K M Cooper

R J Mccracken

M Buurman

D G Kennedy

Residues of nitrofuran antibiotic parent compounds and metabolites in eyes of broiler chickens

Keywords: Veterinary drug residues -antimicrobials, Veterinary drug residues -semicarbazide nitrofurans, broiler chickens, eyes, metabolites, depletion, semicarbazide .manuscriptcentral

Nitrofuran antibiotic residues in food continue to be of international concern. The finding of sources of semicarbazide (SEM), other than through the misuse of nitrofurazone, present a challenge to the use of SEM as a definitive marker residue for this drug. Detection of intact (parent) nitrofurazone would avoid confusion over the source of SEM residues. Broiler chickens were fed sub-therapeutic nitrofurancontaining diets and tissues analysed for parent compounds and metabolites by LC-MS/MS. Depletion half-lives in muscle were longer for tissue-bound metabolite residues, 3.4 days (AOZ, AMOZ) to 4.5 days (SEM), than total metabolite residues, 2.0 days (AOZ) to 3.2 days (SEM). Metabolite concentrations were higher in eyes than muscle. Metabolite half-lives in eyes ranged from 8.5 days (AHD) to 20.3 days (SEM).

Nitrofuran parent compounds were also detected in eyes.. Furaltadone was detected in single eyes after 21 days withdrawal of a 6 mg kg -1 furaltadone diet. When 50 eyes from broilers containing metabolites in muscle close to the 1 µg kg -1 MRPL were pooled into single samples, 1.2 ng furazolidone and 31.1 ng of furaltadone were detected, but nitrofurazone was not detected due to the long depletion half-life of SEM in muscle. Further studies are required to improve LC-MS/MS nitrofurazone sensitivity and refine the sample size necessary to use nitrofurazone detection in pooled eyes as a complement to SEM detection in muscle.

Introduction

The monitoring of residues of nitrofuran metabolites in the food chain has been a major focus in the international control of veterinary drug residues for several years.

Nitrofuran antibiotics were banned within the European Union (EU) (Commission Regulation 1995) due to concerns over their carcinogenicity and mutagenicity (Van [START_REF] Van Koten-Vermeulen | Report of the 40 th Meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA)[END_REF]) and should not be used in food-producing animals or be present in foods produced in, or imported into, the EU. However, improved liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmatory analytical methods developed under the EU funded research project FoodBRAND (http://www.afsni.ac.uk/foodbrand; Cooper et al. 2005b) were instrumental in uncovering the widespread abuse of nitrofurans in various world-wide food industries during 2001-02. These included primarily the poultry and aquaculture industries in S.E. Asia and S. America, in addition to isolated findings of nitrofurans in European pork [START_REF] O'keeffe | Nitrofuran antibiotic residues in pork: the FoodBRAND retail survey[END_REF]) and poultry (Food Standards Agency 2003), and egg powders and honey from various countries (European Commission RASFF). These findings disrupted trade relations between the EU and some exporting countries. The measures taken included the adoption of Decisions that required the mandatory preimport testing of aquaculture products from Vietnam (Commission Decision 2002a), poultry and aquaculture products from Thailand (Commission Decision 2002b) and poultry products from Brazil (Commission Decision 2002c). In the wake of improved analytical techniques and subsequent changes to national legislation and monitoring programmes instigated by some exporting countries, the above decisions were revoked and the frequency of EU Rapid Alerts for nitrofurans gradually fell. The 18-month period from January 2005 produced 73 Rapid Alerts for nitrofuran metabolites (European Commission RASFF). Shrimps from S.E. Asia and India continue to be the most commonly violative foodstuff (58 Alerts), the remaining food sources being fish (5), honey (4), poultry meat (3), bovine (1), porcine (1) and baby food (1). The majority of cases concern findings of semicarbazide (SEM, 48 Alerts), the remaining being 3-amino-2-oxazolidinone (AOZ, 22) and 3-amino-5-morpholinomethyl-2oxazolidone (AMOZ, 3).

The nitrofuran antibiotics are unstable in vivo, breaking down in edible tissues within hours [START_REF] Mccracken | Determination of furazolidone in porcine tissue using thermospray liquid chromatography-mass spectrometry and a study of the pharmacokinetics and stability of its residues[END_REF]. However, they give rise to stable tissue-bound metabolites, which persist in muscle and liver for many weeks [START_REF] Hoogenboom | The use of pig hepatocytes to study the inhibition of monoamine oxidase by furazolidone[END_REF]Cooper et al. 2005b). These metabolites, for example SEM which is the metabolite of nitrofurazone (NFZ), are the marker residues used to indicate the use of, or contamination with, their parent compounds in animal tissues. However, the validity of SEM as a unique marker residue for NFZ has been called into question following recent evidence that SEM in food may arise from sources other than from this illegal antibiotic. In 2003 SEM was detected in baby food (EFSA, 2003). Subsequent investigations [START_REF] Stadler | Semicarbazide is a minor thermal decomposition product of azodicarbonamide used in the gaskets of certain food jars[END_REF]) demonstrated that the SEM was a breakdown product of azodicarbonamide; the blowing agent used in the manufacture of plastic gaskets inside the jar lids. Azodicarbonamide is also a flour treatment agent used in the bread making process, which may account for SEM found in breaded chicken but not in the chicken meat [START_REF] Kennedy | Azodicarbonamide, a flour treatment agent, produces semicarbazide residues in bread[END_REF][START_REF] Pereira | Implications of the use of semicarbazide as a metabolic target of nitrofurazone contamination in coated products[END_REF]. Carrageenan, a widely used thickening agent derived from red seaweed, has also been implicated in the production of SEM under certain conditions [START_REF] Hoenicke | Formation of semicarbazide (SEM) in food by hypochlorite treatment: is SEM a specific marker for nitrofurazone abuse?[END_REF]. Low levels of SEM, possibly of natural origin or a by-product of boiling, have also been observed in cooked crayfish samples (Saari and Peltonen 2004). Consequently, the presence of SEM can no longer be used as definitive proof of administration of NFZ to an animal and some national authorities have become reluctant to take enforcement action on the basis of SEM detection.

One approach to overcoming this uncertainty surrounding SEM would be to find a depot of intact NFZ in tissues of NFZ-treated animals. This laboratory has demonstrated that the four major nitrofurans furazolidone (FZD), furaltadone (FTD), nitrofurantoin (NFT) and NFZ all accumulate in chicken eggs and are not broken down rapidly as in muscle and liver (McCracken and Kennedy 2006). Our previous finding of nitrofuran metabolites, including SEM, at high concentrations (mg kg -1 ) in the retina/choroid layer in eyes of pigs suggested that at least some of these residues may be present in the form of their parent nitrofuran drugs (Cooper and Kennedy 2005). We subsequently demonstrated that NFZ does indeed accumulate in whole eyes of laying hens (Cooper et al. 2005a). In birds fed only 0.3 mg NFZ per kg of feed (approximately 0.1% of a therapeutic dose; National Office Of Animal Health Limited, 1992) the mean NFZ detectable by LC-MS/MS in a single eye was 0.5 ng eye -1 . The mean tissue-bound SEM concentration in muscle of these chickens was 3.3 µg kg -1 . This suggested that, if multiple eyes could be pooled from chickens of a single flock, a test for parent NFZ could be developed with sensitivity comparable to that of tissue- The current study aimed to assess the suitability of testing for nitrofuran parent compounds in eyes of broiler chickens by addressing the following objectives: 1. To demonstrate the depletion profile of nitrofuran metabolite residues in broiler eyes, muscle and liver tissues following drug withdrawal. 2. To demonstrate the presence of nitrofuran parent compounds in eyes of nitrofuran-medicated broiler chickens.

