Angelo Visconti

Giancarlo Perrone

Giuseppe Cozzi

Michele Solfrizzo

risk in the grape-wine food chain

Keywords: ochratoxin A, black aspergilli, mycotoxins, grape, wine, food safety 23

Managing ochratoxin A risk in the grape-wine food chain

Penicillium naturally occurring in a variety of food commodities prior to harvest or more 4 commonly during storage. Numerous animal studies have shown that OTA is a potent 5 nephrotoxin with the degree of renal injury depending on both toxin, dose and exposure time; 6 decreasing nephrotoxic sensitivity was observed from pig to rat, to mice. OTA is 7 immunotoxic, neurotoxic in vitro and in vivo in rats, teratogenic in mice, rats and rabbits 8 [START_REF] Jecfa | Joint FAO/WHO Expert Committee on Food Additives, 56th Meeting[END_REF]. Based on renal carcinogenicity shown in rats and mice, the IARC 9 (International Agency for Research on Cancer) has classified OTA as a Group 2B carcinogen, 10

i.e. carcinogenic to animals and possible carcinogenic to human (IARC 1993). Studies on the 11 genotoxicity of OTA remain controversial. Recent scientific evidence indicates that the site-12 specific renal toxicity as well as the DNA damage and genotoxic effects of OTA, measured in 13 various in vivo and in vitro studies, are most likely attributable to cellular oxidative damage 14 [START_REF] Efsa | European Food Safety Authority. Opinion of the Scientific Panel on 4 contaminants in the Food Chain of the EFSA on a request from the Commission related 5 to ochratoxin A in food[END_REF]. Various studies in humans have associated OTA with an endemic kidney 15 disease observed in the Balkans (Balkan Endemic Nephropathy and related Urinary Tract 16 Tumors), but convincing epidemiological evidence associated with OTA exposure is currently 17 lacking. It has been frequently found in human blood, urine and milk and has been 18 demonstrated a widespread individual exposure to low levels of OTA in Europe and other 19 continents [START_REF] Efsa | European Food Safety Authority. Opinion of the Scientific Panel on 4 contaminants in the Food Chain of the EFSA on a request from the Commission related 5 to ochratoxin A in food[END_REF]. 20 21 The widespread human exposure to OTA is well documented by a number of surveys 22 reporting the occurrence of OTA in a variety of food products. An assessment of the dietary 23 intake of OTA by the population of the European Community has been performed showing 24 that the main contributors to OTA exposure are cereals and cereal products (European 25 Commission 2002). Wine, coffee and beer were identified as significant contributors to 1 human OTA exposure. Dried vine fruit and grape juice contribute to a significant extent to the 2 OTA exposure of vulnerable groups of consumers such as children. The EFSA Scientific 3 Panel on contaminants in the food chain has recently adopted an updated scientific opinion 4 relating to OTA in food, taking into account new scientific informations and derived a 5 tolerable weekly intake (TWI) of 120 ng/kg body weight [START_REF] Efsa | European Food Safety Authority. Opinion of the Scientific Panel on 4 contaminants in the Food Chain of the EFSA on a request from the Commission related 5 to ochratoxin A in food[END_REF]. 6 7 Based on the available scientific toxicological and exposure data, the European Union 8 established maximum permitted limits for OTA in a variety of food products that have been 9

updated with the EC Regulation 1881/2006 (European Commission 2006). Maximum levels 10 have been set at 2 µg/kg for wine, fruit wine, grape juice, grape nectar and grape must 11 intended for direct human consumption, and at 10 µg/kg for dried vine fruit (currants, raisins 12 and sultanas). OTA was detected in wine for the first time in 1996 [START_REF] Zimmerli | Ochratoxin A in table wine and grape juice: occurrence and risk 24 assessment[END_REF]. [START_REF] Ponsone | Ochratoxin a and ochratoxigenic 14 Aspergillus species in Argentinian wine trapes cultivated under organic and non-organic 15 systems[END_REF] Thereafter several surveys have been conducted, mainly in Europe, on the occurrence of the 14 toxin in wine and related products, showing it as a problem mainly for Southern Europe. The 15 results of several reports from different countries are reported in Table 1 for a total of 3512 16 samples. A high incidence of contamination (from 40% to 87%) was reported in all surveys 17 but the Australian one having an incidence of 15% [START_REF] Hocking | Occurrence of ochratoxin A in 7 Australian wine[END_REF], with maximum 18 level at 15.6 µg/kg recorded in Italy (European Commission 2002). OTA levels showed a 19 decreasing gradient from red to rosé, to white wines, and the same trend was observed for the 20 grape juice. Wines from southern and warmer regions of Europe showed incidence and levels 21 of contamination (72.3%, mean value 0.64 µg/kg, n = 635) higher than those from northern 22 European areas (incidence 50.3%, average 0.18 µg/kg, n = 835). Wines produced in Southern 23 Italy, where the climatic conditions favour the growth of OTA producing fungi in grapes, 24 generally show incidence and levels of contamination higher than wines produced in Northern 25 positive correlation between high levels of OTA and resveratrol-related compounds (Perrone 2 et al. 2007b). Considering that a significant reduction of hepatic and renal damage caused by 3 OTA was reported in mice fed with grape juice contaminated with OTA [START_REF] Jeswal | Antidotal effect of grape juice (Vitis vinifera) on ochratoxin A caused 15 hepatorenal carcinogenesis in mice (Mus musculus)[END_REF], there 4 may be some possibility that toxic levels of OTA in red wine are, at some extent, 5 counterbalanced by the beneficial effects of resveratrol derivatives. 6 7 In the following headings different aspects of OTA formation in grapes and strategies for 8 prevention and control in the field are reported together with OTA distribution and possible 9 corrective actions during wine processing. Good agriculture and manufacturing practices are 10 currently under discussion by Codex Alimentarius for the adoption of a code of practice for 11 the prevention and reduction of OTA contamination in wine, based on a code of sound 12 vinicultural practices adopted by the Organisation Internationale de la Vigne et du Vin (OIV) 13 in 2005(FAO/WHO 2007). 14 15 Fungi responsible for OTA accumulation in grapes: taxonomy and ecology. 16 Fungi responsible for OTA accumulation in cereals, i.e., Aspergillus ochraceus and 17

Penicillium verrucosum, were firstly thought to be involved also in OTA formation in grapes. 18 However, a number of studies performed during the last decade provide evidence of a quite 19 different situation showing that all fungi responsible for OTA in grapes belong to Aspergillus 20 section Nigri, the so called "black aspergilli" (Battilani et al. 2003) 6-8 °C and max 47 °C). This is one of the most common group of species in a wide range of 1 fresh and dry fruits, cereals, etc., and is used in food processing as "GRAS" (generally 2 regarded as safe). OTA production by A. niger "aggregate" normally occurs at 20-25 °C, and 3 a w 0.95/0.98 (Esteban et al. 2004). A low percentage of OTA producing strains (5-10%) were 4 detected among the A. niger "aggregate" (Perrone 2006a). 5 6 Among the uniseriate group, A. aculeatus and A. japonicus were often isolated from grapes, 7 but were not proven to produce OTA although they grow at similar conditions as for A. 8 carbonarius. Recently the Aspergillus "uniseriate" population from grapes in Europe were 9 characterized as a population molecularly quite different from A. japonicus and A. aculeatus 10 (Perrone et al. 2006b). This population is being described as a new species to be called A. 11 uvarum (Perrone et al. 2007 submitted). 12 13 Several reports from South America claiming the production of OTA by strains of A. 14 japonicus or that A. niger is the major responsible for OTA accumulation in grapes are based 15 on morphological identification of the producing strains [START_REF] Dalcero | Detection of 19 ochratoxin A in animal feeds and capacity to produce this mycotoxin by Aspergillus 20 section Nigri in Argentina[END_REF]Chulze et al. 16 2006;[START_REF] Ponsone | Ochratoxin a and ochratoxigenic 14 Aspergillus species in Argentinian wine trapes cultivated under organic and non-organic 15 systems[END_REF]). These data have not been confirmed by molecular identification 17 of the species and should be regarded with ultimate care to avoid confusion in the literature. 18

In our survey of about 600 strains of black aspergilli, representative of 3-year sampling, 5% 19 of A. niger "aggregate" strains (360) resulted OTA producers, while all A. carbonarius strains 20 (200) and none of the A. "uniseriate" strains (50) were positive to OTA production (Cozzi et 21 al. 2007). OTA is produced in vineyards and is normally absent up to early veraison. Bunches without 13 visible symptoms can also contain OTA although berries with visible black moulds normally 14

show higher contamination levels. The absence of OTA at early growth stages can be 15 explained by major difficulties encountered by fungi in berry penetration [START_REF] Cozzi | Epidemiology of ochratoxin A 13 producing fungi in Apulian vineyards[END_REF]. 16 The distribution of black aspergilli in vineyards can be summarized as follows: i) A. niger 17 "aggregate" is the principal group at all growth stages; ii) A. carbonarius incidence is 2-3 18 times less than A. niger "aggregate" and increases from ripening to harvest; iii) Aspergillus Meteorological conditions as well as closeness to the sea have been shown to play a major 10 role in determining OTA occurrence in grapes [START_REF] Cozzi | Epidemiology of ochratoxin A 13 producing fungi in Apulian vineyards[END_REF]). A 3-year survey (2004-11 2006) performed on 8 vineyards located in the Salento peninsula of Southern Italy, showed a 12 wide variability of OTA levels between different cultivation years. In particular, the 2005 13 crop was the most conducive to black aspergilli contamination due to the higher relative 14 humidity and rainfall levels, that were associated with hot temperatures at ripening and 15 harvest time (late August-September). Figure 1 shows the occurrence of black aspergilli and 16

