 MRL, MRL, and 5 MRL (50, 100 and 500 µg kg -1 ). The intra-assay CVs were all below 7% (4.90, 6.84 and 0.25%, 300 respectively). The inter-assay CVs were all below 8% (7.22, 0.45 and 3.57%, respectively).

302

In the same way was calculated I'screen Neomycin precision. A blank sample fortified 304 with amounts of analyte at 0.66 MRL and MRL levels (1000 and 1500 µg kg -1 ) was tested. The intra-assay CVs were always below 5% (3.50 and 1.93, respectively); inter-306 assay CVs were 22.99 for and 18.74 % respectively. 308 Tissue samples. A successful tissue extraction method should make the tissue-bound aminoglycosides soluble, remove most of the proteins, eliminate other matrix 310 interferences and provide satisfactory and reproducible recoveries. Homogenization of samples with TCA solutions is commonly used to precipitate tissue proteins and to 312 obtain high recoveries of analytes. This method was applied in the analysis of twenty blank muscle samples (ten bovine and ten swine).

314

The limits of decision and the detection capabilities were established as for milk testing. Accuracy in tissue testing was evaluated in the range 100-1000 µg kg -1 for neomycin 326 and in the range 50-500 µg kg -1 for gentamicin. The correlations between spiked and measured concentration were quite good for both analytes (r 2 > 0.95). The slope was 328 close to 1 in case of gentamicin, lower in case of neomycin (see Figure 4). S y.x values were also quite low: 27.78 and 46.66 µg kg -1 for gentamicin and neomycin, 330 respectively. In case of gentamicin the recovery at the MRL was 160±40%, in case of neomycin it was 94 ± 9%. [Insert Figure 4 about here] 332

The precision of I'screen Gentamicin and I'screen Neomycin assays in tissue testing 334 was studied analysing three times the same fortified muscle sample. Gentamicin was spiked at MRL (50 µg kg -1 ) and 2 MRL (100 µg kg -1 ), obtaining intra assay CVs (n=3 336 duplicates) of 5.35 and 6.84%, respectively and inter-assay CVs (n=3 days) of 12.33 and 14.56%, respectively. In the same way neomycin was spiked at MRL (500 µg kg using the Youden's approach (Youden and Steiner 1975;2002/657/EC). The method implies the deliberate introduction of minor reasonable simultaneous variations of 344 parameters in the test and the observation of their consequences. In both tests, variations in seven parameters in respect to established procedure were introduced: the enzyme 346 conjugate dilution factor, the enzyme conjugate batch, the assay temperature, the assay incubation time, the development incubation time, the number of washings and the kit 348 storage temperature. According to Youden's approach, the effect of each variation was determined by a set of eight combinations, where the parameters were alternated in their 350 "control mode" (according to the established procedure) and "changed mode". For each parameter, the difference between the mean result obtained in the "control mode" and 352 the mean result obtained in the "changed mode" was determined (D i ). The analyses were carried out with a muscle sample, spiked with 100 µg kg -1 of gentamicin and 1000 354 µg kg -1 of neomycin. As shown in Table V, the effect of each parameter is given by the calculated difference D i . The standard deviation of the differences (S Di ) for each set of When the calculated value for the standard deviation of the differences is significantly larger than the inter-assay precision, the test is considered not to be robust. Furthermore, 362 the application of a t-test for each variable gives the opportunity to identify the most disturbing variation factors [START_REF] Scortichini | ELISA qualitative screening of chloramphenicol in muscle, eggs, honey and milk: 484 method validation according to the Commission Decision 2002/657/EC criteria[END_REF]; the experimental t was calculated where n (n=4) is the number of experiments carried out for each parameter in the "control mode" or in the "changed mode", and CV is the inter-assay precision (n=9) 370 [START_REF] Forti | Determination of 446 chloramphenicol in honey by liquid chromatography-tandem mass spectrometry[END_REF].

372

As shown in Table V, the standard deviations of the differences (10.79% for I'screen Gentamicin and 14.70% for I'screen Neomycin) were lower than the inter-assay 374 precisions obtained for the spiked level considered (14.56% and 15.58% respectively), indicating that both EIAs are robust. Moreover, experimental t values were always 376 lower than the two-tailed t critical value (t crit =2.3, υ=9-1, 95% confidence level).

[Insert Table V about here] 378

From the results obtained it can be concluded that none of the changes introduced in the 380 considered parameters negatively influenced the results of the tests; however, results shown in Table V indicate that kits storage temperature is the most critical factor for the 382 performance of I'screen Gentamicin and I'screen Neomycin and should be strictly controlled.

384

Conclusions

386

Immunoassays previously reported in the literature for neomycin and gentamicin detection in milk and meat did not provide data to estimate the percentage of false 388 positive and false negative results at or close to the MRL [START_REF] Haasnoot | 452 Immunochemical detection of aminoglycosides in milk and kidney[END_REF][START_REF] Loomans | Neamin as an immunogen for the development of a generic ELISA detecting gentamicin, 470 kanamycin and neomycin in milk[END_REF]. It has been shown that commercially available kits are easy to use 390 and very sensitive, but quite inaccurate [START_REF] Jànosi | Comparison of three ELISA kits for aminoglycoside detection in milk[END_REF]). Moreover, the approach employed by test kit manufacturers was not adequate for regulatory controls of 392 registered antimicrobials. In order to determine the appropriate decision limits to be applied, as far as we are aware no validation study was performed. In accordance with 394 the European Decision 657/2002, screening methods have to be sensitive enough to detect 95% of non-compliant samples, but, on the other hand, it is also necessary that 396 false positive rates are kept low. Without an appropriate limit of decision, the rate of false non-compliant samples could be excessive and therefore a high number of 398 confirmatory analyses would be required. Confirmatory analyses for aminoglycosides are quite expensive and a very small number of laboratories are accredited to perform it. Two different calibration curves were applied depending on the samples: the milk standard curve in case of milk and the buffer standard curve in case of tissue. Two different calibration curves were applied depending on the samples: the milk standard curve in case of milk and the buffer standard curve in case of tissue.

  closely-related antibiotics produced by Streptomyces spp. and were determined. The decision limit (CCα) and recovery for milk and tissue samples in both assays were also investigated. , neomycin trisulfate salt hydrate, gentamicin solution, neomycin solution, streptomycin sesquisulfate, kanamycin, bovine serum albumin (BSA), 100 horseradish peroxidase (HRP), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) were purchased from Sigma. 102 N-hydroxysulfosuccinimide (sulfo-NHS) was obtained from Pierce. Protein A 104 Sepharose CL-4B was obtained from GE Healthcare Europe Gmbh. The EIA kits I'screen Gentamicin and I'screen Neomycin (Tecna Srl, Trieste, Italy) contain all the 106 necessary materials and methods for the assay.108

128

  All the absorbance values were transformed into relative signal (OD/OD of zero 130 standard = B/B 0 ). Results were elaborated through the software "Magellan" (Tecan).The four parameter logistic was chosen as algorithm to fit the calibration curve 132 according to the following formula: y = (A -D) / [1+ (x/C) B ] + D, where A is the maximal absorbance, D is the minimum absorbance, C is the concentration that 134 produces a response halfway between A and D, while B is the slope at the inflection point of the sigmoidal curve. 136 Gentamicin in tissue extracts was determined through ready-to-use buffer standard 138 solutions, while for milk samples a calibration curve in matrix was used. Milk standard solutions were freshly prepared at each analysis.

152

  Cross-reactivities determinationCalibration curves of different aminoglycosides antibiotics (gentamicin, neomycin, 154 streptomycin, kanamicin) were prepared using the test kit dilution buffers. The crossreactivities values were calculated from the calibration curves obtained, according to the 156 following equation: (IC 50 of gentamicin or neomycin / IC 50 of the tested compound) X 100; IC 50 is the concentration producing the 50% of the maximal absorbance (B/B 0

  sample was mixed head over head for 30 minutes, then centrifuged 10 168 minutes at 2000g at 4°C. The extracts were diluted 2 times with dilution buffer and pH was adjusted to 7.4 with a 0.1M NaOH solution. 170

  shows similar sensitivity but lower repeatability. [Insert Figure 2 218 and Table II about here] 220 According to EU Regulations (EC No 2377/90; EC No 1960/2000; EC No 1181/2002)

  224 calibration curve. Considering the ten folds dilution factor of samples (see experimental), these calibration curves allow the detection of amounts of neomycin in 226 the range of 100-10 000 µg kg -1 in tissue and 500-10 000 µg L -1 in milk. The use of two different calibration curves allows therefore the identification of samples contaminated 228 with levels of antibiotic around MRLs; moreover, MRLs were in both cases in the linear part of the calibration curves.