3. To demonstrate the depletion profile of the nitrofuran parent compounds in broiler eyes following drug withdrawal. 4. To determine if monitoring nitrofuran parent compounds in pooled broiler eyes is comparable to monitoring their tissue-bound metabolites in muscle tissue.

Materials and methods

Materials and instrumentation

Internal standards D 4 -3-amino-2-oxazolidinone, D 5 -3-amino-5-morpholinomethyl-2oxazolidinone, 13 C 3 -1-aminohydantoin and 13 C 15 N 2 -semicarbazide were supplied by Witega Laboratorien Berlin-Adlershof (Berlin, Germany). 3-Amino-2-oxazolidinone (AOZ), semicarbazide (SEM), furazolidone (FZD), furaltadone (FTD), nitrofurantoin (NFT) and nitrofurazone (NFZ) were supplied by Sigma-Aldrich (Poole, UK). 3-Amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), and the nitrofuran parent compound internal standards D 4 -furazolidone, D 5 -furaltadone and 13 C 15 N 2nitrofurazone were supplied by Chemical Synthesis Services (Belfast, UK). 1-Aminohydantoin (AHD) was a gift from Proctor and Gamble Pharmaceuticals USA. Unless stated, all other chemicals and solvents were obtained from Sigma-Aldrich and were analytical and HPLC grade, respectively.

An Agilent 1100 Series HPLC system (Agilent Technologies, USA) coupled to a Quattro Ultima® Platinum tandem mass detector (Micromass/Waters, Manchester, UK), both operating under MassLynx® software, were used for tissue analyses. The mass spectrometer operated in electrospray positive and negative modes and data acquisition was in multiple reaction monitoring mode (MRM). The HPLC system was equipped with a Columbus C18(2) 3 µm, 2.0 x 150 mm column (Phenomenex, UK). A binary gradient mobile phase was used at a flow rate of 0.2 ml/min, solvent A being 0.5 mM ammonium acetate and methanol (80:20 v/v mix), solvent B being 100% methanol. Commercial crumbed feed for broiler chickens was supplied by McLarnons Ltd. (Randalstown, UK). Muscle and liver were minced in a Mini Prep Plus domestic food processor (Waring, Torrington, CT, USA) then were homogenised in a SL2 laboratory homogeniser (Silverson Machines Ltd, Chesham, England). Whole chicken eyeballs were homogenised in self-contained, disposable FOODcon7 homogenisation units using a FASTH21 high throughput, automated tissue homogenisation system (ConsulAR SA, Switzerland).

Production of incurred broiler tissues

Two-week old broiler chickens, purchased from a local supplier, were housed in deep litter pens in 4 groups of 43 birds. Birds were allowed ad libitum access to fresh water and feed at all times. Commercial broiler chick crumb feed was used to prepare four medicated diets containing NFT at 50 mg kg -1 feed, FZD at 10 mg kg -1 feed, FTD at 6 mg kg -1 feed and NFZ at 1 mg kg -1 feed. These concentrations were designed to produce tissue-bound nitrofuran metabolite residues in muscle close to the EU MRPL of 1 µg kg -1 after a drug withdrawal period of between 1 to 3 weeks. Approximate calculations were based on the limited literature data available for dietary nitrofuran levels and muscle residues in chickens [START_REF] Zuidema | Metabolism and depletion of furazolidone and furaltadone in broilers[END_REF][START_REF] Mccracken | The occurrence of nitrofuran metabolites in the tissues of chickens exposed to very low dietary concentrations of the nitrofurans[END_REF]Cooper et al. 2005a) and pigs (Cooper et al. 2005b). The four groups were fed a medicated diet for 7 days after which the medicated diets were withdrawn and replaced with conventional unmedicated broiler grower crumb feed for a further 21 days' withdrawal period. Birds (3 per group) were euthanised by cervical dislocation at intervals during the withdrawal period. Breast muscle and liver were sampled and stored at -20°C. Whole, intact eyeballs were removed and stored at -80°C. During the withdrawal period, muscle samples were analysed immediately for total nitrofuran metabolite concentration as described below. [START_REF] Mccracken | The prevalence and possible causes of bound and extractable residues of the furazolidone metabolite 3-amino-2oxazolidinone in porcine tissues[END_REF] demonstrated that approximately 60% of total AOZ residues in kidney of FZD treated pigs was present in the form of tissue-bound residues. Unpublished data from this laboratory demonstrates that approximately 80% of total SEM residues in muscle of NFZ treated chickens is present in the form of tissue-bound residues. Therefore, when total muscle metabolite concentrations had fallen to approximately 1 to 3 µg kg -1 in the present study, 25 birds from each group were euthanised and muscle, liver and eyes were sampled. These groups of 25 birds provided 50 eyes that could be pooled for analysis of nitrofuran parent compounds. The remaining birds continued on the withdrawal rations and were euthanised (3 per group) and tissues sampled every 3 to 4 days. Tissue-bound nitrofuran metabolites were measured in 1 g samples of chicken muscle and liver essentially as described by Cooper et al. (2005b). Briefly, samples were homogenised and extractable residues were removed by repeated washing in methanol, ethanol and diethyl ether. Tissue-bound metabolites were hydrolysed in mild acid and derivatised with 2-nitrobenzaldehyde before extraction into ethyl acetate. Sample preparation and LC-MS/MS analysis were as described by Cooper et al. (2005b) with the exceptions that a Columbus C18 HPLC column and an additional 13 C 3 -AHD internal standard were used. Highly concentrated samples were diluted as required for analysis.

Tissue analysis by LC-MS/MS

Total nitrofuran metabolites were measured in 1 g samples of chicken muscle as described by Cooper and Kennedy (2006). This method omits the sample solvent prewashing steps and employs minor differences in reagent volumes compared to the tissue-bound metabolite method.

Total nitrofuran metabolites were measured in single whole chicken eyes as described by Cooper et al. (2005a). Eyes were cut into four pieces and placed in FOODcon7 homogenisation units while still frozen in order to retain the vitreous humour. If allowed to defrost, the retina, which contains the vast majority of the ocular nitrofuran residues, partially disintegrates and is dispersed with the humour when the sclera is cut open.