OTA in 8 vineyards of the two major varieties (Negroamaro and Primitivo) cultivated in the 17 area during the three grape-harvest seasons [START_REF] Cozzi | Epidemiology of ochratoxin A 13 producing fungi in Apulian vineyards[END_REF] Despite the widespread occurrence of OTA in various types of wine, there is limited 1 information on the ability of black aspergilli to infect berries and produce OTA in different 2 grape varieties [START_REF] Battilani | Ochratoxin A 6 production by Aspergillus carbonarius on some grape varieties grown in Italy[END_REF]). Grape varieties were shown to affect the incidence of 3 black aspergilli and the level of OTA contamination in in vitro experiments. Three out of the 4 twelve tested varieties, namely "Bianco di Alessano", "Pampanuto" and "Uva di Troia", 5

showed low OTA contamination after artificial infection with a mixture of 5 OTA producing 6 strains, whereas the most susceptible variety (Cabernet Sauvignon) contained over 200 µg/kg 7 OTA and about 80% incidence of colonized berries [START_REF] Battilani | Ochratoxin A 6 production by Aspergillus carbonarius on some grape varieties grown in Italy[END_REF]). 8 9The role of the cropping system was monitored in a 2-year survey carried out on four different 10 cropping systems, namely spur-pruned cordon, bower system, head (or small tree) system 11 and espalier, in eight vineyards in Apulia. In both years the espalier cropping system 12 produced the most contaminated grapes in terms of A. carbonarius infection and OTA 13 accumulation, as shown in Figure 2. This can be explained by the closeness of bunches to the 14 soil, which is the most important source of inoculum of A. carbonarius, as compared to spur 15 pruned cordon and bower system. The higher humidity occurring in the espalier cropping 16 system with respect to the head system can explain the different contamination level despite 17 the similar distance of bunches from the soil for both systems. [START_REF] Cozzi | Epidemiology of ochratoxin A 13 producing fungi in Apulian vineyards[END_REF]). Black aspergilli are opportunistic fungi (saprophytes) mainly responsible for secondary rot of 1 grape berries developing through entry sites favoured by berry wounds or splitting caused by 2 either biotic or abiotic factors (insects, fungi, birds, rainfall, hail). Lobesia botrana 3 (Lepidoptera: Tortricidae) is the major grape berry moth in vineyards of Southern Europe, 4 and can complete three to four generations a year, depending on weather conditions during 5 late summer. Generally the first generation larvae of L. botrana damage flowers, while the 6 following generations damage berries at different ripening stages. A good correlation between 7 pest damage and OTA content has been found in grape berries, due to the contribution of L. 8 botrana to berry wounds and fungal spore dissemination [START_REF] Cozzi | Effect of Lobesia botrana 10 damages on black aspergilli rot and ochratoxin A content in grapes[END_REF]). Larvae can either 9 contribute to spore dispersal or act as spore vectors, by trapping conidia in the cuticle 10 ornamentation, then facilitating a rapid fungal penetration by tunnelling into berries as 11 demonstrated for Botrytis cinerea [START_REF] Fermaud | Association of Botrytis cinerea with Grape Berry Moth 20 Larvae[END_REF][START_REF] Cozzi | Effect of Lobesia botrana 10 damages on black aspergilli rot and ochratoxin A content in grapes[END_REF]). Grape berry 12 damage by L. botrana has been shown to increase considerably the contamination level of 13 black aspergilli and the consequent OTA accumulation in grapes. In Figure 3 a comparison 14 between groups of intact berries, black rot berries with and without L. botrana damages is 15 reported. All samples of berries damaged by L. botrana showed considerable levels of OTA 16 contamination (up to 1000 µg/kg OTA) and black aspergilli infection higher than 10 6 CFU. 17

OTA was not detected in the group of intact berries, while it was found at levels below 1 18 µg/kg in ca. 42 % of black rot berries without grape moth damages [START_REF] Cozzi | Effect of Lobesia botrana 10 damages on black aspergilli rot and ochratoxin A content in grapes[END_REF] The most effective treatment was observed at 21 days before harvesting and a previous 7 treatment at veraison was suggested in high risk conditions. This mixture of active ingredients 8 applied against black aspergilli with the same combination and schedule, both in dosage and 9 timing, is effectively used against grey mould, caused by Botrytis cinerea. Moreover, the 10 insecticide treatment against L. botrana in combination with the fungicide contributes 11 significantly to reduction of OTA level in the field particularly in crop years at high 12 contamination risk [START_REF] Kappes | A crop protection 17 approach to Aspergillus and OTA management in Southern European vineyards[END_REF]. Promising results were also obtained by using yeast 13 as biological control agents which were isolated from grapes in Greece and in Italy. In 14 particular, good results were obtained with two strains of Cryptococcus laurentii and 15

Aureobasidium pullulans, respectively, in Greece [START_REF] Dimakopoulou | Chemical and biological 22 control of sour rot caused by black aspergilli in the grapevine variety agiorgitico of 23 Korinth region. International Workshop on Ochratoxin A in Grapes and Wine: 24 Prevention and Control held in Marsala (TP) Italy, 2005 October 20-21; p. 77 (Poster) 25 incubation time on production of ochratoxin A by black aspergilli[END_REF], and with a 16 strain of Hanseniaspora uvarum in Italy using weekly or two-weekly treatments. 17 Therefore, the availability of reliable analytical methods for OTA determination in must, wine 22 and relevant by-products is important for an adequate risk management of OTA 23 contamination in the wine food chain. The availability of rapid methods would be necessary 24 in winery for screening the whole production in order to take prompt corrective actions. 25 of OTA concentration in wine after the first racking (separation of lees) while a significant 9 reduction of OTA concentration was observed after spontaneous malo-lactic fermentation 10 occurring between the first and the second racking. In contrast, [START_REF] Rousseau | Ochratoxin A in wines: current knowledge[END_REF] reported that 11 OTA content in must increases after crushing grapes, and reaches maximum levels during 12 malo-lactic fermentation. Leong et al. (2006b) reported that 24% of OTA originally present in 13 crushed red grape passed into free run wine (must) and a 72% OTA reduction was recorded in 14 wine after the first racking. 15 16 The different approaches used by these authors to conduct these studies could explain the 17 controversial results. Due to unavailability of naturally contaminated grape some of these 18 studies were performed either by artificially inoculating grape with toxigenic A. carbonarius 19 (Leong et al. 2006b) or by spiking uncontaminated grapes with OTA [START_REF] Fernandes | Fate of ochratoxin A during 22 a vinification trial[END_REF]. 20

These materials are different from naturally contaminated grapes which comprise both 21 contaminated and uncontaminated berries. Spiking uncontaminated grapes with OTA 22 produces an apparent reduction of OTA concentration in the resulting wine since most of the 23 spiked OTA is adsorbed by the grape pomaces and biomass (grape skins, pulp, yeasts released 24 in must) [START_REF] Fernandes | Fate of ochratoxin A during 22 a vinification trial[END_REF]). On the contrary, when using naturally contaminated berries 25 the source of OTA in wine [START_REF] Solfrizzo | Fate of ochratoxin A during vinification of 3 naturally contaminated primitivo and negroamaro grapes[END_REF]). Moreover, in most of these studies OTA 2 was only monitored in liquid fractions and no measurements were performed on pressed 3 pomaces and lees thus the distribution of OTA between solid and liquid fractions obtained 4 during vinification was not measured [START_REF] Grazioli | The role of processing on ochratoxin A content in 4 Italian must and wine: A study on naturally contaminated grapes[END_REF][START_REF] Fernandes | Fate of ochratoxin A during 22 a vinification trial[END_REF]; Rousseau 5 2004). Another critical point is the sample preparation of liquid fractions (must and wines 6 before racking), which contain suspended biomass, before OTA analysis. The separation or 7 inclusion of the biomass in the sample to be analysed has a significant effect on the measured 8 OTA concentration due to the high amount of OTA reversibly bound to the biomass. Indeed 9 Leong et al. (2006b) included the biomass when unsedimented liquid fractions (must and 10 wines before racking) were analysed for OTA. Consequently the OTA concentrations found 11 in these fractions were much higher than that found in the same liquid fraction analysed after 12 spontaneous sedimentation of biomass (first racking). 13 14 The fate of OTA and its distribution in wine and winery by-products during vinification of 15 naturally contaminated Negroamaro and Primitivo grapes has been recently reinvestigated at 16 laboratory (microvinification) and industrial level by [START_REF] Solfrizzo | Fate of ochratoxin A during vinification of 3 naturally contaminated primitivo and negroamaro grapes[END_REF]. Samples of must 17 (before and after maceration), grape pomaces, wine and lees (after the first and second 18 racking) were analysed for OTA in order to evaluate its levels at each step of vinification. 19 Before analysis the liquid fractions were centrifuged in order to separate the biomass and 20 measure the soluble OTA. Results of microvinification experiments showed that only 4% of 21 the OTA present in grapes remains in the wine whereas 95% of OTA originally present is 22 retained on pressed grape pomaces (98% in the skin and 2% in the seeds) and 1% is retained 23 on the lees. Leong et al (2006b) found that 9% of OTA originally present in grapes passed 24 into wine. The use of these wine by-products as food ingredients should be therefore avoided 25 or checked for OTA contamination. OTA concentration in must remained nearly constant 1 after maceration, pressing, juice clarification, alcoholic fermentation and lees separations 2 (after first and second racking). The same OTA concentrations were found in wine samples 3 analysed after one year. The results obtained with the microvinification were also confirmed 4 at industrial level. An increase of OTA concentration in must was observed during maceration 5 of Primitivo crushed grapes highly contaminated with OTA. This increase could be explained 6 by the high concentration of OTA in the grapes that required a longer time for toxin to 7 equilibrate between must and grape pomaces. 8 9