  230

  values, nor the calibration curve IC 50 were significantly different from t 0 , meaning that the kit is stable at +4°C after 1 year storage (data not shown) Decision 657/2002, for the validation of I'screen Gentamicin 248 and I'screen Neomycin the following performance characteristics were determined: Decision Limit (CCα), specificity, Detection Capability (CCβ), recovery, precision, and 250 ruggedness.

  252

  256"negative" (compliant). The cut-off values, i.e. the Limit of Decision (CCα), should be established taking into account both requirements. In order to keep the false non-258 compliant occurrence at a low rate, CCα values were determined as the B/B 0 value of MRL spiked samples + 1.64 SD.260To calculate the probability of false positive and false negative results, 20 blank 262 samples were employed; these samples were fortified at MRL concentrations (100 µg kg -1 of gentamicin and 1500 µg kg -1 of neomycin), as well as at lower concentrations.264TableIIIshows B/B 0 % values for blank and fortified samples, false compliant rates (β errors) and false non-compliant rates (α errors). [Insert TableIIIabout here] At the established Limit of Decision, in I'screen Gentamicin no false non-compliant 268 sample was obtained (0% α error) evaluating the blank milk samples, as well as spiked at 33 µg L -1 (0.33 MRL) and at 50 µg L -1 (0.5 MRL), indicating an assay specificity of 270 100%.272Regarding I'screen Neomycin, no false non-compliant were obtained testing blanks or 0.33 MRL spiked samples, but the specificity decreased with samples fortified at 0.66 274 MRL, generating 50% of false non-compliant results.276 Sensitivity evaluated at MRL contamination values for both kits was 95% (5% β error).

280

  Accuracy was studied by fortification of 20 blank milk samples with four 282 concentrations of both analytes above and below respective MRLs (50, 100, 250 and 500 µg L -1 of gentamicin and 500, 100, 1500 and 2000 µg L -1 of neomycin). Results are 284

Page

  27). [Insert Figure 3 about here] 290 Mean recovery at the MRL was 128±14% for gentamicin and 132±22% for neomycin. Both assays overestimated the true content of analyte, particularly in the lower part of 292

  316TableIVshows B/B 0 %, and α and β errors for both kits. Evaluating the blank muscle established limits of decision, no false non-compliant occurred by both 318 immunoassays (0% α error). No false non-compliant incurred even testing blanks spiked at 0.25 MRL (12.5 µg kg -1 of gentamicin and 125 µg kg -1 of neomycin) by both 320 kits. The false compliant rate (β error) was 0% testing the samples spiked at the MRLs with both test kits, indicating a 100% sensitivity at the MRLs. Considering the results322shown, detection capabilities (CCβ) were 50 µg kg -1 for I'screen Gentamicin, and 500 µg kg -1 for I'screen Neomycin. [Insert

400Page

  From the results presented, we can claim that the two I'screen test kits can satisfy both 402 the specificity and the sensitivity requirements. For milk, as well as for meat testing, CCα have been established close enough to the EU MRLs to minimize false non-404 compliant results. The α error was actually very low even with food samples contaminated at concentrations lower than violating levels. At the same time, violating 406 samples, contaminated at the MRL, have been correctly classified as non-compliant in 95% of milk samples, and in 100% of meat samples.408The use of two different calibration curves in the analysis of milk or tissue samples and 410 a moderate dilution factor contribute to the accuracy of the assays, which, at the MRL level, was in the range between 94% and 160%.412Taking into account the matrices tested and the procedures of their preparation, 414 screening by the test kits here presented meets the quality criteria of EU Decision 657/2002, guaranteeing, at the same time, a low rate of false compliant results. The 416 EIA kits indicates that they can be therefore used as easy and costeffective screening methods in the analysis of aminoglycosides in milk and

Figure 3 .

 3 Figure 1. I'screen Gentamicin EIA calibration curves (mean ± SD, n=10). Two different calibration curves were applied depending on the samples: the milk standard curve in case of milk and the buffer standard curve in case of tissue. 79x55mm (600 x 600 DPI)

Figure 4 .Figure 1 .

 41 Figure 4. Recoveries studies to assess the accuracy of muscle samples testing (mean ± SD, n=20). 79x74mm (600 x 600 DPI)

Figure 2 .

 2 Figure 2. I'screen Neomycin EIA calibration curves (mean ± SD, n=10).

Figure 3 .

 3 Figure 3. Recoveries studies to assess the accuracy of milk samples testing (mean ± SD, n=20).

Figure 4 .

 4 Figure 4. Recoveries studies to assess the accuracy of muscle samples testing (mean ± SD, n=20).

Table I

 I

		F o r
		P
		e
		e r
		R
		e
	182 184	v i e summarizes the parameters of these calibration curves; limits of detection (LOD), calculated as the zero standard OD minus 3 standard deviations (SD), were so low that w
	186	spiking ranges could start at a few ng/ml. Slopes and R 2 values show a high quality of calibration. The low values for standard deviations of LOD, IC 50 and R 2 show the high O n l repeatability of assay results. [Insert Figure 1 and Table I about here] y
	188
		According to European Commission Regulation No 2377/90 and followings (EC No
	190	2377/90; EC No 1960/2000; EC No 868/2002), maximum residue limits of gentamicin
		(MRL) are 50 µg kg -1 for muscle, 750 µg kg -1 for kidney and 100 µg L -1 for milk. On
	192	the basis of LODs reported in Table I, the calibration ranges chosen were 2.5-250 µg L -1

  Table IV about here]

	324

Table I

 I Parameters of gentamicin EIA. Reported values are the mean ± standard deviation relative to 10 calibration curves run on different days

	Page 27 of 33	Food Additives and Contaminants
	Table II Table III Table IV Table V
	Ruggedness test results
	Parameters of neomycin EIA. Reported values are the mean ± standard B/B 0 values of blank and spiked milk samples (n = 20) B/B 0 values of blank and spiked muscle samples (n = 20) I'screen Gentamicin	I'screen Neomycin
	Parameter deviation relative to 10 calibration curves run on different days Buffer calibration curve Milk calibration curve Parameter Buffer calibration curve Milk calibration curve parameter sample I'screen Gentamicin I'screen Neomycin blanks 106.95 ± 4.11 96.16 ± 2.82 Parameter Sample I'screen Gentamicin I'screen Neomycin blanks 98.53 ± 2.61 93.39 ± 2.96 Parameter Difference (D i ) in % recovery (absolute value) t-value Difference (D i ) in % recovery (absolute value)	t-value
	OD max (zero st.) LOD a , ng ml -1 IC 80 , ng ml -1 IC 50 , ng ml -1 slope R 2 a LOD: zero standard OD -3 standard deviations 1.32 ± 0.23 3.02 ± 0.08 8.17 ± 1.18 36.60 ± 8.75 -0.953 ± 0.117 0.9995 ± 0.0005 F o r OD max (zero st.) 1.13 ± 0.19 LOD, ng ml -1 10.20 ± 0.40 IC 80 , ng ml -1 22.54 ± 1.67 0.33 MRL 93.81 ± 3,92 0.5 MRL 85.77 ± 3.70 0.66 MRL ND a 0.25 MRL 95,03 ± 2.40 MRL 84.04 ± 3.07 Enzyme conjugate dilution factor 8.30 B/B 0 (%) Enzyme conjugate batch 4.44 B/B 0 (%) CCα 89.07 Assay temperature 0.01 IC 50 , ng ml -1 96.53 ± 10.80 slope -0.925 ± 0.088 R 2 0.9998 ± 0.0003 F o r MRL 76.63 ± 2.53 CCα 80.78 β error (%) MRL 5 blanks 0 0 Assay incubation time 8.89 β error (%) MRL blanks 0 Development incubation time 2.61 α error (%) 0.25 MRL 0 Number of washings 3.01 Kit storage temperature 14.97 0.33 MRL 0 0.5 MRL 0 α error (%) 0.66 MRL ND a a ND: not determined F o r r o F S Di 10.79%	1.19 ± 0.21 6.53 ± 0.11 12.32 ± 2.17 64.16 ± 19.26 -0.774 ± 0.143 0.9990 ± 0.0006 1.54 ± 0.14 48.47 ± 3.32 91.35 ± 19.35 81.60 ± 3.58 ND a 69.86 ± 3.25 82.64 ± 1.96 67.87 ± 2.07 1.05 0.56 0.00 71.26 334.07 ± 80.67 -0.971 ± 0.242 0.9988 ± 0.0014 63.17 ± 4.54 70.61 5 0 1,13 0.33 0 0.38 0 1.90 0 ND a 50 0	9.93 12.45 2.30 4.92 8.55 10.73 16.92 14.70%	0.90 1.13 0.21 0,45 0.78 0.98 1.54
		P P P P
		e e e e
		e r e r e r e r
			R R R R
			e e e e
			v i e v i e v i e v i e
			w w w w
				O n l O n l O n l O n l
				y y y y