Nitrofuran parent compounds were extracted from single whole chicken eyes essentially as described by Cooper et al. (2005a). Sliced eyes were homogenised in FOODcon7 units with 5 ml water fortified with 10 ng per eye mixed internal standard Dried extract was finally reconstituted in 100 µl of methanol : water (50 : 50 v/v) and centrifuged at 13 000 rpm. LC-MS/MS conditions for the analysis of the four nitrofuran parent compounds were as described by McCracken and Kennedy (2006).

Nitrofuran parent compounds were also analysed in pooled samples comprising 50 pooled eyes, homogenising 5 eyes in 6 ml water in each of 10 FOODcon7 units, fortifying with 1 ng per unit mixed internal standard. After transferring the contents to glass tubes and rinsing the units with 2 ml water, samples were extracted into ethyl acetate (9 + 7 ml) and reconstituted in acetonitrile (3 ml). The samples were washed with hexane (3 + 3 ml) before combining the ten acetonitrile extracts, evaporating to dryness and reconstituting the single combined extract (50 eyes) in 100 µl of methanol : water (50 : 50 v/v) and centrifuging at 13 000 rpm.

Results and discussion

Nitrofuran metabolites in edible tissues

Table I presents the concentrations of total nitrofuran metabolites in broiler breast muscle during the first week of drug withdrawal. Depletion half-lives, calculated on the basis of linear regression of metabolite concentrations over this 7 day period, ranged from 2.0 days (AOZ) to 3.2 days (SEM). This is the first publication of total metabolite half-lives for all four of the nitrofuran drugs in broiler chickens. [START_REF] Zuidema | Metabolism and depletion of furazolidone and furaltadone in broilers[END_REF] metabolite concentrations in the present study, that broilers were euthanised on days 8, 9 and 10 in order to produce muscle samples with tissue-bound nitrofuran metabolite concentrations close to the MRPL of 1 µg kg -1 . Groups of birds were euthanised on different days due to the varying concentrations of dietary nitrofurans employed and the variable metabolite half-lives observed.

[Insert TABLE I about here] Whilst European legislation does not specify whether nitrofuran metabolites should be monitored in the form of tissue-bound or total fractions, analysis of tissue-bound metabolites became the norm in this laboratory, initially because of the cleaner extracts obtained following solvent washing. This approach was reinforced by AFSSA, the Community Reference Laboratory for nitrofuran drugs, which, following the discovery that SEM could arise from degradation of azodicarbonamide, used as a flour treatment agent, issued a statement in 2003 that "when testing composite food, only analyse the part of the product which is of animal origin, for example, only the meat part of breaded products...In the case of a non-compliant sample for total SEM, a sample must be reanalysed for the bound residues of SEM only" [START_REF] Sanders | Community Reference Laboratory web site[END_REF]. Given that chicken muscle is the world's most popular meat, and that nitrofuran residues have been an issue of concern in the global poultry industry, there is a need for data on the depletion half-lives of the tissue-bound nitrofuran metabolites in edible broiler tissues. Figures 1 and2 illustrate the depletion profiles and half-lives of the four major tissue-bound nitrofuran metabolites in muscle and liver respectively. (Note: the nitrofuran drugs were fed at different dietary concentrations, so absolute metabolite concentrations cannot be compared meaningfully across drug groups). Depletion half-lives for all metabolites were longer in muscle (ranging 3.4 to 4.5 days) than in liver (ranging 2.2 to 3.1 days), possibly reflecting the lower metabolic rate and slower cellular turnover of muscle tissue. This tissue difference was also evident in our previous studies of nitrofuran depletion in porcine tissues (Cooper et al. 2005). SEM exhibited the longest half-life in broiler muscle (4.5 days) and was detectable after 3 weeks withdrawal of a low contamination level diet (1 mg kg -1 , normal therapeutic dose being 200-300 mg kg - 1 ). Total nitrofuran metabolites in muscle (Table I) exhibited shorter half-lives than their corresponding tissue-bound metabolites (Figure 1), possibly due to the faster clearance rate of the "free" (solvent-extractable) residues portion compared with the tissue-bound residues.

[Insert FIGURE 1 and FIGURE 2 about here]

Nitrofuran metabolites in broiler eyes

The recent controversy over sources of SEM in foods, other than through abuse of NFZ, led this laboratory to investigate eyes as a potential depot of nitrofuran residues.

We demonstrated that total nitrofuran metabolites were detectable at parts per million concentrations in retina of pigs fed therapeutic nitrofuran doses (Cooper and Kennedy 2005). We then proceeded to show that NFZ-treated laying hens had similarly high concentrations of total SEM in whole eyeballs (Cooper et al. 2005a) and that SEM was even detectable in a single eye when feeding a diet containing NFZ at only 0.03 mg kg -1 . Figure 3 shows that AOZ, AMOZ, AHD and SEM residues can be detected by LC-MS/MS in single eyes of broiler chickens fed sub-therapeutic nitrofuran doses.

The depletion half-lives of total metabolites in eyes are considerably longer (ranging 8.5 to 20.3 days) than the depletion half-lives for tissue-bound metabolites in muscle and liver (Figures 1 and2). As is the case with some other xenobiotic residues such as the β-agonist clenbuterol [START_REF] Malucelli | Tissue distribution and residues of clenbuterol, salbutamol, and terbutaline in tissues of treated broiler chickens[END_REF], ocular tissue, specifically the retina, appears to be a metabolic "dead end" tissue where nitrofuran metabolites accumulate to high concentrations and where they are not subject to the higher cellular turn-over and clearance rates of other tissues such as muscle and liver. The pigment melanin has been identified as the site of binding of various drugs in pigmented ocular tissues [START_REF] Ings | The melanin binding of drugs and its implications[END_REF]).

[Insert FIGURE 3 about here] The concentrations of nitrofuran metabolites are also significantly greater in eyes (measured as ng per eye; each eye weighing approximately 1 g) than in muscle and liver (measured as ng per g tissue). The MRPL for the nitrofurans is 1.0 µg kg -1 in poultry meat and aquaculture products (Commission Decision 2003). Commission Decision 2005/34/EC has recently harmonised the standards applied for monitoring banned substances, including the nitrofurans, in foods. The MRPL is now to be considered a "reference point(s) for action irrespective of the matrix tested." Furthermore, "where the results of analytical tests on products are below the MRPL… the products will not be prohibited from entering the food chain." Where recurrent sub-MRPL findings are observed, "the Commission shall bring the matter to the attention of the competent authority in the country of origin and shall make appropriate proposals" (Commission Decision 2005). Given the higher concentrations and longer half-lives of nitrofuran metabolites in eyes compared to muscle and liver, and the possibility of applying the MRPL as an reference point for action irrespective of the matrix tested, it is now possible, in principle at least, that a chicken may be condemned as unfit for human consumption on the basis of metabolites detected in its eyes long after the metabolite concentration in muscle or liver has fallen below the MRPL, further diminishing the temptation to misuse these drugs.