Removal of OTA 10

Several fining agents have been tested for their ability to remove OTA from contaminated 11 must/wines [START_REF] Castellari | Removal of ochratoxin A 1 in red wines by means of adsorption treatments with commercial fining agents[END_REF]Leong et al. 2006c). Oenological decolourising carbon has 12 been reported to remove the highest amount of OTA, although carbon also removes 13 anthocyanins and other coloured polyphenols from wine. The effectiveness of the treatment 14 with oak wood fragments depended upon the quantity of wood chips and powder used 15 [START_REF] Savino | Reduction of ochratoxin A contamination in 19 red wines by oak wood fragments[END_REF]). Removal of OTA from grape juice, must and wine by using oenological 16 yeast strains has been reported [START_REF] Bejaoui | Ochratoxin A removal in synthetic and 12 natural grape juices by selected oenological Saccharomyces strains[END_REF]Garcia Moruno et al. 2005;Cecchini et al. 17 2006). The removal of OTA during fermentation is based on adsorption mechanism other than 18 degradation. However, the efficacy of yeasts for OTA reduction at industrial level as well as 19 their impact on wine quality parameters (phenol compounds) has not been shown. In our 20 laboratory we have confirmed OTA reduction by yeasts or inactivated yeast walls while a 21 consistent reduction of colour index (expressed in terms of Folin Ciocalteu index) occurred. 22

23

The results obtained in our laboratory on the efficacy of selected adsorbent materials to 24 remove OTA from contaminated red wine are reported in Table 2. The best results in terms of 25 The main source of OTA in the wine production chain is the infection by "black aspergilli" 6 occurring in the field. A. carbonarius is the main species responsible for OTA accumulation 7 in grape berries occurring from early veraison to ripening. OTA production is influenced by: 8 climatic conditions/geographic areas; grape varieties/crop systems; berries damage caused by 9 insects, fungal infection or excessive irrigation/rainfall. Fungicidal and insecticidal treatments 10 reduce OTA contamination. Susceptibility to infection can vary from different years and 11 regions. Developing of risk maps based on critical control points can help to prevent and 12 control OTA accumulation in grapes. Availability of rapid methods for OTA analysis is also 13 important for preventive and corrective intervention at some critical control points. After 14 maceration of (red) grapes OTA remains stable during vinification and after one year aging. 15 During vinification of (red) grapes only 4% OTA remains dissolved in the wine, while 96% is 16 retained by solid winery by-products (grape pomace and lees). Carbon reduces OTA 17 concentrations in wines, but negatively affects wine quality. Good Agriculture Practices 18 (balanced soil tillage, irrigation, nitrogen fertilization, pruning) and Good Manufacturing 19 Practices (reduced harvest to vinification time, segregation of rot bunches) help considerably 20 to reduce OTA contamination risk. Main critical control points as well as preventive and 21 corrective actions are summarized in Table 3

  OTA) is a major mycotoxin produced by several species of Aspergillus and 3

  . Recently, a survey of retailed wine samples from Southern Italy showed a 1

22 23 Black

 23 aspergilli and OTA occurrence in the vineyards: role of environmental, ecological 24 and agronomical factors.[START_REF] Castellari | Removal of ochratoxin A 1 in red wines by means of adsorption treatments with commercial fining agents[END_REF] Black aspergilli cause a black rot disease of grape due to high fungal sporulation on berries 1 which renders them completely deprived and dry. The incidence of colonised berries is related 2 more closely to seasonal conditions during the year of cultivation than to the grape growth 3 stage(Battilani et al. 2003; Cozzi et al. 2007; Leong et al. 2006a). Fungal conidia are usually 4 present on berry skin from setting and increase in number from early veraison to harvest, with 5 a peak at ripening. Black aspergilli overwinter in soil, and frequent soil cultivation can favour 6 fungal infection in vineyard. The severity of aspergillus rot is influenced by excessive 7 irrigation prior to ripening causing berry splitting. Rain prior to harvest is a common cause of 8 berry damage favouring Aspergillus infection (Cozzi et al. 2007; Leong et al. 2006a). Berry 9 damage caused by insects, birds or other fungal infection, is the primary factor affecting the 10 disease development and OTA accumulation in berries. 11 12

  19 uniseriate is the least represented group, sporadically occurring in Portugal, Greece and Spain, 20 and more frequently in Italy, France and Israel (Battilani et al. 2006). 21 22 Based on the results of the Wine-Ochra Risk project carried out in six Mediterranean 23 countries, the incidence of berries infected by black aspergilli at harvesting is significantly 24 correlated with the latitude and longitude, with a positive West -East and North -South 25 et al. 2006). Following the geostatistical approach described by Battilani et 1 al. (2006), data on incidence of A. carbonarius were run with ArcView and a predictive map 2 was drawn. The incidence of A. carbonarius was significantly correlated with geographic 3 coordinates showing a positive gradient going towards the South of Europe. Based on the 4 combination of the degree-day and rainfall parameters in late August -early September in 5 several countries of the Mediterranean basin, discriminant analysis gave promising 6 perspectives for predicting OTA presence in vineyards by the development of thermo-wetness 7 maps (Battilani et al. 2006). 8 9

18 19All

 18 the above ecoagronomical factors play individual roles in developing A. carbonarius on 20 grapes and the consequent OTA accumulation although the final result in relation to OTA risk 21 is better represented by a combination of all these factors. Developing of risk maps based on 22 critical control points can help to prevent and control OTA accumulation in grapes. 23

25 Page

 25 Field 19 trials, performed in 2004 and 2005 using both biological and conventional insecticidal 20 treatments, confirmed that a successful control of the third generation of L. botrana reduces 21 the inoculum of Aspergilli and the formation of OTA in grapes (Perrone et al. 2007a; Kappes 22 et al. 2005). It is therefore important to ensure an adequate insect control in combination with 23 fungicide treatment in order to obtain an effective pest management. 24 Chemical and biological control of OTA producing fungi. 1 The following chemicals have been shown to be active in reducing at different extent both 2 fungal growth and OTA levels in grape bunches: mepanipyrim, pyrimethanil, fluazinam, 3 iprodione and the mixture cyprodinil and fludioxonil. The latter mixture was confirmed as 4 effective in several field trials carried out in several Mediterranean countries including 5 France, Spain, Greece and Italy (Bellì et al. 2007; Kappes et al. 2005; Tjamos et al. 2004). 6

18

  Distribution of OTA in wine and winery by-products and its fate during vinification of 19red grapes.20 OTA occurring in grapes is transferred to wine and relevant by-products during vinification. 21

4 (

 4 are available for OTA analysis in food products that need to be adapted 1 for their application to wine and by-products[START_REF] Visconti | Fitness for purpose -Ochratoxin A analytical 15 developments[END_REF]. The AOAC 2 official method 2001.01 for OTA determination by HPLC in wine[START_REF] Visconti | Determination of ochratoxin A in wine and beer by 20 immunoaffinity column cleanup and liquid chromatographic analysis with fluorometric 21 detection: collaborative study[END_REF]) can 3 also be used for must providing that solid fraction is previously separated by centrifugation[START_REF] Solfrizzo | Determination of ochratoxin A in 24 grape and winery by-products by immunoaffinity column cleanup and 25 Exposition[END_REF]). The fate of OTA during vinification of grape has been studied by 5 different authors with contrasting results. Fernandes et al. (2003) observed an increase of 6 OTA concentration in must during maceration of crushed grapes and a consistent reduction of 7 OTA during pomaces and lees separations. Grazioli et al. (2006) found little or no reduction 8

  grape pomaces and solid biomass have high OTA concentration and represent 1

4 5

 45 obtained with carbon or commercial preparations containing carbon 1 (Mikofree, Myco AD A-Z, Standard Q/FIS). On the other hand the efficacy in removing OTA 2 was proportional to the reduction of polyphenol content of treated wines. 3Conclusions

••••Food

  Figure 1

.

  Aspergillus niger aggregate 18was predominant from early veraison to ripening representing 80-85% of contamination. A. 19 carbonarius increased from veraison to ripening. OTA contamination of processed berries 20 was assessed and results were correlated with the incidence of black aspergilli population, in 21 particular with the increasing CFU values of A. carbonarius. The incidence of A. carbonarius 22 increased from ripening to harvest when vineyard relative humidity usually increased, with 23 high risk of OTA accumulation always associated with hot temperature[START_REF] Cozzi | Epidemiology of ochratoxin A 13 producing fungi in Apulian vineyards[END_REF]).