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Acknowledgements

AAFVG (Associazione Allevatori del Friuli Venezia Giulia, Codroipo, Italy), for 422 providing milk samples. 424


Mariel G Pikkemaat

Patrick P J Mulder

J W Alexander Elferink

Michel Nielen

Angela De Cocq

Harry J Van Egmond

An improved microbial

Keywords: Veterinary drug residues -fluoroquinolones, Veterinary drug residues -oxolinic acid Antibiotic residues, screening method, inhibition test, LC-MS/MS, quinolones, poultry, egg, Premi  Test

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Quinolones are a class of synthetic antibiotic drugs that are commonly used in human and veterinary medicine. In poultry they are used to treat respiratory and intestinal infections caused by Gram negative bacteria. Quinolone antibiotics act by inhibiting the bacterial DNA gyrase, causing inhibition of DNA replication. Since their introduction in veterinary medicine in the early 1990s, a significantly increased incidence of quinolone resistant E. coli and salmonella has been reported [START_REF] Hopkins | Mechanisms of quinolone resistance in Escherichia coli and Salmonella: Recent developments[END_REF]. Although none of the quinolones licensed for clinical use is approved for veterinary use, they all share a common mechanism of action and are therefore likely to induce the similar of modes of resistance. This is of great concern regarding the transfer of resistance to human pathogens (Bogaard and Stobberingh 2000). A significant correlation has been observed between the licensing of enrofloxacin for veterinary use and decreased susceptibility to ciprofloxacin in salmonella isolated from humans (Trelfall et al. 1997). Quinolones were therefore chosen as one of the priority groups of residues within EU-project "New technologies to screen multiple chemical contaminants in foods" (acronym BioCop).

To protect the consumer from exposure to residue levels that might constitute a health risk, the European Union has introduced legislation with regard to authorisation of veterinary medicine. The approval of veterinary medicinal products can only occur after an extensive safety and residue evaluation and subsequent registration in Annex I, II or III of Council Regulation 2377/90 (EC 1990). For substances included in Annex I and III the registration includes establishment of Maximum Residue Limits (MRLs). In practice for economic reasons veterinary drugs are only developed for "major" food-producing species. As a result only very few substances have MRLs established in eggs, so only a very limited number of products are allowed to be used in animals from which eggs are produced for human consumption. This situation provokes 'off label' and illegal administration of medicinal products to laying hens, making quinolones an important class of drugs for the monitoring of residues in poultry products.

Food surveillance programs intended to maintain legislation concerning the presence of drug residues rely heavily on the availability of fast and accurate screening methods.

Analytical methods for the determination of quinolone residues in animal products are widely available (Munns et al.1998;Yorke and Froc 2000;[START_REF] Schneider | Multiresidue determination of fluoroquinolone antibiotics in eggs using liquid chromatography-fluorescence-mass spectrometry[END_REF][START_REF] Berendsen | Multi-analyte method for the quantitave LC-MS/MS determination of quinoles in poultry muscle[END_REF]). These procedures however are technically complicated, expensive and time-consuming and often detect only a limited number of analytes simultaneously. When the aim is to screen large numbers of samples rapidly and at relatively low cost, microbiological screening methods are more suitable. Microbial inhibition assays are generally applied as a first qualitative screening step, primarily developed to sift out large numbers of compliant results, reducing the number of samples that need to be analyzed by a quantitative confirmatory method. Throughout the EU the most common screening method for antibiotic residues in animal tissue is probably the EU Four Plate Test (Bogaerts and Wolf 1980) or derivatives of this concept. Since this method was developed a decade before the introduction of quinolones in veterinary medicine, it does not comprise a test plate sufficiently sensitive for quinolone residues.

For the detection of quinolones Ellerbroek introduced in the early '90s an assay using Escherichia coli as an indicator strain (Ellerbroek 1991), which has been included in several other multiplate screening methods since then [START_REF] Nouws | A microbiological assay system for assessment of raw milk exceeding EU maximum residue levels[END_REF][START_REF] Myllyniemi | A microbiological six-plate method for the identification of certain antibiotic groups in incurred kidney and muscle samples[END_REF][START_REF] Okerman | Inhibition tests for detection and presumptive identification of tetracyclines, beta-lactam antibiotics and quinolones in poultry meat[END_REF][START_REF] Gaudin | Validation of a microbiological method: the STAR protocol, a five-plate test, for the screening of antibiotic residues in milk[END_REF][START_REF] Ferrini | Combined Plate Microbial Assay (CPMA): A 6plate-method for simultaneous first and second level screening of antibacterial residues in meat[END_REF]). The use of E. coli as an indicator strain however has its shortcomings. It has difficulty detecting oxolinic acid and flumequine residues at sufficiently low levels, while especially flumequine is commonly used in poultry. Enrofloxacin, on the other hand, can be detected extremely sensitive with respect to MRL values. Therefore, the use of E. coli for screening purposes will easily lead to false compliant results with respect to flumequine, but will also yield many false non-compliant results when enrofloxacin is present in a sample.

The Premi  Test (DSM Nutritional Products, Geleen, the Netherlands) has been introduced several years ago as an attractive on-site alternative for the traditional multiplate systems. It is a fast microbial assay based on the inhibition of Bacillus stearothermophilus and applicable for a variety of matrices, among which egg and poultry muscle. However, this test organism is relatively insensitive to quinolone antibiotics, so additional testing will be required to ensure the whole antibiotic spectrum is adequately covered. This paper presents an improved microbiological screening assay for the detection of quinolone residues in poultry muscle and egg, that is better suitable for testing in compliance with MRL values.

It is of essential importance to validate microbiological drug residue screening systems with fortified or incurred tissue, since the influence of matrix components seriously affects the detection capacity of an inhibition assay. Tissue binding of drug residues or

Material and methods

Antibiotic standards

For preparation of antibiotic stock solutions drug standards of known purity with certificate of analysis were used. Flumequine and oxolinic acid were obtained from Sigma-Aldrich (Zwijndrecht, Netherlands), enrofloxacin from Bayer (Leverkusen, Germany), danofloxacin from Pfizer (Groton, USA) and difloxacin from Fort Dodge Animal Health (Naarden, the Netherlands). Stock solutions were prepared by dissolving 5 mg of the antibiotic in 5 ml 0.1 M NaOH and diluting to 100 ml with demineralized water. These stock solutions were diluted with demineralized water to concentrations suitable for preparation of the spiked samples. Naarden, the Netherlands), enrofloxacin (Baytril, 10% solution, Bayer, Mijdrecht, the Netherlands) or flumequine (Flumequine, 50% water-soluble powder, Dopharma, Raamsdonksveer, the Netherlands). Another group of 75 animals remained untreated to provide blank reference material. Medication was administered through the drinking water: prior to the experiment the avarage daily water uptake was determined and the required drug concentrations were calculated taking into account an intended dose of 30 mg/kg total body weight. The birds were kept on their respective drinking water treatments for 5 consecutive days, during which the (medicated) water was refreshed daily. On day 6 the animals were euthanized by electrocution and breast muscle material was collected. Breast samples were individually packed and stored at -20°C. The drug residue concentration in each breast sample was determined using the microbiological assay. Samples containing drug concentrations around the MRL were obtained through a cryogenic grinding and mixing procedure. Frozen poultry breasts were roughly diced, after which their temperature was decreased further using liquid nitrogen. From this point on the material remained deep frozen by adding liquid nitrogen on regular intervals. The pieces were first roughly ground using a UMC 5 Electronic cutting mill (Stephan Machinery, Hameln, Germany), then smaller portions were blended to a fine powder 8 using a Grindomix GM200 (Retch GmbH, Haan, Germany), after which the material was sieved through a 1.25 mm sieve. Batches of 1.5 kg containing around 100 µg/kg enrofloxacin, 300 µg/kg difloxacin or 400 µg/kg flumequine were prepared by combining the proper amount of incurred and blank reference material. After collecting the sieved material it was homogenized by additional stirring for 10 minutes under liquid nitrogen.