Nitrofuran parent compounds in broiler eyes

The major attraction of testing chicken eyes is their ability to retain the intact nitrofuran parent compounds in contrast to edible tissues in which the parent compounds are broken down inside a matter of hours [START_REF] Mccracken | Determination of furazolidone in porcine tissue using thermospray liquid chromatography-mass spectrometry and a study of the pharmacokinetics and stability of its residues[END_REF], Cooper et al. 2005b).

We previously demonstrated that NFZ was detectable in eyes of laying hens (Cooper et al. 2005a). Table II now demonstrates that FZD, FTD and NFT are also detectable in single broiler chicken eyes when no withdrawal period is observed. Whilst direct comparisons cannot be made between the absolute concentrations of the four nitrofuran parent compounds in eyes because of the differing dietary concentrations administered, we have shown that accumulation of nitrofuran parent compounds in broiler eyes is not restricted to NFZ. The failure to detect NFZ in single eyes in this study (Table II) may have been due to the fact that SEM has a long depletion half-life in muscle and therefore it was necessary to feed NFZ at a very low dietary level (1 mg kg -1 ) to achieve SEM muscle concentrations close to the MRPL within the confines of a short term study. NFZ was also the least sensitive of the nitrofuran parent compounds under LC-MS/MS ionisation. However, monitoring compliance with the prohibition on the use of NFZ in food-producing animals may still be feasible by measuring NFZ in the retina of larger food-producing animals (e.g. cattle and pigs).

[Insert TABLE II about here]

NFT was still detectable in a single eye after 3 d withdrawal of a 50 mg kg -1 NFT diet.. FTD, by contrast, when fed at only 6 mg kg -1 feed, was still detectable (0.08 ng eye -1 ) in a single eye after 21 days withdrawal (Table II; depletion half-life 3.4 days). The ability to detect FTD over such a period is due partly to FTD being the most sensitive of the nitrofuran parent compounds under the LC-MS/MS method described above. ocular tissues, given that in pigs fed 400 mg kg -1 FTD, the parent compound was detected in isolated retina at mg kg -1 concentrations, much higher than the µg kg -1 concentrations found in FZD treated pigs (Cooper et al. 2005a).

Twenty-five broilers from each group were euthanised after 8, 9 or 10 days withdrawal when it was estimated (based on real-time analysis of total metabolites) that tissuebound metabolite concentrations in muscle would be close to the 1 µg kg -1 MRPL.

Table III shows that two groups were below (AHD 0.8 µg kg -1 , SEM 0.4 µg kg -1 ) and two groups above the MRPL (AOZ 1.4 µg kg -1 , AMOZ 1.6 µg kg -1 ) on these dates.

Processing and pooling 50 whole eyes into a single extract reconstituted in 100 µl of solvent proved to be easier than expected. Eyes were split across 10 groups of 5 eyes, each independently extracted and washed with hexane to remove the considerable orange oily fraction present in intact eyeballs, before combining to a single extract which could be dissolved in 100 µl of 50% methanol. Ultra-centrifugation further clarified the samples such that extracts of 50 eyes were of a similar clarity to extracts of a single eye and suitable for injection onto a HPLC column. However, greater ionisation suppression was evident during tandem MS analysis of extracts of 50 eyes compared to single eye samples (peak areas reduced by a factor of approximately 4).

Comprehensive validation of the LC-MS/MS method is hindered by the enormous number of eyes required for the sample compositing process, however, the authors estimate that in a fortified sample of 50 broiler eyes, nitrofuran parent compounds can be detected down to approximately 4 ng NFZ, 2 ng NFT, 1 ng FZD and 0.2 ng FTD per pooled sample.

[Insert withdrawal of a diet containing 10 mg kg -1 FZD (5% of a therapeutic dose). This equates to 0.02 ng eye -1 , which would be undetectable in a single broiler eye using current LC-MS/MS techniques. In contrast, the stability of FTD was reinforced by the detection of 31.1 ng in 50 pooled eyes 10 days after withdrawal of a diet containing only 6 mg kg -1 FTD. This equates to 0.62 ng eye -1 , which is consistent with the detection of 0.6 ng FTD in single eyes at 7 days withdrawal (Table II). Pooled eyes were unavailable for testing for NFT. However, on the basis of the concentrations of NFT in single eyes (Table II), it can be estmiated that NFT would have been detected in 50 pooled eyes at approximately 10 ng. Possibly the most significant result was the inability to detect NFZ in 50 pooled eyes after 8 days withdrawal of a 1 mg kg -1 NFZ diet. This can again be attributed primarily to the long depletion half-life of SEM in muscle (4.5 days) that results in levels of SEM close to the MRPL being detected in muscle even following withdrawal of a low contamination level diet. This was compounded by the poor sensitivity of NFZ under the applied LC-MS/MS conditions.

It may be possible that, if MS/MS sensitivity can be improved and samples analysed when SEM in muscle is closer to the 1 µg kg -1 MRPL rather than the 0.4 µg kg -1 in the current test samples, NFZ may be detectable in a 50 eyes pooled sample. However, a larger sample size may also be considered to improve test sensitivity. Given the scale of commercial broiler production facilities, obtaining 50 or 100 eyes from a chicken house (in which all birds receive the same feed and are therefore suitable for pooling into a single sample) would present little difficulty. However, it must be recognised that the testing of eyes for nitrofuran parent compounds is unlikely to replace the testing of edible tissues for nitrofuran metabolites as the routine method of nitrofuran residue monitoring. Imported poultry meat is rarely accompanied by the eyes from the source bird. Nevertheless, the testing of nitrofuran parent compounds in pooled eyes from a poultry flock would be a valuable tool within a monitoring programme. It would remove any doubt over the source of nitrofuran metabolites and enable poultry producers to police their own supply chains and competent national authorities to carry out comprehensive investigative work within their domestic primary production facilities. Ultimately it would assist EU Member States to enforce fully the ban on the use of nitrofuran drugs in primary production. Furthermore, the higher concentrations of nitrofuran metabolites detected in eyes may permit laboratories lacking LC-MS/MS technologies to monitor nitrofuran abuse in their primary production using HPLC-UV detection as an alternative to monitoring tissue-bound metabolites in edible tissues, albeit at a level which would not meet EU confirmatory criteria.

This study has provided useful data on the depletion profiles and half-lives of the nitrofuran metabolites in broiler chicken muscle and liver. It has also demonstrated that higher concentrations of metabolites with longer half-lives are detectable through the analysis of whole eyes of broilers. This study, in conjunction with our previous work (Cooper et al., 2005a), has now shown that all four major nitrofuran parent antibiotics, as opposed to their metabolites, are detectable in broiler eyes, with FTD, in particular, being detectable even after 3 weeks withdrawal of a contamination level diet. This was the first attempt to measure nitrofuran parent compounds in pooled samples of eyes from broilers whose muscles contained nitrofuran metabolites close to the EU MRPL.