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	24
	25
	10

Table 1 . Occurrence of ochratoxin A in wine

 1 Figure 1. Occurrence of black aspergilli and OTA in 8 vineyards of Primitivo and 2Negroamaro varieties in Apulia during three grape-harvest seasons(2004)(2005)(2006). Logarithmic graph of the distribution of OTA concentration in grape berries in 9 relation to black aspergilli contamination levels in groups of berries: In: intact berries, Ar: 10 Aspergillus rotten berries, and Lb: Aspergillus rotten berries with L. botrana larvae damages.

	.

Table 2 . Percent removal of ochratoxin A (OTA) from red wine containing 10 µg/L OTA and treated with different amounts of adsorbent.

 2

	Adsorbent dosage (g/L)

a Percent reduction of polyphenol content (Folin Ciocalteu index) is reported in parenthesis b not tested c used on red wine containing 8.5 OTA µg/L

Table 3 Main critical control points, preventive and corrective measures to reduce ochratoxin A contamination in grapes and wine.

 3

	Risk factors	Preventive and corrective measures
	Field and pre-harvest

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


Fernando Ramos
email: fjramos@ci.uc.pt

Maria Lurdes Baeta

Jorge Reis

Maria Irene Noronha Da Silveira

Noronha Da Silveira

Maria Irene

M L Baeta

M I N Silveira

Fernando Jorge

Ramos

Evaluation of the illegal use of clenbuterol in feed, drinking water, urine, and hair samples collected in bovine farms in Portugal

Keywords: Clenbuterol, drinking water, feeds, urine, hair 34

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 36

Food with high fat content is considered one of the main causes of cardiovascular diseases. 37

For that reason, consumers in developed countries have been avoiding the consumption of 38 meat with higher fat content. So, animal producers have used some anabolic 39 substances which curtail the production of this kind of meat. β-agonists were utilized 40 because they result in animal carcasse meat with less fat tissue and, at the same 41 time, increased muscle protein, which provides a very attractive meat to consumers' 42 eyes [START_REF] Asato | Repartitioning agents: 5-[1-310 Hydroxy-2-(isopropylamino) ethyl] -anthranoline and related phenethanolamines; Agents for 311 promoting growth, increasing muscle acreation and reducing fat deposition in meat-producing 312 animals[END_REF][START_REF] Mersmann | Acute metabolic effects of adrenergic agents in swine[END_REF]Inkster, Hovell, Kyle, Brown, & Lobley, G., 43 1989). The search for food sources has increased, and consequently so has competition in the 44 animal diseases. This pressure has also led to the illegal use of pharmacological agents, 48 specifically anabolic compounds, to promote the growth of animals (Directive nº 2003(Directive nº /74, 49 2003)). Clenbuterol, a β-agonist, is an authorised drug in the veterinarian therapeutics of 50 bovines, equines and companion animals as bronchodilator or as tocolitic agent for cows. The 51 use of β-agonists as growth promoters is a public health risk; for example, animal-derived 52 foods containing clenbuterol have caused several outbreaks of food poisoning in Spain 53 [START_REF] Martinez-Navarro | Food poisoning related to consumption of illicit ß-agonist in 378 liver[END_REF][START_REF] Garay | Intoxicación por clenbuterol: datos clínicos y analíticos de un brote 359 epidémico en Móstoles, Madrid[END_REF], France [START_REF] Pulce | Intoxication alimentaire collective due à la présence de residus de clenbutérol dans du 389 foie de veau[END_REF], Italy (Maistro, 54 Chiesa, Angeletti, & Brambilla, 1995;Brambilla, Loizzo, Fontana, Strozzi, Guarino, & 55 Soprano, 1997;Brambilla et al., 2000), China [START_REF] Shiu | A cluster of clenbuterol poisoning associated with pork 419 and pig offal in Hong Kong[END_REF] and Portugal (Barbosa et 56 al., 2005). In addition, the European Union (EU) has prohibited the use of growth-promoting 57 drugs in animals destined for human consumption, and the use of such prohibited substances 58 also constitutes an unfair practice in the market of animal production (Directive nº 96/22, 59 1996). 60 61 To implement the EU regulations, the EU Commission issues guidelines describing National 62

Residue Control Plans for all Member States and these Plans involve sampling animals before 63 and after slaughter (Directive nº 96/23, 1996). In the case of β-agonists in Portugal, the 64 Residue Control Plan stipulates the collection of liver and muscular tissue samples from 65 animals after slaughter, as well as samples from urine, drinking water, and feed from live 66 animals. GC-MS was used as the analytical method for determining the presence of β-67 agonists, in agreement with the parameters established by Commission Decision nº 68 2002/657/CE (2002). The present paper is intended to contribute to a better understanding of 69 (Kurien, Everds & Scofield, 2004). 50 120 ml of urine was the minimum quantity of each sample. 121

Hair samples were collected using scissors and hair was cut as close as possible to the skin of 122 the animal. A quantity of 20 g, at least, was considered appropriate. Of the 37 hair samples 123 initially collected, only 5, one per farm B, C, D, E and F, were from animals that urine 124 samples had also been collected. After six months, on farm B, hair and urine samples were 125 simultaneously collected from 56 animals. 126 127 Table 1 shows the number of each sample matrices collected on each farm. 128 Urine and water samples were frozen at -20 ºC ± 2 ºC and hair and feed samples were stored 129 at 4ºC ± 2ºC until further analysis. 130 131

Sample preparation 132

Drinking water-Samples were thawed at room temperature and, after homogenization, 15 ml 133 were transferred to a centrifuge tube. After 10 min centrifugation at 4000g, an aliquot of 10 134 ml of clarified sample was transferred to another container and 100 µl of 1 µg/ml IS and 5 ml 135 of 0.1 M phosphate buffer (pH 6.0 ± 0.1) were added. Finally, pH was adjusted to 6.0 ± 0.3 136 with 1 M potassium hydroxide [START_REF] Boyd | Methods for the determination of ß-agonists in 319 biological matrices. Analyst, 121, 1R -10R. 320 of contaminated beef meat in Italy[END_REF][START_REF] Ramos | β 2 -Agonist extraction procedures for chromatographic analysis[END_REF]. 137 138 Feed-5.0 g of ground and sieved sample were weighed into a centrifuge tube and 25 ml of 139 chloroform and 100 µl of 1 µg/ml IS were added. After a 30 min agitation period at 420 rpm, 140 everything was filtered into a decantation funnel already containing 10 ml of 1 M 141 hydrochloric acid. The decantation funnel was mixed manually for 1 min and allowed to rest 142 until complete phase separation was achieved. The organic phase was discarded. Another 10 143 ml of chloroform were added to the decantation funnel and the previous step repeated. The 144 Urine-Urine samples, collected each one for a different animal, were thawed at room 148 temperature and, after homogenization, 15 ml were transferred into a centrifuge tube. After 10 149 min centrifugation at 4000g, pH was adjusted to 5.2 ± 0.3 with glacial acetic acid. Then, 10 150 ml of clarified sample were taken and the following were added: 100 µl of 1 µg/ml IS, 4 ml of 151 0.2 M acetate buffer (pH 5.2 ± 0.1) and 50 µl of Helix pomatia. The mixture was vortexed for 152 20 sec, and then incubated overnight in an oven at 48 ºC ± 2 ºC. Samples were left to cool at 153 room temperature. Then 5 ml of 0.1 M phosphate buffer (pH 6.0 ± 0.1) were added and the 154 pH was adjusted to 6.0 ± 0.3 with 1 M potassium hydroxide [START_REF] Boyd | Methods for the determination of ß-agonists in 319 biological matrices. Analyst, 121, 1R -10R. 320 of contaminated beef meat in Italy[END_REF]. 155 156Hair-Hair was washed with an aqueous solution of 5% Tween 80. The sample was then 157 rinsed with purified water, as often as necessary, until the washing water was clear. Hair 158 samples were dried overnight in an oven at 80 ºC, and subsequently cut into small pieces 1-2 159 mm in length. Pieces with a total mass of 1.0 g were transferred to a centrifuge tube to which 160 3 ml of 2 M sodium hydroxide and 100 µl of 1 µg/ml IS were added. Following 161 homogenization, the sample was digested at 80 ºC for 1 h, with agitation every 15 min (Fente, 162 Vázquez, Franco, Cepeda, & Gigosos, 1999;[START_REF] Ramos | Optimization of diphasic dialysis procedure for clenbuterol residues 404 extraction in bovine retina and hair[END_REF]. After cooling to room 163 temperature, 4 ml of 0.2 M acetate buffer (pH 5.2 ± 0.1) were added and the pH was adjusted 164 to 5.2 ± 0.3 with 2 M hydrochloric acid. The mixture was centrifuged for 5 min and the 165 supernatant was filtered. The sediment was resuspended in 5 ml of acetate buffer and 166 centrifuged again for 5 min. The second supernatant was also filtered and mixed with the 167 previous one (Cristino, Ramos, & Silveira, 2003). Then, 50 µl of Helix pomatia were added, 168 the mixture was vortexed for 20 sec, and incubated overnight at 48 ºC ± 2 ºC. Samples were 169 The sample extracts prepared as described above were purified by SPE using mixed-phase 175 columns. A 500 mg column was used for urine and water samples, while a 1000 mg column 176 was used for feed and hair; the volumes used for the former were one-half the volumes used 177 for the latter. Sorbent was activated with methanol, water, and phosphate buffer (2 or 4 ml of 178 each solvent), after which the extract was loaded. Acetic acid (1 ml or 2 ml) was added to the 179 columns and left for 10 min before being eluted. The washing step was performed using 6 or 180 12 ml of methanol. After each of the previous two steps, the sorbent was dried using 181 centrifugation at 4000g. Analyte elution was carried out using 5 or 10 ml of ethyl acetate: 182 32% ammonia (97:3, v/v) [START_REF] Montrade | Multi-residue 384 analysis for _-agonistic drugs in urine of meatproducing animals by gas chromatography-385 mass spectrometry[END_REF]Ramos, 183 Bañobre, Castilho, & Silveira, 1999) 184 185 Derivatization 186