Incurred eggs were obtained from a group of eighty 30-week old Lohman brown laying hens. Eggs were collected during a period of 4 weeks, during which the first 2 weeks untreated reference eggs were obtained. After 2 weeks the medication was started: each half of the group was exposed to either oxolinic acid (Sigma-Aldrich, Zwijndrecht, Netherlands) or flumequine (Flumequine 50% WSP, 50% water-soluble powder, Dopharma, Raamsdonksveer, the Netherlands). Medication was provided through the drinking water: daily water uptake of a group was determined and the required drug concentrations were calculated taking into account an intended dose of 30 mg/kg total body weight. The hens were kept on their respective drinking water treatment regimes for nine consecutive days, during which the (medicated) water was refreshed daily. Eggs were collected daily and pooled in 3 or 4 portions consisting of eggs laid on the same day. Egg samples were homogenized using an Ultra-Turrax T18 (IKA ® Werke GmbH, Staufen, Germany) and the residue concentration of each batch was estimated using the microbiological assay. Egg samples were then diluted with the untreated reference eggs to concentrations around 50 µg/kg for oxolinic acid and 200 µg/kg for flumequine.

Sample preparation

Incurred and fortified egg samples could be applied directly onto the test plate. Analysis of poultry muscle required some additional sample preparation. Fortified poultry muscle samples were prepared by mixing 195 g of roughly chopped material and 5 ml of the appropriate antibiotic spike solution. This mixture was let to rest for at least one hour at room temperature and subsequently blended in a rotary hatcher. Both spiked and incurred poultry tissue were further treated the same. Approximately 30 g of the blended or powder material was heated in a centrifuge tube for 20 minutes at 64 °C, after which the sample was centrifuged for 10 minutes at 27000 x g. The supernatant (meat fluid) was applied directly onto the test plate.

Microbiological screening assay

Although the principle of the assay (microorganism and test agar) is the same for egg and poultry muscle samples, the test plate was optimized for each specific matrix. The test plate for egg samples contained per liter: 23.5 g Plate Count Agar (Difco), 5% of a 1 M phosphate buffer pH 6.5 and 1.0 g Tween 80. The test plate for poultry muscle samples contained per liter: 15.7 g Plate Count Agar (Difco) and 5% of a 1 M phosphate buffer pH 6.5. The media were sterilized for 15 min at 121 °C and after cooling down to 48°C the pH was adjusted to 6.5 ± 0.1 if necessary. The media were seeded with Yersinia [START_REF] Barker | Detection of 4-quinolone residues in rainbow trout muscle using a bioassay[END_REF]) at 10 6 CFU/ml agar and immediately poured as a layer of approximately 2.2 mm. After solidification holes with a diameter of 14 mm were punched into the plate, with a maximum of 9 holes in 120*120*17 mm plates or 36 holes in 245*245*20 mm plates. A sample volume of 250 µl was applied and plates were incubated for 16-18 hours at 30°C.

Interpretation of the test plate results

The presence of antibiotics is shown by the formation of growth inhibition zones around the hole. The test plate is not vulnerable to naturally occurring antimicrobial compounds, so any visible inhibition is considered positive. The diameter of the zones was measured with a precision of 0.1 mm using a vernier calliper. Quinolone concentrations in incurred samples were estimated from calibration curves comprising five calibrators. Residue concentrations in these fortified poultry muscle or egg samples were in the range of 25-400, 50-600, 100-600 and 50-150 µg/kg for enrofloxacin, difloxacin, flumequine and oxolinic acid, respectively. Calibration curves were obtained as a best fit regression line calculated by the method of least squares, using the diameter of inhibition zones vs. the logarithm of the antibiotic concentrations.

Premi  Test

The Premi  Test (DSM Nutritional Products, Geleen, the Netherlands) was carried out according to the manufacturers instructions. Samples of 100 µl meat fluid or egg were applied to an ampoule. Prior to the incubation at 64ºC, ampoules containing egg samples

LC-MS/MS measurements

To the egg samples (1 g) 10 ml water was added and the samples were extracted for 15 min with a rotary tumbler. Muscle tissue samples were first minced and homogenized with a Moulinette meat mincer and further treated as the egg samples. After centrifugation at 3000 rpm (10 min) the supernatant was passed through a 0.45 µm membrane filter. A 2-ml aliquot was passed through an Ultracel YM-30 Centricon ultrafilter (Millipore, Bedford, MA, USA) by centrifugation at 4500 rpm for 45 min. The filtrate was transferred to a 500 µl HPLC vial and analysed by LC-MS/MS. A Waters Alliance 2690 HPLC coupled to a Micromass Quattro Ultima tandem mass spectrometer (Waters, Milford, MA, USA) was used. The quinolones were separated by gradient elution on a Waters Symmetry ® C 18 150 x 3.0 mm column (Waters, Milford, MA, USA), The gradient was run with a flow of 400 µl min -1 starting at 100% 5 mM formic acid for 1 min and then changed to 5 mM formic acid in acetonitrile in 10 min.

After an isocratic hold for 2 min the solvent composition was changed in 1 min to the starting conditions. Total run was 19 min. The column effluent was split 1:2 before entering the mass spectrometer. The mass spectrometer was operated in positive electrospray mode with the capillary voltage set at 2.7 kV, cone voltage: 20 V, source temperature: 120 o C, desolvation gas temperature: 300 o C, cone gas flow: 200 l min -1 , desolvation gas flow: 500 l min -1 , collision gas: argon, at 2.2 10 -6 bar. For each analyte Ib.

Of each incurred batch 10 randomly chosen samples were processed and analysed in duplicate. The homogeneity of each batch was verified by applying the methodology presented by [START_REF] Fearn | A new test for 'sufficient homogeneity[END_REF]. No outliers were identified by applying Cochran's test and all for all batches the determined sampling variance (s all 2 ) was below the calculated critical value (c) for the test. It could be concluded that all batches are sufficiently homogeneous.

[Insert Table Ia and Ib about here]

Results

Detection capability

ccording to EU commission decision 2002/657/EC the detection capability (CCβ) of a method is defined as "the smallest content of the substance that may be detected, identified and/or quantified in a sample with an error probability of β" (EC 2002). The β error is set at 5% and at least 20 investigations for one concentration level have to be [Insert Table II about here] [Insert Figure 1a and 1b about here]

Incurred samples

The accuracy of the method was examined by a semi quantitative determination of the residue concentration in incurred poultry samples containing enrofloxacin, difloxacin or flumequine, and incurred egg samples containing oxolinic acid or flumequine. For each matrix/residue combination a calibration curve was generated, using a relevant set of five matrix calibrators. Residue concentrations in the incurred sample were then calculated from the diameter of the inhibition zones and compared with the values determined by LC-MS/MS. To verify that the homogenization under liquid nitrogen has no effect on the antibiotic concentration obtained in meat juice after sample preparation, we compared calibration lines obtained from fortified samples subjected to the cryogenic procedure with those obtained from the conventional procedure. This resulted in virtually identical lines for all three quinolones (data not shown). to the biologically active residues ciprofloxacin and sarafloxacin, respectively. The antimicrobial activity of the primary metabolite of flumequine, 7-hydroxy-flumequine, is negligible. Except for the lowest enrofloxacin concentration, the microbiologically determined residue levels in muscle were indeed found to be somewhat higher. This could however not entirely be attributed the presence of ciprofloxacin and sarafloxacin, since the LC-MS/MS analyses showed that these compounds account for less than 10%

of the total residue level. It can not be excluded that other unkown microbiologically active metabolites are responsible for the effect. LC-MS/MS analysis on the extracts of incurred and matrix calibrator samples prepared for the microbiological assay, also confirmed the observed discrepancy, excluding the possibility that the differences were caused by the slightly different sample preparation procedures of the two methods.

The Premi  Test screening result was negative for all tested samples. Indicative data on the sensitivity of the test provided by the manufacturer do not claim detection levels for quinolones in egg, in poultry they are presumed to be >600 µg/l for enrofloxacin and >100 µg/l for flumequine. Our results indicate that the detection level for flumequine is at least >500 µg/kg. It can be concluded that the Premi  Test is not suitable for screening poultry for compliance with MRLs with respect to quinolone residues.