Measuring FZD in 50 pooled eyes was shown to be an alternative to measuring tissuebound AOZ in single muscle samples. FTD was detectable in single eyes of broilers with MRPL concentrations of AMOZ in their muscle. However, NFZ could not be detected in 50 pooled eyes of broilers with 0.4 µg kg -1 SEM in their muscle. Further FZD (10 mg kg -1 ), FTD (6 mg kg -1 ), NFT (50 mg kg -1 feed) and NFZ (1 mg kg -1 ) respectively for 7d. Mean ± SE (n=3).

Figure 2. Depletion, during 3 weeks withdrawal, of tissue-bound nitrofuran metabolites AOZ, AMOZ, AHD and SEM from liver of broiler chickens fed FZD (10 mg kg -1 ), FTD (6 mg kg -1 ), NFT (50 mg kg -1 feed) and NFZ (1 mg kg -1 ) respectively for 7d.

Mean ± SE (n=3).

Figure 3. Depletion , during 3 weeks withdrawal, of total nitrofuran metabolites AOZ, AMOZ, AHD and SEM from single whole eyes of broiler chickens fed FZD (10 mg kg -1 ), FTD (6 mg kg -1 ), NFT (50 mg kg -1 feed) and NFZ (1 mg kg -1 ) respectively for 7d. Mean ± SE (n=3).

Table I. Depletion, during first week of withdrawal, of total nitrofuran metabolites AOZ, AMOZ, AHD and SEM from breast muscle of broiler chickens fed FZD (10 mg kg -1 ), FTD (6 mg kg -1 ), NFT (50 mg kg -1 feed) and NFZ (1 mg kg -1 ) respectively for 7d. Mean (n=3) ± SE. Table II. Detection of nitrofuran parent compounds in single whole eyes of broiler chickens fed FZD (10 mg kg -1 ), FTD (6 mg kg -1 ), NFT (50 mg kg -1 feed) and NFZ (1 mg kg -1 ) for 7d. ND = not detected. Mean (n=3) ± SE. Depletion half-life of FTD was

  bound SEM in muscle. The current methodology for statutory monitoring of NFZ abuse in animal tissues is detection of tissue-bound SEM in muscle or liver and regulatory laboratories are required by EU legislation to set in place confirmatory methods which can quantify SEM at 1.0 µg kg -1 or less (the Minimum Required Performance Limit, MRPL, set down in Commission Decision 2003/181).

  ethyl acetate, reconstituted in acetonitrile and washed with hexane.

  published total AOZ and AMOZ depletion data in broilers fed therapeutic doses. These graphs suggested AOZ and AMOZ half-lives were in the region of 4 days. It was on the basis of estimates made from the measurements of the total

  to improve NFZ sensitivity under LC-MS/MS and to refine the sample size necessary to enable NFZ detection in pooled eyes to be applied as an alternative to SEM detection in muscle. Such a test would circumvent the problem of alternative sources of SEM in the food chain and would help protect both the consumer from eating meat of birds that have received a banned substance, and of poultry producers from being wrongly accused of administering nitrofurazone on the basis of semicarbazide detection in muscle.

Figure 1 .

 1 Figure 1. Depletion, during 3 weeks withdrawal, of tissue-bound nitrofuran metabolites, AOZ, AMOZ, AHD and SEM from breast muscle of broiler chickens fed,

  Detection of nitrofuran parent compounds in 50 pooled eyes of medicated broiler chickens when tissue-bound nitrofuran metabolites in breast muscle (n=5) were close to the MRPL of 1 µg kg -1 .

  Figure 1

  Figure 2

  Figure 3

TABLE AND FIGURE CAPTIONS:

 AND

Table I Total nitrofuran metabolites (µg kg -1 muscle)

 I

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	Withdrawal	AOZ	AMOZ	AHD	SEM
	(days)
	0	31.9 ± 6.6	19.5 ± 4.3	18.0 ± 1.6	4.8 ± 0.3
	3 7 Half-life (days) F o 7.5 ± 1.0 2.8 ± 0.5 2.0 r	8.9 ± 1.0 3.0 ± 0.2 2.6	4.9 ± 0.5 2.6 ± 0.5 2.5	1.9 ± 0.2 1.0 ± 0.2 3.2
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Table II Nitrofuran parent compound (ng per eye)

 II

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	Withdrawal (days)	FZD	FTD	NFT	NFZ
	0	8.3 ± 7.4	10.4 ± 1.7	1.8 ± 0.7	ND
	3 7 14 F o r ND ND ND	2.0 ± 0.2 0.6 ± 0.2 0.2 ± 0.1	0.4 ± 0.1 ND ND	ND ND ND
	17 21	ND ND P	0.3 ± 0.2 0.1 ± 0.0	ND ND	ND ND
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Table III

 III

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	Nitrofuran	mg kg -1 feed	Withdrawal (days)	Tissue-bound metabolite	µg kg -1 muscle ± SE	Nitrofuran parent	ng per 50 pooled eyes
	NFT	50	9	AHD	0.8 ± 0.1	NFT	Not tested
	FZD	10	10	AOZ	1.4 ± 0.1	FZD	1.2
	FTD	6	10	AMOZ	1.6 ± 0.1	FTD	31.1
	NFZ	1	8	SEM	0.4 ± 0.1	NFZ	Not detected

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Food Additives and Contaminants

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

Acknowledgements

Thanks are expressed to Jim Gillespie and David Johnston (AFBI) for broiler husbandry and the department of Agriculture & Rural Development for funding this work.


Keywords: pesticides, plant protection products, GC-MS, grapevine

Although the list of pesticides used in integrated pest management (IPM) in grape growing and their annual application rates are limited, we are still confronted with the problem of pesticide residues in grapes. This paper presents the results of pesticide monitoring of 47 samples of wine grapes (Vitis vinifera L.) from the 2006 vintage from the vineyards included in IPM. The grape samples were analysed for the presence of 67 pesticides. Among them, 20 were allowed in IPM in 2006. Grapes were sampled at harvest. Two internal analytical methods were used for determination of pesticides: GC-MS method for determination of dithiocarbamates and a multi-residue GC-MS method. One grape sample (2.1%) contained no residues or they were below limit of detection, 28 samples (59.6%) contained residues lower or equal than maximum residue levels (MRLs) and 18 samples (38.3%) exceeded national MRLs for cyprodinil (concentration range was 0.03-0.40 mg kg -1 of cyprodinil) and fludioxonil (concentration was 0.03 mgkg -1 of fludioxonil). Multiple residues were found in 41 samples (87.2%). The highest number of pesticides detected per sample was seven. No violation of pesticides allowed in IPM was observed. Folpet (97.9%), cyprodinil (51.1%), dithiocarbamates (44.7%), chlorothalonil (23.4%), chlorpyriphos (19.1%) and pyrimethanil (14.9%) were the most frequently found pesticides in grapes. The risk assessment showed that the exceeded concentrations of cyprodinil and fludioxonil did not represent any risk for consumer's health (National Estimate of Short Term Intake in % of Acceptable Daily Intake was below 100%).