Eluates were evaporated to dryness under a nitrogen stream at 65 ºC. The residue was 187 dissolved in methanol and transferred to a derivatization vial. Methanol was also evaporated 188 to dryness under the conditions mentioned above for the eluates, and the resulting dry residue 189 was redissolved with 50 µl of derivatization reagent. After vial encapsulation, derivatization 190 was performed at 65 ºC for 20 min [START_REF] Ramos | Clenbuterol food poisoning diagnosis by gas chromatography-mass spectrometric 400 serum analysis[END_REF]. Table 2 shows m/z ions detected for each β-agonist, as well as for the IS (clenbuterol-d6). 200 A total of 168 urine samples were tested. All of the 24 samples found to be positive had been 272 collected in 3 farms, and they constituted 58.5% of such samples. 273 A high number (15 of 22) of drinking water samples were positive in the farms where the β-274 agonist was detected. All positive samples were collected from individual troughs of intensive 275 bovine production, where the animals were normally maintained in cattle-sheds during the 276 greater part of the day. It was also observed that clenbuterol was only put in the watering 277 system inside each cattle-shed. Thus, clenbuterol was pumped from a 50-liter water barrel into 278 the pipe that supplies bovine individual troughs. Indeed, clenbuterol water solubility allows an 279 easy and homogeneous mixture in the water that was supplied by the farms' wells and then 280 adequately managed and stored. The pump system was automatically activated by animals 281 when they drank water and the system could control clenbuterol concentration in drinking 282 water, according to animal size, age and feed and forage intake that were more or less 283 uniformly allocated in each cattle-shed. It was also confirmed that this pump system was easy 284 to make inoperative in order to difficult authority inspections. 285 286 Our data suggests that a new methodology for illegal clenbuterol administration to the 287 animals is possible to use and should be inspected by authorities. However, clenbuterol was 288 also illegally used in extensive bovine growth promotion. Farm A that performs extensive 289 bovine growth promotion presented clenbuterol-negative drinking water samples. Samples 290 from farm A were collected from two different paddocks and the four animals that were 291 declared positive for urine analysis were from the same paddock. Clenbuterol administration 292 to these animals could not be found, because drinking water and feed samples collected in the 293 same paddock were negatives. Our data for urine and drinking water samples reinforces the usefulness of these matrices for 296 screening β-agonists in live animals, which is confirmed by the Canadian Food Inspection 297 Agency [START_REF] Dickson | Determination of β-agonist 336 residues in bovine urine using liquid chromatography-tandem mass spectrometry[END_REF] or by the National Plans for Residue 298 Control in EU countries (Directive nº 96/23, 1996). Nevertheless, and with respect to live 299 animals and to the Portuguese Residue Control Plan, the hypothesis of analyzing hair samples 300 should also be considered. As it was observed in the present study, it would be relevant to 301 include hair samples. So, the present data will be shared by all the Portuguese authorities 302 involved in the official residue control plan in order to discuss the eventual inclusion of hair 303 samples for controlling β-agonists in bovines suspected of illegal fattening. Finally, this study 304 shows that clenbuterol still remains one of the most illegally used β-agonist substances on 305 farms that practice bovine growth promotion. 306

  , cimaterol, clenproperol, clenpenterol, bromobuterol, and 77 hydroxymethylclenbuterol were kindly offered by Prof. François André (Laberca, Nantes, 78 France); salbutamol and terbutaline were acquired from Sigma (Madrid, Spain); and 79 clenbuterol and hexadeuterated clenbuterol (clenbuterol-d6) as an internal standard (IS) were 80 supplied, respectively, by Interchim (Montluçon, France) and by RIVM (Bilthoven, the 81 Netherlands). N-methyl-N-trimethylsilyl-trifluoracetamide (MSTA; Sigma, Madrid, Spain), 82 1,4-Dithioerythritol (DTE; Merck, Lisbon, Portugal), and trimethyliodosilane (TMIS; Sigma) 83 in the proportion of 5 ml:10 mg:10 µl (MSTA:DTE:TMIS) were used as the derivatization 84 reagent. Purified water was obtained from a Milli-Q Plus system (Millipore, Bedford, MA, 85 USA) and all other reagents were from Merck, including Helix pomatia β-glucuronidase (30 86 U/ml) and arylsulfatase (60 U/ml). 87 88 For storage and sample preparation, the following materials were used: an Electrolux freezer 89 (Coimbra, Portugal), a Mettler AG285 balance (Zurich, Switzerland), a Moulinex mixer 90 (Lisbon, Portugal), a 40-mesh Endecotts sieve (London, UK), vortex agitators from 91 UnimagZX (Reagente 5, Oporto, Portugal) and Edmund Bülher (KL2 model, Tübingen, 92 Germany), and a Memmert oven (Schwabach, Germany)extraction (SPE) was performed using a vacuum manifold system involving 95 Clean Screen DAU mixed-phase (C8+Benzyl sulfonic acid) 500 and 1000 mg columns 96 (Interchim, Montluçon, France). Centrifugation was done on a Sigma 3-16K centrifuge 97 (Reagente 5, Oporto, Portugal), and pH determination was done with a WPA CD7400 98 potentiometer (Cambridge, UK). Material for derivatization was supplied by Chromacol (Dias 99 de Sousa, Lisbon, Portugal). β-agonist determination was achieved by GC-MS using Agilent 100 Technologies (AT) equipment (Soquimica, Lisbon, Portugal), comprising an AT6890N gas 101 chromatograph equipped with a HP-5MS column (30 m x 0.25 mm I.D. x 0.25 µm of film 102 thickness), an AT7683B automatic injector, an AT5975 MSD detector, an HP Compaq 103 computer, and an HP LaserJet 2420d printer. Nitrogen and helium gases were supplied by 104 Sogafer (Coimbra, Portugal). 105 106 Samples 107 Samples of feed, drinking water, urine, and hair were collected in 16 cattle farms during the 108 period between February 28 and August 24, 2007. 109Drinking waters were collected from troughs/tanks in minimum amounts of 100 ml per 110 sample. From farms that perform extensive growth enhancement (A, D, E, G, I and K), 111 samples were collected from the pasture tanks. For intensive farms (B, C, F, H, J, L, M, N, O 112 and P), samples were collected from individual bovine troughs from the cattlecollected from the bovine mangers or the feed storage silos. When 116 sampling was made in the storage silos, appropriate equipment was used in order to collect 117 sample aliquots at different depths. At least, 500 g of each feed were collected. Urine 118 sampling was always taken from male animals in disposable plastic bags adequately attached 119 s back, as well as by specialized technicians

  2 µl was injected into the GC-MS system in splitless mode (1 min), using 194 helium as carrier gas with a constant flow of 1.4 ml/min through the column. The column 195 was submitted to the following temperature gradient: 100 °C (0.4 min) -------› 10 °C/min ----› 196 200 °C (3 min) ----› 10 °C/min ----› 300 °C (5 min). Injector and detector temperatures were 197 270 and 280 ºC, respectively, and detection was done in electron impact (EI) mode with 198 selective ion monitoring (SIM). 199

Figures 1 -

 1 Figures 1 -4 show chromatograms of a standard solution and of positives drinking water, 201

Figure 2 -Figure 3 -Figure 4 -

 234 Figure 1 -TIC chromatogram of β-agonist standards

Table 1 -

 1 Number of samples collected on each farm

	Page 23 of 25	Food Additives and Contaminants
	Farm	Water	Feed	Urine	Hair
	A	2	3	8 (4)	2 + 41
	B	7 (6)	2	22 (14) + 56	13 (1) + 56
	C	4 (2)	2	11 (6)	1 (1)
	D	1	2	1	5
	E	3	2	6	1
	F	11	11	25	6
	G	2	4	1	9
	H	8	8	14	-
	I	1	1	6	-
	J	14	-	5	-
	K	2	3	13	-
	L	11 (7)	-	-	-
	M	8	-	-	-
	N	6	-	-	-
	O	18	-	-	-
	P	8	-	-	-
	TOTAL 106 (15)	78 (0)	168 (24)	134 (2)
	Note 1 -Data in brackets are the number of clenbuterol positive samples
	Note 2 -Data separated by a + mark indicate that samples were collected with a interval of six
	months

http://mc.manuscriptcentral.com/tfac Email: tfac@csl.gov.uk

Table 2 -

 2 Detected m/z ions for each β-agonist

	Time window	β -agonists	m/z ions

Table 3 -

 3 Performance characteristics of the method for clenbuterol

	Page 25 of 25
	Matrices	CCα	CCβ	Accuracy (n=6)	Repeatability (n=6)	Reproducibility (n=18)
		[µg/l] or [µg/kg] [µg/l] or [µg/kg]	[%]	[% (CV)]	[% (CV)]
	Urine	0.12	0.25	98.45	2.31	9.93
	Feed	0.73	1.92	92.10	6.97	12.23
	Hair	1.05	2.53	93.31	5.82	13.55

http://mc.manuscriptcentral.com/tfac Email: tfac@csl.gov.uk

Food Additives and Contaminants

intensive meat production sector. The demand for higher productivity, preferably at lower

improvement of animal feeding and lodging, the use of genetic breeding and the control of

the use (or misuse) of β-agonist substances in live animals in Portugal. The data obtained

the relevance of sampling drinking water and hair samples for Portuguese plan for clenbuterol

aqueous phase was recovered and 5 ml of 0.1 M phosphate buffer (pH 6.0 ± 0.1) were added.