[Insert

Discussion

We succeeded in developing a microbiological inhibition assay that is capable of efficient screening for quinolone residues in incurred tissue samples. The sensitivity of a microbiological screening method is often determined using standard antimicrobial solutions (Ellerbroek 1991;Calderon et al. 1996;Currie et al.1998;Tsai and Kondo 2001;[START_REF] Ferrini | Combined Plate Microbial Assay (CPMA): A 6plate-method for simultaneous first and second level screening of antibacterial residues in meat[END_REF]. Such an approach however does not provide a clear view on the true potential of a test, since matrix compounds may significantly affect the sensitivity of a test system. This is an aspect often neglected in method development,

while it has been shown before that assay sensitivity with meat samples for example is likely to be much lower [START_REF] Okerman | Detection of antibiotics in muscle tissue with microbiological inhibition tests: effects of the matrix[END_REF]. Our results indicated that the sensitivity of the presented microbial assay increased approximately two-fold when standard antimicrobial solutions were analysed . When such an insufficiently characterized screening method is implemented in food surveillance programs, this may have serious implications for consumer safety, since it is likely to yield false compliant screening results.

A potential strategy to account for the effect of tissue factors is the use of fortified tissue fluid. Routine screening for antibiotic residues in meat, however, is often performed 17 using small meat disks that are directly applied on an agar plate. Using fortified tissue fluid ignores the possibility of binding of the antibiotic to the tissue. To account for this effect we developed a procedure involving fortification of a relatively large amount of tissue and applying extracted fluid on the test plate. The fact that our calibrator lines resulted in an accurate estimation of the residue levels in the incurred samples, indicates that this approach is valid for quinolone antibiotics. For practical reasons we used a sample preparation procedure involving a heating step followed by centrifugation, since this yields the largest quantity of tissue fluid. Alternatively, the meat extract can also be obtained using a meat press or by applying a freeze-thaw cycle, which is very convenient for on-site use. Additional heating does not affect the detection capability.

Although matrix components often tend to decrease the sensitivity of a test, the opposite also can occur: egg samples notoriously cause false non-compliant results in microbiological screening methods because of the presence of natural inhibiting factors.

Many inhibition assays rely on Bacillus subtilis or close relatives like B.

stearothermophilus or B. cereus as a test organism, which makes these screening systems vulnerable to lysozyme activity. This problem can be reduced by applying a heat inactivation step. In contrast to for example the Premi  Text, the inhibition assay presented in this paper however allows direct analysis of egg samples without a pretreatment step. Key factors contributing to the robustness and the sensitivity of the presented test plate appeared to be the choice of the test agar (Plate Count Agar, Difco) and the addition of phosphate. We found addition of Tween80 to be an effective cure against coagulase zones that can complicate the analysis of egg samples. The microbial inhibition assay reported here, slightly overestimated the quinolone residues present in incurred tissue. In practice this should not be a problem since the method was developed to function as a screening method and should therefore primarily avoid false compliant results. Since it was shown that the bioassay can be applied semiquantitatively, the test plate allows the definition of a so called "action-level", an inhibition zone above which samples should be considered "suspect" and require additional quantitative analysis. For flumequine the highest limit of detection is obtained, however relative to MRL values oxolinic acid is detected least sensitive. Therefore, we propose that 50 µg/kg oxolinic acid should be used as a reference determining the actionlevel for poultry muscle.

Since no MRLs for quinolones have been established in eggs, in principle a zero tolerance policy should be applied. However, establishing an MRL for a specific compound implies that a certain exposure level poses no threat on the consumer's safety.

The calculation of the MRL value is based on the acceptable daily intake (ADI) which assumes an average intake per person of 500 g of meat, 1.5 l of milk, 100 g egg and 20 g of honey. One could argue that the ADI definition legitimates higher residue levels in eggs compared to meat. This is an ongoing discussion for which no consensus exists among the different members of the European Union. Applying a zero tolerance policy implies an exponential increase in the costs of a monitoring system, since only chemical methods are appropriate. This situation calls for defining a pragmatic approach that is compatible with reasonable risk management.

F

  compounds promoting microbial growth may significantly decrease sensitivity of a test plate. On the other hand naturally occurring antimicrobial compounds may cause false positive results. Many of the published antibiotic screening assays have only been characterized using standard antibiotic solutions, which makes it difficult to evaluate their practical applicability. The presented screening assay is validated using fortified poultry and egg samples and is able to detect quinolone residues below EU MRLs. The accuracy of the assay is shown with incurred samples around MRL for which residue concentrations were established with high performance liquid chromatographytandem mass spectrometry (LC-MS/MS). In 2005 the test plate was succesfully implemented in the routine analysis of poultry within the framework of the Dutch national residue monitoring plan.

  The animal experiments were approved by the Institutional Animal Experiment Commission under permission nr 2005021 and 200502 respectively. Incurred poultry muscle samples were obtained by dosing three groups of 25 three-week old Ross 308 broilers with either difloxacin (Dicural, 10% solution, Fort Dodge Animal Health,

  muscle and egg samples was established by the procedure described by[START_REF] Fearn | A new test for 'sufficient homogeneity[END_REF]: 10 randomly taken samples from a batch were analysed in duplicate by LC-MS/MS (see below).

  10 minutes at 80ºC to inactivate natural inhibitors like lysozyme. The incubation was continued until the negative control turned yellow (3-4 hours).

  MS fragmentation conditions were optimized (TableI). The system was run in multiple reaction monitoring (MRM) mode. Product ion 1 was used for quantification and product ion 2 was used for confirmation purposes. Quantification was carried out against blank muscle and egg material fortified before extraction at 6 concentrations: difloxacine and flumequine: 25-1000 µg/kg, enrofloxacin and oxolinic acid: 10-500 µg/kg, ciprofloxacin and sarafloxacin: 5-200 µg/kg. The performance characteristics of the LC-MS/MS method are summarized in Table

  To determine the detection capability of the newly developed bioassay, fortified samples were analyzed. For each quinolone residue concentration at least 20 samples were analyzed on 5 different days using at least 10 individual plates. When the experiments fulfilled the "at least 19 out of 20 samples non-compliant" standard, the detection capability was regarded to be smaller or equal to this particular concentration.TableIIsummarizes the detection capability of the new screening assay. From the results it is shown that the detection capability for all of the quinolones lies well below their corresponding MRL values in poultry muscle, also for the traditionally "difficult" compounds flumequine and oxolinic acid. The detection capability of quinolone residues in egg is comparable to the detection in muscle. To illustrate the importance of using fortified tissue samples for validation of a microbiological screening method, figure1aand 1b show a comparison of inhibition zones obtained with some of the quinolone standard solutions and fortified poultry muscle or egg samples containing the same concentrations. The figures indicate that in general the sensitivity of the assay decreases about two-fold when matrix samples are analysed.Due to the low detection capability of the assay, the risk of obtaining false-compliant results is minimized. Furthermore, the assay appears also sufficiently resistant towards naturally occurring growth inhibiting compounds; since the introduction of this test in routine screening of poultry by the Dutch Food and Consumer Product Safety Authority in 2005 over 600 samples were analysed without yielding any false non-compliant results. Routine analysis data for egg are not available yet, but screening of over 20 eggs with respect to poultry breed and husbandry system gave no false noncompliant results.

Figure 1 .

 1 Figure 1. Relation between residue concentration and size of the inhibition zone. Open symbols represent antibiotic standard solutions, the closed equivalents indicate the same residue in fortified samples. Figure 1a shows fortified poultry muscle, figure 1b fortified egg samples

Table

  IIIa shows the results of the residue analysis in incurred poultry muscle samples and Table IIIb the results of incurred egg samples. The estimates obtained from the

	enrofloxacin and difloxacin, since these quinolone species are partly metabolized in vivo
	F o
	r
	P
	e e r
	R e v i e w
	O n
	l y
	microbiological method and the chemically determined values appeared to correspond
	well. As microbiological assays do not differentiate between the target compound and
	any biologically active metabolites, we expected a potential overestimation in case of
	15

  Table IIIa and IIIb about here]

	Page 15 of 29	Food Additives and Contaminants
	Specificity of the assay was tested using MRL concentrations of a relevant spectrum of
	licensed tetracyclines, macrolides, aminoglycosides, sulphonamides and β-lactam
	antibiotics. None of these antibiotics showed growth inhibition on the quinolone
	screening test plate.
	F o
	r
	P
	e e r
		R e v i e w
		O n
		l y
	Cross-reactivity
		16

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Table Ia .

 Ia It effectively detects quinolone residues in poultry muscle as well as in eggs. This quinolone residue test can be implemented in existing multi-plate screening systems, but also serve as an addition to commercial screening methods like the Premi  Test, which do not adequately detect quinolones in poultry and eggs. LC-MS/MS fragmentation conditions.