Introduction

Numerous analytical methods for determining pesticide residues in grapes have been published until now. GC and HPLC are the two most powerful techniques. GC was cited in the literature on many occasions. [START_REF] Oliva | Determination of chlorpyrifos, penconazole, fenarimol, vinclozolin and metalaxyl in grapes, must and wine by on-line microextraction and gas chromatography[END_REF] developed an on-line micro-extraction for determination of chlorpyrifos, penconazole, fenarimol, vinclozolin and metalaxyl in grapes.

For determination, electron capture detection and mass selective detection were used (Oliva et al. 1999). The method was extended to some other organophosphorus insecticides [START_REF] Oliva | Multiresidue method for the rapid determination of organophosphorus insecticides in grapes, must and wine[END_REF]. [START_REF] Oliva | Multiresidue method for the rapid determination of organophosphorus insecticides in grapes, must and wine[END_REF] used on-line micro-extraction technique, followed by nitrogenphosphorus or electron-capture detection and conformation by mass spectrometric detection to determinate fungicide residues in grapes. An automated matrix solid-phase dispersion method (MSPD) with gas chromatography-mass spectrometry detection for eleven pesticides in grapes was developed by Maria [START_REF] Kristenson | Miniaturized automated matrix solid-phase dispersion extraction of pesticides in fruit followed by gas chromatographic-mass spectrometric analysis[END_REF]. [START_REF] Sandra | Multi-residue screening of pesticides in vegetables, fruits and baby food by stir bar sorptive extraction-thermal desorption-capillary gas chromatography-mass spectrometry[END_REF] described stir bar sorptive extraction technique followed by gas chromatography-mass spectrometry for determination of pesticides in grapes. Recently, methods using classical liquid-liquid extraction were published. Fernández [START_REF] Gonzáles | Determination of fungicide residues in white grapes for winemaking by gas chromatography with mass spectrometric detection and assessment of matrix effects[END_REF] described organic solvent extraction with dichloromethane-acetone followed by gas chromatography with mass spectrometric detection used for determination of fungicide residues in grapes. [START_REF] Angioni | Gas chromatographic ion trap mass spectrometry determination of zoxamide residues in[END_REF] described extraction with hexane and analysis of zoxamide with mass spectrometry. Sharif et al. (2006) described extraction of grape samples with ethyl acetate, clean-up with three different sorbents and final determination by gas chromatography and electron-capture detection.

HPLC covers polar and thermally labile compounds which cannot be determined by GC. [START_REF] Miliadis | High-performance liquid chromatographic determination of benzoylurea insecticides residues in grapes and wine using liquid and solid-phase extraction[END_REF] clean-up by solid-phase extraction (SPE) on silica sorbent and determination with a UV diode array detector for benzoylurea insecticides in grapes. [START_REF] Taylor | Multi-residue method for rapid screening and confirmation of pesticides in crude extracts of fruits and vegetables using isocratic liquid chromatography with electrospray tandem mass spectrometry[END_REF], [START_REF] Mol | Determination of polar organophosphorus pesticides in vegetables and fruits using liquid chromatography with tandem mass spectrometry: selection of extraction solvent[END_REF] and Jansson et al. (2004) We used a multi-residue method for the determination of 66 pesticide residues, which included extraction with mixture of acetone, petroleum ether and dichloromethane, clean-up on gel permeation chromatography and GC/MS detection. The other method was used for determination of dithiocarbamates. In this method samples are heated in a two-phase system isooctane/stannous (II) chloride in diluted hydrochloric acid. The produced carbon disulfide is dissolved in the organic phase (isooctane) and determined by GC/MS. In Slovenia one half (8554 ha) of registered vineyards were included in IPM. The main restrictions in that production system rely on grapevine fertilization, plant protection and vineyard soil management. In IPM the use of some environmentally dangerous pesticides are not allowed and only those from the lists found in Technical Guidelines for IPM in Grape Growing, published by the Government, can be used. The main goal of IPM is a proper use of pesticides regarding the concentrations at application, the maximum number of applications, the consideration to pre-harvest interval and the certification of sprayers. Consideration of prognostic forecasts and economic losses in the case of plant infection is a very important step in the decision making process for the application of pesticides. In IPM the quantity of copper applied must not exceed 5 kgha -1 year -1 and the use of some derivates of iso-cyclic compounds (tolylfluanid, dinocap), dithiocarbamates (mancozeb, metiram, propineb), inhibitors of the ergosterol biosynthesis (IBE fungicides: myclobutanil, penconazole, tebuconazole), fungicides against grey mould (boscalid, fenhexamide, iprodione, fludioxonil, cyprodinil, pyrimethanil), insecticides, folpet and chlorothalonil is limited (Technical Guidelines for IPM in Grape Growing in 2006Growing in , 2006)). The main benefits of IPM grape growing are lower incidence and lower levels of residues in grapes and consequently also in wines.

The results of monitoring are used for: determination of harmonisation with the legally prescribed MRLs, determination of harmonisation of IPM with good agricultural practice, determination of origin or cause of residues determined and risk assessment for samples which exceeded MRLs.

The incidence and concentrations of pesticide residues in harvested grapes depends on the pests and diseases of grapevine typical for each vine growing region, type of grape production (conventional, IPM, organic), pesticide concentrations at application, time period and climatic conditions from the last spraying until harvest. There are only a few papers dealing with a wide range of pesticide residues in wine grapes. On the other hand, more data exist for dissipation rates of some pesticides in wine grapes [START_REF] Navarro | Dissipation of chlorpyrifos, fenarimol, mancozeb, metalaxyl, penconazole, and vinclozolin in grapes[END_REF][START_REF] Cabras | Pesticide residues in grapes, wine, and their processing products[END_REF] and from grapes to wine (Cabras et al. 1999;[START_REF] Navarro | Evolution of residual levels of six pesticides during elaboration of red wines, Effect of wine-making procedures in their disappearance[END_REF]. It is also interesting that there is a lack of results concerning pesticide residues in grapes and wines for different types of grape growing systems, either for IPM or for organic viticulture. These results are obviously needed to support consumer confidence in the growing market of wines from sustainable viticulture. A fifteen years old review of [START_REF] Farris | Pesticide residues in food processing[END_REF] describes the results of pesticide residues in grapes. Most of the pesticides listed in the paper are still registered but some of them were forbidden for use in IPM in Slovenia (i.e. triadimefon, dimethoate, carbaryl, deltamethrin, thiabendazole). The results of the studies mentioned above are more closely examined and compared with our results in the Results and Discussion section of this paper. Many authors also showed that a proper use of pesticides does not cause an exceedance of MRLs in grapes (Cabras et al. 1999;[START_REF] Farris | Pesticide residues in food processing[END_REF][START_REF] Navarro | Evolution of residual levels of six pesticides during elaboration of red wines, Effect of wine-making procedures in their disappearance[END_REF]). However, [START_REF] Soleas | Pesticide residues in unfermented grape juices and raw wines: a 5-year survey of more than 3000 products[END_REF] assayed 26 pesticides in 1827 raw grape juices prior to fermentation, in which grapes were the source of the contamination. Pesticides found most frequently in their samples were folpet, captan, guthion, carbaryl and dimethoate. Only the first one is allowed for use in IPM of grape growing in Slovenia. The goal of the present paper was therefore to investigate the incidence and concentrations of pesticide residues in grapes from vineyards included in IPM and to examine the important part of safety assurance in grape growing, which is the first stage of the winemaking process.