[START_REF] Ramos | Clembuterol em 408 alimentos compostos para animais por cromatografia gasosa-espectrometria de massa[END_REF]

http://mc.manuscriptcentral.com/tfac Email: tfac@csl.gov.ukFood Additives and Contaminants

left to cool to room temperature. Then 5 ml of 0.1 M phosphate buffer (pH 6.0 ± 0.1) were

added and pH was adjusted to 6.0 ± 0.3 with 1 M potassium hydroxide[START_REF] Boyd | Methods for the determination of ß-agonists in 319 biological matrices. Analyst, 121, 1R -10R. 320 of contaminated beef meat in Italy[END_REF]

[START_REF] Ramos | β 2 -Agonist extraction procedures for chromatographic analysis[END_REF].

hair samples. The results for the positive samples are shown in Table1.

Loizzo, A. (2000). Clinical and pharmacological profile in a clenbuterol epidemic poisoning

review. LaboratoryAnimals, 38,

Ramos, F., Bañobre, M.C., Castilho, M.C. & Silveira, M.I.N. (1999). Solid phase extraction

Chromatography & Related Technologies, 22, 2307-2320.


Geraldine ; Dowling

Pasquale Gallo

Serena Fabbrocino

Luigi Serpe

Liam Regan

Determination of ibuprofen, ketoprofen, diclofenac and phenylbutazone in bovine

Keywords: Ibuprofen, Ketoprofen, Diclofenac, Phenylbutazone, Bovine Milk, Method Validation

Introduction

Phenylbutazone (PBZ), ibuprofen (IBP), ketoprofen (KPF) and diclofenac (DCF) are non-steroidal anti-inflammatory drugs (NSAIDs). The molecular structure of these compounds is shown in PBZ is a pyrazole derivative, IBP and KPF are propionic acid derivatives and DCF is an aniline derivative.

In the USA a survey was carried out involving 2000 veterinarians whose practices dealt 50% of the time with food producing animals [U.S Code of Federal Regulations, 1988]. The survey showed that 93 % of veterinarians use NSAIDs on a regular basis.

Dairy practitioners reported more frequent use of NSAIDs than did beef practitioners.

Overall, veterinarians indicated that NSAIDs were an important group of compounds which were routinely used for the treatment of food producing animals [Kopcha, et al., 1992]. According to EU law, all substances for veterinary use need to be included in Annexes 1-3 of Regulation 2377/90 [European Commission, 1990]. This regulation establishes lists of compounds that have a fixed MRL (Annex I), that need no MRL (Annex II) or that have a provisional MRL (Annex III). PBZ and IBP are compounds that have not been included in Annexes 1-3 and have no maximum residue limit (MRL) established. Substances that have no MRL established are prohibited for use in food producing animals. DCF is listed in Annex I and a provisional MRL has been set for different animal species and target matrices. DCF is however prohibited for use in milk producing animals. KPF is listed in Annex II and is allowed for the application to species that produce milk but are not intended for human consumption. NSAIDs can cause adverse health effects in humans such as aplastic anaemia, gastrointestinal disorders and agranulocytosis [START_REF] Insel | Goodman and Gilman's The Pharmacological Basis of Therapeutics[END_REF] and changes in renal function [START_REF] Goodman | Goodman and Gilman's The Pharmacological Basis of Therapeutics[END_REF]. Longterm exposure to PBZ has caused kidney tumors in mice and liver tumors in rats [START_REF] Kari | [END_REF]. The widespread use of NSAIDs presents a potential risk to the consumer if food containing residues enter the food chain so there is a need for the development of methods to monitor compliance with legislation in the EU in a variety of animal tissues and products.

In the determination of NSAIDs in animal tissues PBZ has been shown to be extracted from equine plasma using acetonitrile [Hardee, et al., 1982;Neto, et al., 1996] or ethyl acetate [Grippa, et al., 2000], from equine urine with methanol [Stanley, et al., 2007] or a mixture of dichloromethane:ethanol solution [Neto, et al., 1996]. PBZ has been shown to be extracted from bovine plasma using acetonitrile [Miksa, et al., 2005;Fiori, et al., 2004] or a straight forward ultra-centrifugation procedure [De Veau, et al., 1999]. IBP, KPF and DCF has been shown to be extracted from bovine plasma using acetontrile [Miksa, et al., 2005]. PBZ has been shown to be extracted from ovine, equine, and porcine muscle using a mixture of ethyl acetate: methanol and DL-dithio-threitol [Clarke, et al., 2002]. PBZ has been shown to be extracted from bovine kidney using a mixture of water: ammonium hydroxide [Clarke, et al., 2002]. PBZ has been shown to be extracted from bovine milk with a mixture of ethanol: ammonia [Martin, et al., 1983]. KPF has been shown to be extracted from bovine milk using acetonitrile [Daeseleire, et al., 2003]. The incorporation of a hydrolysis step at the beginning of the extraction procedure for the determination of NSAIDs is also reported in the literature. This hydrolysis step allows the deactivation of plasma bound proteins and the release of the protein bound NSAID residues. After hydrolysis PBZ has been shown to be extracted from bovine plasma using dichloromethane [START_REF] Singh | [END_REF], from equine plasma with a mixture of dichloromethane:n-hexane:diethyl ether solution [Hines, et al., 2004], porcine, ovine, bovine and caprine urine were extracted with chloroform [Igualada and Moragues, 2005] and equine urine was extracted with dichloromethane [START_REF] Singh | [END_REF] or diethyl ether [Gonzalez, et al., 1996] for the determination of PBZ. After hydrolysis or solvent extraction, solid phase extraction [SPE] is often used in the purification of extracts containing NSAIDs. Bovine plasma has been shown to be purified using C 18 SPE for the determination of PBZ and DCF [START_REF] Gowik | [END_REF], IBP, KPF, DCF and PBZ [Vinci, et al., 2006] and IBP, KPF and DCF [De Jong, et al., 1989]. Plasma from pigs and rabbits was purified using this approach to determine IBP, KPF, DCF and PBZ [Vinci, et al., 2006]. Equine plasma was purified using C 18 for the determination of IBP, KPF, DCF and PBZ simultaneously [Vinci, et al., 2006] or PBZ alone [Taylor and Westwood, 1995] or the C 18 was substituted for an Oasis HLB [Quintana, et al., 2004] for the determination of PBZ. Bovine, equine and porcine muscle was purified using a Florisil SPE cartridge. Bovine muscle extracts were applied to an Oasis HLB TM cartridge for the removal of interferences in the determination of PBZ and KPF [Van Hoof, et al., 2004]. Bovine kidney extracts were applied to silica cartridges [Clarke, et al., 2002] and bovine plasma extracts were purified using affinity columns [Fiori, et al., 2004] for the determination of PBZ.

A few methods have been reported for the analysis of NSAIDs in animal tissues using LC-UV [Hardee et al., 1982;Grippa et al., 2000;De Veau, 1999;Martin, et al., 1983;[START_REF] Singh | [END_REF][START_REF] Gowik | [END_REF]Taylor and Westwood, 1995;Quintana et al., 2004;Moragues, 2005;Vinci, et al., 2006;Van Hoof, et al., 2004] and GC-MS [Neto et al., 1996;[START_REF] Singh | [END_REF]Hines et al., 2004;Gonzalez, et al., 1996;Taylor and Westwood, 1995]. There are few methods that analyse for IBP, KPF DCF and PBZ in animal products such as milk and those available are based on LC-UV [Martin, et al., 1983] for the determination of PBZ or LC-MS detection [Daeseleire, et al., 2003] for the determination of KPF and FLU. NSAIDs have been derivatised for GC-MS using the following derivatisation reagents; Methelut [Neto et al., 1996], Bis(trimethylsilyl) trifluoroacetamide [START_REF] Singh | [END_REF], n-trimethylsulfonium hydroxide [Hines, et al., 2004] and N-methyl-N-(trimethylsilyl) fluoroacetaminde and Trimethylanilium hydroxide [Taylor and Westwood, 1995]. Gonzalez et al. monitors for IBP, KPF, DCF and PBZ simultaneously in equine plasma and urine by GC-MS in SIM mode used Methyl iodide/anhydrous potassium carbonate [Gonzalez, et al., 1996] as derivatisation agents.