	Conclusion Bogaerts R, Wolf F. 1980. A standardized method for the detection of residues of anti-Tsai C, Kondo F. 2001. Improved agar diffusion method for detecting residual
	Component	Precursor ion	Product ion 1	Product ion 2	Collision energy
	bacterial substances in fresh meat. Die Fleischwirtschaft 60:672-674. antimicrobial agents. Journal of Food Protection 64:361-366. (m/z) (m/z) (m/z)	(eV)
	The microbial screening assay reported here allows rapid and inexpensive monitoring of Enrofloxacin 360.2 316.1 245.1 23
	o r P e e r R e v i e w O n l y Calderon V, Gonzaz J, Diez P, Berenguer JA. 1996. Evaluation of a multiple bioassay Yorke JC, Froc P. 2000. Quantitation of nine quinolones in poultry tissues by high-Ciprofloxacin 332.2 288.1 245.1 21 large numbers of samples. F o r P e e r R e v i e w O n l y technique for determination of antibiotic residues in meat with standard solutions of antimicrobials. Food Additives and Contaminants 13:13-19. Currie D, Lynas L, Kennedy DG, McCaughey WJ. 1998. Evaluation of a modified EC four plate method to detect antimicrobial drugs. Food Additives and Contaminants 15:651-660. EC 1990. Council Regulation (EEC) No 2377/90 of 26 June 1990: laying down a Community procedure for the establishment of maximum residue limits of veterinary medicinal products in foodstuffs of animal origin. Official Journal of the European Communities L224:1-8. EC 2002. Commission Decision 2002/657/EC of 12 august 2002: implementing Council Directive 96/23/EC concerning the performance of analytical methods and the y interpretation of results. Official Journal of the European Communities L221:8-36. F o r P e e r R e v i e w O n l y F o r P e e r R e v i e w O n l y performance liquid chromatography with fluorescence detection. Journal of Difloxacin 400.2 356.2 299.1 21 F o r e e e l y l n O n O w w e i v i e v R R r e r e e P P Chromatography A 882:63-77. Sarafloxacin 386.2 342.2 299.1 23 F Flumequine 262.1 244.1 202.0 24 o r Oxolinic acid 262.1 244.1 216.0 25
	Ellerbroek L. 1991. The microbiological determination of the quinolone carbonic acid
	derivatives enrofloxacin, ciprofloxacin and flumequine. Fleischwirtschaft 71:187-189.
						19 20 23

Table Ib .

 Ib LC-MS/MS performance characteristics for the quantification of quinolones in muscle tissue

			Food Additives and Contaminants	Page 24 of 29
		F o r
		P
		e
	Component	Level of	Accuracy e r	Repeatability	Within-lab	LoD / LoQ
		fortification	(%)	(RSD, %) R	reproducibility	(µg/kg)
		(µg/kg)		e	(RSD, %)
	Enrofloxacin Ciprofloxacin Difloxacin Sarafloxacin	100 100 300 100	96 97 101 99		22 28 15 v i e 27 w	25 31 18 27	1 / 2 1 / 3 1 / 2 2 / 4
	Flumequine Oxolinic acid	400 100	114 110		22 25	24 27 O n l	5 / 10 10 / 20
							y

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Table II .

 II The detection capability of the microbial inhibition assay for quinolone residues in poultry muscle and egg samples.

		Food Additives and Contaminants
	Component Flumequine Enrofloxacin Difloxacin Danofloxacin Oxolinic acid	MRL in poultry muscle 400 100 *) 300 200 100 F o r P e e r R e v i e w Detection capability (CCβ) in poultry muscle Detection capability (CCβ) in egg ≤100 ≤150 ≤ 25 ≤15 ≤ 50 ≤50 ≤ 50 ≤15 ≤ 50 ≤50	O n l y

*) Sum of enrofloxacin and ciprofloxacin Page 25 of 29 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Table IIIa .

 IIIa Screening results and residue concentrations in incurred poultry muscle samples (A to I) determined by the microbial inhibition assay and LC-MS/MS. The MRL in poultry muscle is 100 µg/kg for enrofloxacin, 300 µg/kg for difloxacin and 400 µg/kg for flumequine.Concentrations of microbiologically active metabolites are shown between brackets; ciprofloxacin is formed when enrofloxacin was used for medication and sarafloxacin in case of difloxacin use.

			Screening Result	Concentration	Relative Standard	Concentration (µg/kg)	Relative Standard	Deviation between
		Incurred
	Medication		microbiological		(µg/kg) determined	deviation (%) LC-	determined with	deviation (%)	microbiological assay
		material	assay	Premi  Test	with LC-MS/MS	MS/MS method (*)	microbiological assay	microbiological assay	and LC-MS/MS (%)
		A	+	-	48 (3)	14.4	39	30	-24
	Enrofloxacin
		B	+	-	96 (6)	2.8	102	4	0
		C	+	-	169 (11)	5.8	240	4	+33
		D	+	-	166 (5)	3.0	239	6	+40
	Difloxacin	E	+	-	126 (11)	9.8	187	7	+36
		F	+	-	318 (14)	7.4	519	6	+56
		G	+	-	83	6.9	94	16	+13
	Flumequine	H	+	-	312	3.8	378	7	+21
		I	+	-	562	4.1	703	4	+25

*) for the principal component

Table IIIb .

 IIIb Screening results and residue concentrations in incurred egg samples (A to F) determined by the microbial inhibition assay and LC-MS/MS.

			Screening Result	Concentration	Relative Standard	Concentration (µg/kg)	Relative Standard	Deviation between
		Incurred
	Medication	material	microbiological assay	Premi  Test	(µg/kg) determined with LC-MS/MS	deviation (%) LC-MS/MS method	determined with microbiological assay	deviation (%) microbiological assay	microbiological assay and LC-MS/MS (%)
	Oxolinic acid	A	+	-	25	5.0	23	34	-8
		B	+	-	46	6.7	40	17	-13
		C	+	-	144	6.8	109	2	-24
	Flumequine	D	+	-	67	6.1	68	23	+1
		E	+	-	151	5.5	161	13	+6
		F	+	-	334	4.0	350	25	+5

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants


Franz Berthiller

Ulrike Werner

Michael Sulyok

Rudolf Krska
email: rudolf.krska@boku.ac.at

Marie-Theres Hauser

Rainer Schuhmacher

LC/MS/MS determination of phase II metabolites of the mycotoxin zearalenone in the model plant Arabidopsis thaliana

Keywords: mycotoxins, conjugated mycotoxins, masked mycotoxins, zearalenone, metabolism, mass spectrometry, Arabidopsis thaliana

The biotransformation products of zearalenone (ZON), a Fusarium mycotoxin, were elucidated using the model plant Arabidopsis thaliana. After treatment of plant seedlings with 50µM ZON both the liquid media and the plant extracts were analysed with LC/MS/MS. An array of 17 different metabolites, most prominently glucosides, malonylglucosides, dihexoseand hexose-pentose-disaccharides of ZON, α-Zearalenol and β-Zearalenol were detected in the samples. Time courses for the different ZON-metabolites were recorded and give a closer insight into the metabolism kinetic. A scheme proposing the ZON metabolism in A. thaliana is given. The aspect of food safety regarding the (potential) occurrence of masked mycotoxins in agricultural commodities is discussed.

Introduction

Mycotoxins (reviewed in [START_REF] Hussein | Toxicity, metabolism, and impact of mycotoxins on humans and animals[END_REF]Brasel 2001, Bennet andKlich 2003), poisonous secondary metabolites of moulds, can be released into food and feed after the growth of certain fungi on plant commodities. Living plants, however, are able to metabolize these xenobiotics (Wallnöfer et al. 1996, Cole and[START_REF] Cole | Secondary metabolism of agrochemicals in plants[END_REF]. Humans and animals that consume parts of the contaminated plants (e.g. cereals, nuts, raisins, etc.) are therefore not just exposed to the native mycotoxins, but also to altered forms. Little is known about the occurrence, bioavailability and metabolism of these compounds.