Materials and methods

Sampling

The sampling of wine grapes was random and it was carried out in all three vine growing regions in Slovenia (Podravje, Posavje and Primorska). Grape samples were taken directly in the vineyard after the pre-harvest interval of the pesticides elapsed. Agricultural inspectors performed the sampling in autumn 2006.

Analytical methods

Multi-residue method

The following 66 pesticides were analysed using multiresidual method: acephate, aldrin, azinphos-methyl, azoxystrobin, bifenthrin, bromopropylate, bupirimate, captan, carbaryl, carbofuran, chlorothalonil, chlorpropham, chlorpyriphos, chlorpyriphos-methyl, cyhalotrinlambda, cypermethrin, cyprodinil, DDT, deltamethrin, diazinon, dichlofluanid, dimethoate, diphenylamine, endosulfan, endrin, fenitrothion, fenthion, fludioxonil, folpet, HCH-α, heptachlor, heptenophos, imazalil, iprodione, kresoxim-methyl, lindane, malathion, mecarbam, metalaxyl, methamidophos, methidathion, myclobutanil, omethoate, oxydemetonmethyl, parathion, penconazole, permethrin, phorate, phosalone, pirimicarb, pirimiphosmethyl, procymidone, propargite, propyzamide, pyridaphenthion, pyrimethanil, quinalphos, spiroxamine, thiabendazole, tolclofos-methyl, tolylfluanid, triadimefon, triadimenol, triazophos, trifloxystrobin and vinclozolin.

The extraction procedure and determination by GC/MS of the multi-residue method is described elsewhere (Baša [START_REF] Česnik | Multiresidual analytical method for determination of pesticide residues in fruit and vegetables[END_REF][START_REF] Fillion | Multiresidue Method for the Determination of Residues of 251 Pesticides in Fruit and Vegetables by Gas Chromatography/Mass Spectrometry and Liquid Chromatography with Fluorescence Detection[END_REF] [START_REF] Makovi | Extraction with acetone, liquid-liquid partitioning with petroleum ether/methylene chloride[END_REF][START_REF] Thier | Organochlorine, Organophosphorus, Nitrogen-Containing and Other Pesticides[END_REF][START_REF] Thier | Manual of Pesticide Residue Analysis[END_REF]). The multi-residue method was validated on the strawberry matrix. This method was accredited by the French accreditation body COFRAC and covers the validation and accreditation for the whole group

Method used for determination of dithiocarbamates

The extraction procedure and determination by GC/MS and validation of this method is described elsewhere (Baša [START_REF] Česnik | Validation of the method for the determination of dithiocarbamates and thiuram disulphide on apple, lettuce, potato, strawberry and tomato matrix[END_REF].

Quality assurance

Each series of analyses included two spiked samples of commodity analysed. For batch acceptance recoveries had to be 70 -110 %. The recoveries were allowed to be higher for those pesticides which were not found in the samples. The accuracy of methods has been verified since 2002 by participating in the French interlaboratory proficiency scheme BIPEA (Bureau Interprofessionnel d′Etudes Analytiques). Z-scores for eight grape juice samples for 44 measurements from 2003 to 2004 were mainly below ±2. Unsatisfactory results were obtained on five occasions; the highest absolute z-score was 2.54. Taking into account the measurement uncertainty the "false" results fell within the tolerance limits. We have also participated in the CRL European Proficiency Test since 2004. In 2005 our work was verified and accredited by French accreditation body (COFRAC) for a multi-residue method on leafy vegetables, root and tuber vegetables and flour. In 2006 the scope of accreditation was expanded to pome fruit and berries and small fruit. In 2006 we also accredited a method for the determination of dithiocarbamates for the groups: pome fruit, berries and small fruit, leafy vegetables, root and tuber vegetables, fruiting vegetables and cereals.

Results and discussion

Validation of the multi-residue method for 66 pesticides on berries and small fruit matrix showed that limits of detection ranged from 0.0004-0.02 mg kg -1 , limits of quantification range from 0.001-0.08 mg kg -1 , repeatability standard deviations ranged from 5-10% and recoveries at spiking levels from 0.013-0.2 mg kg -1 ranged from 93.4-102.5%. Validation of method for determination of dithiocarbamates on berries and small fruit matrix showed that the limit of detection was 0.02 mg kg -1 , limit of quantification was 0.05 mg kg -1 , repeatability standard deviation was 0.5%, and the recovery at spiking level 2 mg kg -1 was 99.6%.

Out of the total of 47 grape samples analysed, 18 samples (38.3%) exceeded the maximum residue limits (cyprodinil and fludioxonil), 28 samples (59.6%) contained residues lower than maximum residue limits, and in one grape sample (2.1%) residues were not found. Forty-one (87.2%) out of the total of 47 grape samples analysed contained residues of two or more pesticides. Residues of two pesticides were determined in 14 samples (29.8%) of which MRL was exceeded by 4 samples (8.5%) with the values from 0.12 -0.29 mg kg -1 (cyprodinil), residues of three pesticides were determined in 13 samples (27.7%) of which MRL was exceeded by 4 samples (8.5%) with the values from 0.25 -0.40 mg kg -1 (cyprodinil), residues of 4 pesticides were determined in 7 samples (14.9%), of which MRL was exceeded by 7 samples (14.9%) with the values from 0.07 -0.34 mg kg -1 (cyprodinil), residues of 5 pesticides were determined in 5 samples (10.6%) of which MRL was exceeded by one sample (2.1%) at a level of 0.24 mg kg -1 (cyprodinil), residues of 6 pesticides were determined in one sample (2.1%) which exceeded MRL at a level of 0.14 mg kg -1 (cyprodinil), residues of 7 pesticides were determined in one sample (2.1%) which exceeded MRL at levels of 0.10 mg kg -1 (cyprodinil) and 0.03 mg kg -1 (fludioxonil). [Insert table 1 about here] Pesticides sought during the analyses and allowed in IPM were : azoxystrobin, chlorothalonil, chlorphyriphos, chlorpyriphos-methyl, cyprodinil, dithiocarbamates, endosulfan, fludioxonil, folpet, iprodione, kresoxim-methyl, metalaxyl, myclobutanil, penconazole, phosalone, pyrimethanil, spiroxamine, tolylfluanid, triadimenol and trifloxystrobin. In the grape samples we found 14 of the pesticides listed. No violation of pesticides allowed in IPM was observed; we could conclude that viticulturists used only those allowed in the Technical Guidelines for IPM in 2006.