The method developed in this study was based on a method developed by Vinci, et al., (2006) but adapted to include deuterated analogues of IBP, DCF and PBZ. LC-MS detection was replaced by GC-MS/MS coupled with a derivatisation step for detection of IBP, KPF and DCF. The advantage of the additional derivatisation step is the ability to produce two daughter ions for the determination of IBP whereas by LC-MS only precursor (parent) and one daughter ion for these compounds could be obtained [Vinci, et al., 2006]. This method involves the addition of acetonitrile to bovine milk followed by clean-up using Isolute TM C 18 solid phase extraction (SPE) cartridges and analysis by GC-MS/MS. To the best of our knowledge there are no

Materials and Methods

Materials and reagents

Water, ethanol, ethyl acetate, methanol, acetonitrile, acetic acid, hydrochloric acid (37 %), n-hexane and iso-octane (HiPerSolv grade) were obtained from BDH (Merck, UK). Heptafluorobutyric Acid Anhydride was obtained from Pierce, (Pierce, USA).

1,1,1-3,3,3-Hexafluoro-2-propanol was obtained from Sigma (Sigma Aldrich, Ireland). PBZ was purchased from Sigma (Sigma Aldrich, Ireland). d 10 -PBZ was obtained from Cambridge Isotope Labs (Cambridge Isotope Labs, USA). KPF was purchased from Fluka Riedel-de-Haen ( Sigma Aldrich, Ireland). IBP and DCF were purchased from Sigma Aldrich (Sigma Aldrich, Ireland). d 3 -IBP and d 4 -DCF were obtained from CDN Isotopes (CDN Isotopes, Canada). Primary stock standard solutions of PBZ, KPF, IBP, DCF, d-10 PBZ, d 3 -IBP and d 4 -DCF (stable for 12 months) were prepared in ethanol at a concentration of 1 mg mL -1 . Intermediate single standards solutions of all analytes (stable for 6 months) were prepared in methanol at a concentration of 10 µg mL -1 . PBZ, KPF, IBP and DCF standard fortification solution (stable for 6 months) was prepared in methanol at a concentration of 500 ng mL -1 from the 10 µg mL -1 intermediate stock solution. A fortification solution of d 10 -PBZ, d 3 -IBP and d 4 -DCF (stable for 6 months) was prepared in methanol at a concentration of 500 ng mL -1 from the 10 µg mL -1 intermediate stock solution. All standards were stored at 4 o C in the dark. Isolute TM endcapped C 18 solid phase extraction cartridges (6 mL, 1 g) were obtained from Biotage (Biotage, UK).

Methanol: water (10:90, v/v) and 10 mM ascorbic acid were used as solid phase extraction wash solvents. N-hexane: diethyl ether (50:50,v/v) was used as the solid phase extraction elution solvent. Injection solvent was iso-octane.

GC conditions

The GC system consisted of a Varian CP-3800 Gas Chromatograph coupled to a Varian 1200 L Quadrupole MS and a Varian 8400 Autosampler (Varian, CA, USA).

The carrier gas was helium at flow of 1.5 mL min -1 . IBP, KPF, DCF and PBZ was Workstation (version 6.5) chromatographic management software (Varian, CA, USA). The described GC system was shown to be suitable for the analysis of IBP, KPF, DCF and PBZ (Figures 2345).

MS/MS parameters

The analysis was performed using EI positive MS/MS with multiple reaction monitoring (MRM) mode. Two transitions per compound were used and the collision voltages were optimised as shown (Table 2). The MS/MS detector conditions were as follows: electron energy -70 eV; detector voltage 1950 volts; detector setting fixed voltage; collision cell pressure 1.5 mTorr; ion source temperature 250 o C; transfer line temperature 290 o C; and the collision gas was argon.

Milk samples

Milk obtained for use as negative controls was separated into 50 mL aliquots and stored at -20 °C. The milk was obtained from local supermarkets and was analysed in previous batches and milk found to contain no detectable residues of IBP, KPF, DCF and PBZ was used as negative controls.

Sample extraction and clean-up

Milk samples (5 ml) were aliquoted into 50 ml polypropylene tubes. The milk aliquots (5 mL) were fortified with internal standard at levels corresponding to 15 ng mL -1 by adding a 150 µL portion of a 500 ng mL -1 mix solution of d 3 -IBP, d 4 -DCF and d 10 -PBZ. Samples were fortified at levels corresponding to 5, 7.5 and 10 ng mL -1 by adding 50, 75 and 100 µL portions of a 500 ng mL -1 solution of IBP, KPF, DCF and PBZ. After fortification, samples were held for 15 min prior to extraction. Acetonitrile (5 mL) was added and the samples were vortexed (30 sec), centrifuged (3500 rpm, 10 Samples are reconstituted in 50 µL of iso-octane. An aliquot (2 µL) is injected onto the GC column.

Matrix-Matched Calibration

Matrix matched calibration curves were prepared and used for quantification. Control milk previously tested and shown to contain no residues was prepared as above (2.4).

One control milk sample was used for each calibration standard level. Milk samples (5 ml) were aliquoted into 50 ml polypropylene tubes. Samples were fortified with internal standard at levels corresponding to 15 ng ml -1 by adding a 150 µL portion of a 500 ng mL -1 mix solution of d 3 -IBP, d 4 -DCF and d 10 -PBZ. Samples were fortified at levels corresponding to 0, 2, 5, 7.5, 10 and 20 ng ml -1 by adding 0, 20, 50, 75, 100 and 200 µL portions of a 500 ng mL -1 standard solution of IBP, KPF, DCF and PBZ. After fortification, samples were held for 15 min prior to the extraction procedure as described above (2.5). Calibration curves were prepared by plotting the response factor as a function of analyte concentration (0 to 20 ng ml -1 ) to quantify samples For estimation of accuracy, blank milk samples were fortified with IBP, KPF, DCF and PBZ at 5, 7.5 and 10 ng ml -1 . Six replicate test portions, at each of the three fortification levels, were analysed. Analysis of the 18 test portions was carried out on three separate occasions. For the estimation of the precision of the method, repeatability and within-laboratory reproducibility was calculated. The decision limit (CCα) of the method was calculated according to the calibration curve procedure using the intercept (value of the signal, y, where the concentration, x is equal to zero) and 2.33 times the standard error of the intercept for a set of data with 6 replicates at 3 levels. The detection capability (CCβ) was calculated by adding 1.64 times the standard error to the CCα.

Method validation

Results and Discussion

Preliminary experiments

The GC-MS/MS method was developed to provide confirmatory data for the analysis of bovine milk for IBP, KPF, DCF and PBZ. The MS/MS fragmentation conditions were investigated and collision energies were optimised. For a method to be deemed confirmatory four identification points are required. These identification points can be obtained by monitoring one precursor ion (parent mass) and two daughters (corresponding to strong and weak ion).

IBP, KPF and DCF are derivatised with a mixture of HFAA and HFPOH. The reagents form a derivative with the hydroxyl group of the carboxylic acid on IBP, KPF and DCF. PBZ does not have any functional groups that are easily derivatised.

The ions monitored for each analyte generally result from cleavage of the aliphatic chains containing the hexafluoropropyl moiety. IBP, KPF, DCF and PBZ are chromatographed on a Restex-1 MS column with retention times of 6.1, 11.9, 14.

Specificity

The technique of GC-MS/MS itself offers a high degree of selectivity and specificity.

To establish the selectivity/specificity of the method, a variety of milk samples were fortified with the IBP, KPF, DCF and PBZ and d 3 -IBP, d 4 -DCF and d 10 -PBZ and non-fortified samples were also analysed. No interfering peaks were observed at the retention time for IBP, KPF and PBZ (Figures 2345). Additionally samples were fortified with 2.0 ng ml -1 of other NSAIDs which included vedaprofen (VEDA), tolfenamic acid (TOLF), mefenamic acid (MEF), flunixin (FLU), oxyphenylbutazone (OXYPHEN) and suxibutazone (SUXI). No interferences were observed in the retention window of IBP, KPF, DCF and PBZ in chromatograms when fortified with these substances.

Linearity of the response

The linearity of the chromatographic response was tested with matrix matched curves using 6 calibration points in the concentration range of 0 to 20 ng mL -1 . The regression coefficients (r 2 ) for all the calibration curves used in this study were ≥ ≥ ≥ ≥ 0.99. Representative matrix matched calibration curves are shown (Figure 6).

Accuracy

The accuracy of the method was determined using bovine milk samples fortified at 5.0, 7.5 and 10.0 ng ml -1 . Mean corrected recovery (n = 18) of the analyte, determined in three separate assays (Table 2) was between 104 and 112 % for IBP, KPF, DCF and PBZ.

Precision

The usefulness of suitable deuterated standards is demonstrated in the excellent repeatability and within-laboratory reproducibility obtained for IBP, DCF and PBZ (Table 2). No deuterated analogue is available for KPF so poorer precision was obtained for this analyte. The precision of the method, expressed as RSD values for the within-lab reproducibility at the three levels of fortification (5, 7.5 and 10 ng ml - 1 ), for IBP, DCF and PBZ was less than 8 %. The RSD values were less than 28 % for Even by applying correction by means of the I.S of the other analytes to KPF a much higher RSD was achieved and this indicates the necessity for the incorporation of a structurally identical isotopically-labelled IS of KPF in the method. Due to the low precision obtained for KPF without a suitable IS, the method developed was suitable for qualitative confirmation only.