Zearalenone (ZON) is a Fusarium mycotoxin with strong estrogenic activity. The main concern about this substance is caused by its ability to bind to estrogen receptors in (Bennet and Klich 2003). It was shown in 1988 that ZON can be transformed to α-zearalenol (αZOL), β-zearalenol (βZOL) and ZON-4-glucoside (ZON-4-Glc) by plant enzymes in maize cell suspension cultures [START_REF] Engelhardt | Transformation of the Fusarium mycotoxin zearalenone in maize cell suspension cultures[END_REF]. Also αZOL-4-glucoside (αZOL-4-Glc) and βZOL-4-glucoside (βZOL-4-Glc) were detected in such cultures [START_REF] Engelhardt | Metabolism of mycotoxins in plants[END_REF]. A mini-survey of 24 wheat samples revealed that for 22 samples ZON contamination was above the lower limit of quantification and for 10 of these samples (42%) also ZON-4-Glc could be quantified [START_REF] Schneweis | Occurrence of zearalenone-4-β-Dglucopyranoside in wheat[END_REF]. The ZON-4-Glc fraction was in the range of approximately 10-20% of the ZON content. The only plant metabolites of mycotoxins that have been subject to animal studies are ZON-4-Glc and ZON-4-sulfate (ZON-4-Sulf). ZON-4-Glc is completely cleaved to ZON during digestion in swine [START_REF] Gareis | Cleavage of zearalenone glycoside, a "masked" mycotoxin during digestion in swine[END_REF][START_REF] Gareis | Maskierte Mykotoxine[END_REF], while ZON-4-Sulf is easily cleaved by acid and in rats [START_REF] Plasencia | Isolation and characterization of zearalenone sulfate[END_REF]. Gareis coined the term "masked mycotoxins" to emphasize substances that are usually not detected in routine analysis but contribute to the total mycotoxin content. The picture further complicates when non-extractable bound residues are taken into account. Experiments with radiolabelled 14 C-ZON in maize cell suspension cultures indicated that over 50% of the initial radioactivity was incorporated as insoluble "bound residue" in the plant matrix, which still might be partly bioavailable [START_REF] Engelhardt | Metabolism of mycotoxins in plants[END_REF].

Arabidopsis thaliana or mouse-ear cress is a small flowering plant that is widely used as a model organism in plant biology. Arabidopsis is a member of the Brassicaceae family, which includes cultivated species such as cabbage and radish. Arabidopsis is not of major agronomic significance, but it offers important advantages for basic research in genetics and molecular biology (http://www.arabidopsis.org). The plants posses a small genome (about 125Mb) that has been fully sequenced since 2000, a rapid life cycle and they are easy to cultivate also in restricted space. It has been shown that Arabidopsis is susceptible to the cereal ear blight fungal pathogens Fusarium graminearum and Fusarium culmorum [START_REF] Urban | Arabidopsis is susceptible to the cereal ear blight fungal pathogens Fusarium graminearum and Fusarium culmorum[END_REF]. [START_REF] Levsen | Structure elucidation of phase II metabolites by tandem mass spectrometry[END_REF].

This current work reports on an assay for monitoring metabolic transformation of ZON in plants, using A. thaliana as model plant and a QTrap LC/MS/MS (Applied Biosystems) instrument for identification and quantification of the metabolites. After treatment of plant seedlings with ZON in liquid medium, both the medium as well as plant extracts were analyzed for metabolites. The multiple reaction monitoring mode was chosen for analysis because of its high selectivity and sensitivity. Masses of potential analytes were calculated from known metabolic reactions. The identities of detectable metabolites were confirmed with enhanced product ion (MS/MS) and MS 3 scans.

Material and Methods

Chemicals and Materials

Murashige and Skoog basal salt mixture and MS-grade ammonium acetate were purchased from Sigma (St. Louis, US), agar and DMSO from Fluka (Buchs SG, Switzerland). Sucrose was obtained from Agrana (Vienna, Austria), 12% sodium hypochlorite solution from Roth (Karlsruhe, Germany) and solid potassium hydroxide from VWR (Vienna, Austria). LC-grade methanol was purchased from J.T. Baker (Deventer, The Netherlands). Water for use as LC mobile phase was purified successively by reverse osmosis and a Milli-Q plus system from Millipore (Molsheim, France). ZON, αZOL, βZOL, and zearalanone (ZAN) were purchased from biopure Referenzsubstanzen GmbH (Tulln, Austria). ZON-4-Glc was synthesized [START_REF] Scharnhorst | Nachweis von Zearalenon und dem Metaboliten Zearalenon-4-O-β-Glukosid in Mais und Maisprodukten[END_REF] according to a modified protocol from [START_REF] Grabley | Glycosylation of mycotoxins[END_REF]. ZON-4-Sulf was isolated from Fusarium graminearum inoculated rice [START_REF] Plasencia | Isolation and characterization of zearalenone sulfate[END_REF]. Authentic αZOL-4-Glc and βZOL-4-Glc were available from fermentation mixtures after treatment of transgenic yeast with αZOL or βZOL [START_REF] Poppenberger | Identification of Arabidopsis thaliana glucosyltransferases that inactivate the estrogenic Fusarium mycotoxin zearalenone using a novel yeast bioassay[END_REF].

Treatment of A. thaliana with ZON and sample preparation

Seeds of A. thaliana, accession Columbia, were sterilized in aqueous sodium hypochlorite solution (5%) for three minutes, rinsed with sterile water twice, stratified for 48h and germinated on Murashige and Skoog agar plates [START_REF] Murashige | A revised medium for rapid growth and bio-assays with tobacco tissue cultures[END_REF] containing 4.5% (m/v) sucrose at 22°C under continuous light (approx. 70µE/m 2 /s). After ten days approximately 40 seedlings per sample were transferred to 20 mL liquid Murashige and Skoog medium containing 2.5% (m/v) sucrose and shaken at 60 rpm for another three days for acclimatization. In each experiment the ZON concentration was adjusted to 50µmol/L (15.9 mg/L) by adding 100 µl of a 10 mM ZON stock solution (in 10% DMSO, 0.01N KOH) to 20mL liquid medium. Seedlings were harvested at different time periods (0, 30min, 2h, 5h, 12h, 24h, n=3 per time point) after treatment with a single dose of ZON.

After harvest, the seedlings were weighed, rinsed with 20 mL water per sample and homogenized in 4 mL acetonitrile/water (75/25, v/v) using an Ultra-Turrax ® T25 mixer (IKA, Staufen, Germany) at 24000 rpm for 1 min. Extracts were centrifuged at 5300 rpm for 10 min to remove insoluble cell wall components, and then filtered through a paper filter (Roth, Karlsruhe, Germany). Each liquid medium was combined with the washing water obtained after rinsing of the seedlings and diluted 1:1 (v/v) with methanol to ensure that ZON is completely dissolved. Matrix blanks (both plant medium and plant extracts) to which no ZON was added were prepared accordingly. The samples were stored at 4°C in the dark until analysis by HPLC-MS/MS. Quantification was performed using external calibration with mixed standards containing 10, 30, 100, 300 and 1000µg/L of ZON, αZOL, βZOL, ZON-4-Sulf and ZON-4-Glc in mobile phase. Samples were diluted 1:10 (or 1:20 for time point 0) Recoveries for ZON, αZOL, βZOL, ZON-4-Sulf and ZON-4-Glc in the plant medium were elucidated by spiking diluted Murashige and Skoog medium (1:1 (v/v) with methanol) with 10, 30, 100, 300 and 1000µg/L of the mentioned analytes, before measurement with LC-MS/MS.

LC parameters

HPLC separation was achieved on a Thermo Aquasil C18 column (100mm x 4.6mm, 3µm) at 25°C applying a methanol/5mM aqueous ammonium acetate linear gradient from 30% methanol to 90% methanol. After an initial hold time of 0.5min, 90% methanol were reached after 7.5min and kept constant until 10min. Afterwards the column was re-equilibrated with 30% methanol until the end of the run at 15min. 25µL aliquots were injected into the HPLC-MS/MS. A flow rate of 0.75mL/min was chosen.

MS parameters

ZON and its metabolites were detected and characterized with a 2000 QTrap MS/MS instrument equipped with a Turbo Ionspray (electrospray) source. This instrument uses a triple quadrupole ion path and is capable of performing all of the conventional tandem quadrupole modes as well as several high sensitivity ion trap mass spectrometer scans using the final quadrupole as a linear ion trap (enhanced modes). To monitor for assumed metabolites, the instrument was operated in the MRM mode. MRM transitions (dwell time: 25ms) were monitored in a single LC run to screen for numerous conjugates (Tab.1). MS settings were as follows: curtain gas 20psi (137.9kPa), source temperature 400°C, source gas 25psi (172.4kPa), auxiliary gas 65psi (448.2 kPa), collision induced dissociation (CID) gas 6 (arbitrary units, corresponding to the pressure in the collision cell), ionspray voltage -4200V. Default values of DP -30V, and CE -30eV were used to monitor the ZON and ZOL conjugates. In case of detection of chromatographic peaks, MS/MS spectra were recorded in the enhanced product ion (EPI) mode (linear iontrap (LIT) fill time 50ms, LIT scan speed 1000amu/s). Also MS 3 spectra were recorded for confirmation (data not shown).