CCα and CCβ

The decision limit (CCα) is defined as the limit above which it can be concluded with an error probability of α, that a sample contains the analyte. In general, for non-MRL substances an α equal to 1 % is applied. The detection capability (CCβ) is the smallest content of the substance that may be detected, identified and quantified in a sample, with a statistical certainty of 1-β, were β = 5 %. CCα and CCβ were calculated using the intercept (value of the signal, y, were the concentration, x is equal to zero) and the standard error of the intercept for a set of data with 6 replicates at 3 levels (5.0, 7.5 and 10.0 ng ml -1 ). Blank milk was fortified at 1, 1.5 and 2 times the minimum required performance level of 5 ng ml -1 set for IBP, KPF, DCF and PBZ.

CCα is the concentration corresponding to the intercept + 2.33 times the standard error of the intercept. CCα values of 0.59, 2.69, 0.90 and 0.70 ng ml -1 were achieved for IBP, KPF, DCF and PBZ. CCβ is the concentration corresponding to the signal at CCα + 1.64 times the standard error of the intercept (i.e the intercept + 3.97 times that standard error of the intercept). CCβ values of 1.01, 4.58, 1.54 and 1.19 ng ml -1 were achieved for IBP, KPF, DCF and PBZ.

Measurement Uncertainty

According to SANCO/2004/2726 rev 1 the within laboratory reproducibility can be regarded as a good estimate of the combined measurement uncertainty of individual methods [START_REF] Sanco | ) CRLs View on state of the art analytical methods for national residue control plans SANCO[END_REF]. For the calculation of the extended uncertainty a safety factor is required. The within laboratory reproducibility should be multiplied by a value of 2.33 and this should be used when determining the CCα, corresponding to a confidence level of 99 %. As the only source of variation during the validation was the different days and different milk sourced from different animals it was decided to use a safety factor of 3.0 instead of 2.33. The measurement uncertainty of the method was estimated at 17.8, 80.9, 28.2 and 20.2 %. This was determined by calculating the within laboratory reproducibility of the method, followed by multiplication of the within laboratory reproducibility by the safety factor of 3.0. Recently it was proposed by Community Reference Laboratories (CRLs) in Europe that laboratories should be capable of monitoring for KPF, DCF and PBZ in bovine milk at a level of 5 ng ml -1 and IBP at a level of 10 ng ml -1 in EU member states [START_REF] Sanco | ) CRLs View on state of the art analytical methods for national residue control plans SANCO[END_REF]. This study shows that the required sensitivity was achieved for all compounds that easily meets the proposed levels. The method performs very well in terms of accuracy and within-laboratory reproducibility. For KPF improvements can be made if a suitable deuterated analogue becomes available.

The objective of the work to develop, validate and accreditate a method for these residues in bovine milk at low ng ml

Fig. 1 .

 1 NSAIDs are used widely in veterinary medicine in the treatment of food producing animals. Depending on their heterogeneous molecular structures the NSAIDs can be classified into four main sub-classes: (a) salicyclic acid derivatives; (b) propionic acid derivatives; (c) pyrazole derivatives and (d) aniline derivatives including both anthranilic acid derivatives and nicotinic acid derivatives.

  the simultaneous determination of IBP, KPF, DCF and PBZ in bovine milk using GC-MS/MS in multiple reaction monitoring mode.

  column with film thickness of 0.25 µm, diameter 0.25 mm, length 30 m packed with SE-54/CP Sil 8 type material (Restek, UK). The injector temperature was maintained at 275 °C and it was a constant temperature splitless programmable temperature vaporising (PTV) injection. The injection volume was 2 µl. The oven temperature was set at 100 o C for 1 min and ramped to 300 o C in increments of 10 o C per min. The temperature is held at 300 o C for 1 min. The runtime is 22 minutes. Data acquisition and integration were performed using Varian MS

  C) and the supernatant was transferred to a clean polypropylene tube. The sample pellet is re-extracted with 5 mL of acetonitrile and the supernatants are combined. 10 mM ascorbic acid (20 mL) and 1 M hydrochloric acid (0.2 mL) were added to the extracts and the pH of the samples were checked to ensure they were at pH 3 before proceeding to the solid phase extraction (SPE) stage. The sample extracts were purified by SPE using C 18 SPE cartridges. Sample extracts were loaded onto the cartridges (preconditioned with n-hexane:diethyl ether (50:50, v/v) (3 mL), methanol (3 mL) and water (5 mL). The samples were loaded onto cartridges under gravity. The cartridges were washed with 10 mM ascorbic acid (3 mL) and methanol:water (10:90, v/v) (3 mL). The cartridges were dried under vacuum (30 min). The cartridges were eluted with n-hexane:diethyl ether (50:50, v/v) (3 mL). The eluates were reduced to dryness under nitrogen without heat before re-dissolving in 50 µL of heptafluorobutyric acid anhydride (HFAA) and 25 µL 1,1,1-3,3,3-Hexafluoro-2-propanol (HFPOH) and vortexed (1 min). Samples are capped and placed in an oven at (60 o C, 45 min). Samples are allowed to come to room temperature and the derivatisation reagent is evaporated under a gentle stream of nitrogen with no heat.

  4 and 16.4 min respectively. d 3 -IBP is used as I.S for IBP and KPF. d 4 -DCF and d 10 -PBZ were used as I.S for DCF and PBZ.

  method was according to procedures described in Commission Decision 2002/657/EC[European Commission, 2002] covering specificity, calibration curve linearity, recovery (accuracy), precision, decision limit (CCα) and detection capability (CCβ).

  in this study has been used to confirm the presence of IBP DCF and PBZ and for qualitative confirmation of KPF in bovine milk in the National Monitoring Plan inIreland in 2006Ireland in , 2007Ireland in and 2008. . In routine monitoring for these substances at our laboratory it was possible to detect the precursor ion and two daughter ions (at 2 ng mL -1 ) in multiple reaction monitoring mode. Furthermore the relative retention time versus internal standard requirement and the product ion ratio requirement was also met. The method has been carried out by different analysts over a period of three years under varying environmental conditions and the method was shown to be robust. The method was accredited by the Irish National Accreditation Board in 2006 according to ISO17025 standard which is the main internationally recognised standard used by testing and calibration laboratories.ConclusionsA relatively fast, simple and selective GC-MS/MS method for the detection of IBP, KPF, DCF and PBZ in bovine milk has been developed. There is no published confirmatory method for the simultaneous determination of IBP, DCF, PBZ and the qualitative confirmation of KPF in bovine milk by GC-MS/MS that is validated according to Commission Decision 2002/657/EC[European Commission, 2002].

Fig. 2 .

 2 Fig. 2. Chromatogram of Negative Control Milk (A) fortified at 15 ng.ml -1 with internal standard d 3 -IBP and chromatogram of Negative Control Milk (B) fortified with 2 ng.ml -1 of IBP and at 15 ng.ml -1 with internal standard d 3 -IBP.

Fig. 3 .

 3 Fig. 3. Chromatogram of Negative Control Milk (A) fortified at 15 ng.ml -1 with internal standard d 3 -IBP and chromatogram of Negative Control Milk (B) fortified with 2 ng.ml -1 of KPF and at 15 ng.ml -1 with internal standard d 3 -IBP.

Fig. 4 .

 4 Fig. 4. Chromatogram of Negative Control Milk (A) fortified at 15 ng.ml -1 with internal standard d 4 -DCF and chromatogram of Negative Control Milk (B) fortified with 2 ng.ml -1 of DCF and at 15 ng.ml -1 with internal standard d 4 -DCF.

Fig. 5 .Fig. 6 .

 56 Fig. 5. Chromatogram of Negative Control Milk (A) fortified at 15 ng.ml -1 with internal standard d 10 -PBZ and chromatogram of Negative Control Milk (B) fortified with 2 ng.ml - 1 of PBZ and at 15 ng.ml -1 with internal standard d 10 -PBZ.

Table 1 :

 1 MS/MS parameters for determination of IBP, KPF, DCF and PBZ

	Page 21 of 22	Food Additives and Contaminants
			Scan	Collision	Rt
	Compound	Transition	Time [sec]	[V]	[min]
	IBP	356>161	0.20	-18	6.0
		356>252	0.20	-18	6.0
	d 3 -IBP	359>316	0.20	-18	6.0
	KPF	404>327	0.12	-22	11.9
		404>181	0.12	-15	11.9
	DCF	214>151	0.12	-25	14.5
		214>179	0.12	-20	14.5
	d 4 -DCF	218>183	0.12	-20	14.4
	PBZ	308>183	0.20	-21	16.1
		308>252	0.20	-13	16.1
	d 10 -PBZ	318>193	0.20	-21	16.1

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Table 2 :

 2 Intra-and inter-assay variation for accuracy of IBP, KPF, DCF and PBZ from bovine milk

			%RSD
		%RSD	Within	%	MU
	Analyte	Repeatability	Laboratory	Rec	%
	IBP	4.4	5.9	104.1	17.8
	KPF	23.7	28.8	108.3	80.9
	DCF	7.9	9.9	111.6	28.2
	PBZ	4.7	6.7	105.3	20.2

Table 3 :

 3 Calculated CCα and CCβ values based on data from Assay 1, 2 and 3

			Page 22 of 22
	Analyte	CCα	CCβ
		(ng ml -1 )	(ng ml -1 )
	IBP	0.59	1.01
	KPF	2.69	4.58
	DCF	0.90	1.54
	PBZ	0.70	1.19

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Acknowledgements

The authors would like to thank staff at The State Laboratory, Ireland and The Istituto Zooprofilatico Sperimentale del Mezzogiorno, Italy for their practical assistance.