Results and Discussion

A. thaliana was treated with 50µM ZON (about 15.9mg/L) for 0, 0.5, 2, 5, 12 and 24h in triplicates. None of the screened analytes were detected in the matrix blanks (medium and plant extracts), which were treated in the same manner as the samples but to which no ZON has been added.

In the media ZON and its metabolites ZON-4-Glc, ZON-4-Sulf, βZOL and to a lesser extent also αZOL were detected after treatment with ZON. For all of these metabolites standards were available, which showed identical retention times, MS/MS and MS/MS/MS spectra (data not shown) and proved the identities of the detected metabolites. Spiking experiments at different concentrations in blank media revealed no significant matrix effects for the components mentioned above (data not shown). Recoveries for all analytes were close to In the plant extracts also αZOL-Glc (at low levels) and βZOL-Glc were unambiguously identified (besides ZON, αZOL, βZOL, ZON-4-Glc and ZON-4-Sulf), but their concentration are only given in relative values due to the lack of known concentrations of the authentic standards. Fig. 1B shows a MRM chromatogram of a cell extract after 24h ZON exposure to A. thaliana. Furthermore, the chromatographic peaks, corresponding to the MRM transitions of malonyl-glucosides (ZON-MalGlc, αZOL-MalGlc, βZOL-MalGlc), dihexose-(ZON-DiHex, αZOL-DiHex, βZOL-DiHex) and hexose-pentose-disaccharides (ZON-HexPent, αZOL-HexPent, βZOL-HexPent) of ZON, αZOL and βZOL were detected, although often in low levels (especially for the αZOL derivatives). For βZOL also a trihexose conjugate (βZOL-TriHex) was measured. MS/MS (EPI) and MS 3 scans confirmed the presence of ZON or the ZOLs respectively in all metabolites, but it has to be pointed out that no standards were available for the malonylglucosides and the di-or trisaccharides. Further experiments (e.g. using LC-NMR or authentic standards) are therefore required to fully characterize the molecular structures of these compounds.

[Insert figure 1 about here] Since xylose conjugates are usually found as conjugation products of xenobiotics in plants (Vostrowsky and Hirsch 2005) we suggest the hexose-pentose-disaccharides which were detected for the first time in this study to be glucose-xylose conjugates. According to other detected plant metabolites [START_REF] Cole | Secondary metabolism of agrochemicals in plants[END_REF]Edwards 2000, Levsen et al. 2005), the di-and tri-hexose conjugates are assumed to be di-and tri-glucosides respectively. In the present study we could not verify the exact location of the conjugation site by LC-MS/MS, as the added moieties were cleaved first from the molecules after low-energy CID, which is in good accordance with other studies (Zaia 2003). It is therefore not certain whether the dihexose conjugates are 2,4-dihexosides (with one molecule of sugar (glucose) conjugated to each of the phenolic groups of ZON) or oligosaccharides. ZON-2,4-diglucoside has been shown to be a microbial transformation product of ZON (El-Sharkawy 1989). The detection of a trihexose conjugate of βZOL but the absence of a ZON trihexoside derivative suggests that also the non-phenolic hydroxy-group of βZOL could be conjugated to a hexose in the found metabolite. Additional (enzymatic and NMR spectroscopy) experiments are planned to confirm the structures of the dihexoside-and the hexose-pentose-disaccharides.

Time course experiments with points at 0, 30min, 2h, 5h, 12h and 24h were performed as well. While after 0 min all of the added ZON was recovered in the medium, after 24h almost all available ZON (>97%) was metabolized. Fig. 2A summarizes the concentrations of the detected metabolites in the medium over time.

Phase I metabolism led to αZOL and βZOL, which were further conjugated very similarly to ZON in phase II metabolism. Intracellular (plant extract) concentrations (Fig. 2B) of ZON, αZOL and βZOL but also of ZON-4-Glc and ZON-4-Sulf increased during the first few hours (often about 5h) to reach a maximum but then declined as the substances were further processed to ZON-DiHex, ZON-MalGlc and ZON-HexPent. It has to be pointed out here, that matrix effects were not investigated for plant extracts and the yields of the chosen extraction method are unclear. For this reason Fig. 2B The variety of the identified ZON metabolites, in total 17 different substances, is summarized in Fig. 3. The proposed pathway shows metabolic activation (phase I) and conjugation (phase II) processes. The later occurrence of malonylglucosides and disaccharides suggests that they derive from the respective monoglucosides, which is also in agreement with the metabolism of other xenobiotics in plants [START_REF] Cole | Secondary metabolism of agrochemicals in plants[END_REF].

[Insert figure 3 about here] As part of their metabolism, plants are capable of transforming xenobiotics into a huge variety of conjugated forms. Nonetheless, usually just the parent mycotoxin is measured in routine analysis of feed and food. It has been shown that some of these conjugates can be cleaved easily by the gut microflora of swines [START_REF] Gareis | Cleavage of zearalenone glycoside, a "masked" mycotoxin during digestion in swine[END_REF]), releasing the precursor toxin. The total available amount of mycotoxins in foodstuff, therefore, might currently be underestimated.

It has to be stressed that A. thaliana is in no way an agricultural crop. In different plants the metabolism of a given xenobiotic might look very different. Still, the ease of use of A. thaliana plants combined with its known genome sequence allows a more general for certain metabolic pathways can be identified and looked for in other, more economically important, plants. Also a variety of (available) mutants of A. thaliana can be compared in their metabolic behaviour towards various xenobiotics with the wild-type plants using LC-MS/MS. Regarding the aspect of food safety, we want to screen wheat and maize for the occurrence of ZON and other mycotoxin plant metabolites in the very near future. Also the structural identities of the metabolites found will be confirmed by LC-NMR.

Conclusion

This work shows the great capability of plants to metabolize xenobiotic compounds. We utilized a method for the determination of ZON bio-transformation products in A. thaliana.

Seventeen different metabolites of ZON have been identified to occur in ZON treated

Arabidopsis plants. Known transformation products include ZON-4-Glc, ZON-4-Sulf, αZOL, βZOL, αZOL-Glc and βZOL-Glc. Dihexosides, hexose-pentosides and malonylglucosides of ZON, αZOL and βZOL are described for the first time.

  steady advancement of analytical instrumentation during time, nowadays very selective and sensitive techniques are available. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has become state of the art in metabolism research

  if the respective concentration exceeded 1000µg/L and measured again.

  optimized for standard substances, resulting in the following parameters: ZON m/z 317.1 -> m/z 130.9 (declustering potential (DP) -51V, collision energy (CE) -38eV), αZOL and βZOL m/z 319.1 -> m/z 129.9 (DP -71V, CE -48eV), ZAN m/z 319.1 -> m/z 275.1 (DP -56V, CE -16eV), ZON-4-Sulf m/z 397.2 -> m/z 317.1 (DP -46V, CE -18eV), ZON-4-Glc m/z 479.1 -> m/z 317.1 (DP -31V, CE -22eV), αZOL-4-Glc and βZOL-4-Glc m/z 481.1 -> m/z 319.1 (DP -31V, CE -22eV). For all other potential metabolites, ions with m/z ratios corresponding to [M-H] -of the assumed conjugates were allowed to pass the first quadrupole (Q1) for fragmentation in Q2. Q3 was mainly set to the m/z value of deprotonated ZON (m/z 317.1) or αZOL and βZOL (m/z 319.1) respectively.

  concentrations. A typical MRM chromatogram of the medium after treatment ofArabidopsis with ZON for 24h is shown in Fig.1A.

  contains the signal intensities of ZON, αZOL, βZOL, ZON-4-Glc and ZON-4-Sulf divided by the response factors of the respective analytes as measured in standard solution.

  Fig. 2C shows the relative signal intensities of ZON-DiHex, ZON-MalGlc and βZOL-Glc as a

  interactions that occur between plants and mycotoxins. Genes responsible

Fig. 1 .

 1 Figure captions:

Fig. 2 .

 2 Fig. 2. (A) Concentrations of ZON, ZON-4-Glc, ZON-4-Sulf, αZOL and βZOL found in the

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Acknowledgements

The authors thank Prof. Gerhard Adam for the scientific support. We also thank the Austrian Genome Research programme (GEN-AU), the Christian Doppler Society, the Austrian Science Fund (FWF project P16410) and the Lower Austrian Government for funding.