It was expected that among pesticides found the most frequently in grapes would be folpet (97.9% of analysed samples), cyprodinil (51.1%), dithiocarbamates (44.7%), chlorothalonil (23.4%), chlorpyriphos (19.1%) and pyrimethanil (14.9%). Namely, folpet, dithiocarbamates and chlorothalonil belong to the group of contact fungicides against downy mildew (Plasmopara viticola (Berk et Curtis ex. De bary)) Berl. et de Toni) and are frequently used for two or three last applications in a growing season. Cyprodinil and pyrimethanil are anilinopyrimidines used against grey mould (Botryotinia fuckeliana de Bary (Whetzel)).

During growing season, grapes are usually sprayed two or three times with those fungicides and the last application coincides with the beginning of grape ripening (growth stage 35-36 according to Eichhorn-Lorenz scale). Chlorpyriphos is an organophosphorus insecticide used against grapevine moths (Eupoecilia ambiguella Hübn. and Lobesia botrana Den. & Schiff.) and is also applied at the beginning of grape ripening (Technical Guidelines for IPM in Grape Growing in 2006Growing in , 2006)). All pesticides mentioned above, except cyprodinil, were determined within EC or national MRLs (pyrimethanil). Viticulturists accurately considered the guidelines for the use of pesticides, especially their application rates and pre-harvest intervals.

Concentrations of the most frequently found pesticides in wine grapes (Table 1) are comparable to those already reported by [START_REF] Farris | Pesticide residues in food processing[END_REF] (folpet (0.50 mg kg -1 ), dithiocarbamates (0.90-1.60 mg kg -1 ), chlorothalonil (0.23-0.60 mg kg -1 )), [START_REF] Navarro | Dissipation of chlorpyrifos, fenarimol, mancozeb, metalaxyl, penconazole, and vinclozolin in grapes[END_REF] (chlorpyriphos (0.14 mg kg -1 )), [START_REF] Cabras | Fate of some new fungicides (cyprodinil, fludioxonil, pyrimethanil, and tebuconazole) from vine to wine[END_REF] (cyprodinil (1.03 mg kg -1 ), pyrimethanil (1.11 mg kg -1 )) and Rial [START_REF] Otero | Multiresidue method for fourteen fungicides in white grapes by liquid-liquid and solid-phase extraction followed by liquid chromatography-diode array detection[END_REF] ((folpet (0.10-0.60 mg kg -1 ), cyprodinil (0.14-1.45 mg kg -1 ), pyrimethanil (2.33 mg kg -1 )), where all the values were below EC or national MRLs. A comparison of our results with the results of [START_REF] Soleas | Pesticide residues in unfermented grape juices and raw wines: a 5-year survey of more than 3000 products[END_REF] that assayed 26 pesticides in 1827 raw juices prior to fermentation from 9 major wineproducing countries shows that there is some important difference in the list of detected pesticides. Among the same pesticides assayed in the grape samples we did not detect some of the very problematic insecticides i.e. endosulfan (allowed in IPM), dimethoate, malathion, parathion and carbaryl.

The fungicides that exceeded the national MRL in Slovenia are cyprodinil in 18 samples (38.3%) and fludioxonil in one sample (2.1%). The national MRL for both compounds is 0.02 mg kg -1 . The highest content of cyprodinil was 0.40 mg kg -1 and of fludioxonil 0.03 mg kg -1 .

The European Community MRLs for both substances have not been determined yet. The Codex Alimentarius MRLs are 3 mg kg -1 for cyprodinil and 2 mg kg -1 for fludioxonil. The risk assessment performed with Pesticide Safety Directorate (PSD, York, UK), model for acute exposure for cyprodinil at concentration level 0.40 mg kg -1 and Acceptable Daily Intake (ADI) 0.03 mgkg -1 body weight -1 day -1 (Acute Reference Dose for cyprodinil was not determined) showed that National Estimate of Short Term Intake (NESTI) expressed in ADI percentage ranged from 2.5% for 7-10 years old children to 31.6% for adults. The risk assessment performed with PSD model for acute exposure for fludixonil at concentration level 0.03 mg kg -1 and ADI 0.40 mg kg -1 body weight -1 day -1 (Acute Reference Dose for fludixonil was not determined) showed that NESTI expressed in ADI percentage ranged from 0.0% for 1-10 years old children and residential elderly people to 0.2% for adults and vegetarians. ADIs were found on the internet (http://europa.eu.int/comm/food/plant/protection/evaluation/index_en.htm), Status of active substances under EU review (doc. 3010)) as well as the PSD model for acute exposure (http://www.pesticides.gov.uk/approvals.asp?id=1687). The risk assessment showed that the exceeded grape samples did not present any risk for health (NESTI in % of ADI was below 100%) and are therefore safe for the consumers. Perhaps the national MRLs should be reconsidered. Additionally, all the grape samples were wine grapes which were further processed into wine. The published results (Cabras et al., 1999;[START_REF] Navarro | Evolution of residual levels of six pesticides during elaboration of red wines, Effect of wine-making procedures in their disappearance[END_REF] and also our unpublished results showed that pesticide concentrations during the vinification process are importantly reduced. For example, in spite of the fact that the cyprodinil concentration in grapes of another experiment was above (0.25 mg kg -1 ) the national MRL, it was not found in the processed wine and therefore its starting concentration presented no risk for the wine consumers.

Conclusions

Although the list of pesticides used in IPM in grape growing and their annual application rates are limited, we are still confronted with the problem of pesticide residues in grapes. This paper presents the results of pesticide monitoring of 47 samples of wine grapes (Vitis vinifera L.) from vineyards included in IPM for the presence of 67 pesticides. In IPM the use of some environmentally and consumer dangerous pesticides is not allowed and only those from the lists in Technical Guidelines for IPM in Grape Growing can be used. The main goal of IPM is therefore a proper use of pesticides regarding the prescribed concentrations, the maximum number of treatments and the consideration of the pre-harvest interval. Our results for the 2006 vintage showed that aims mentioned above were fully achieved as far as the list of permitted pesticides is concerned. The same applies to the residue concentrations where neither EC nor national MRLs were exceeded except for cyprodinil and fludioxonil. The residues in samples were exceeded due to the very low national MRLs. The risk assessment showed that grape samples that exceeded MRLs do not present any risk for consumer's health (NESTI in ADI percentage was below 100%). It has to be stressed that wine grapes are subjected to the vinification process in which pesticide residues are further reduced or eliminated. The cases of fungicides exceeding the national MRLs should be probably further examined and discussed.

We could conclude that the use of permitted pesticides, the consideration of pesticide concentrations and the pre-harvest interval importantly diminish the health risk for consumers and provide safety assurance in IPM wine grape growing. Therefore, such a type of wine grape production does not represent any cause of concern in Slovenia.

  described methods for determination of pesticide residues in grapes after extraction with ethyl acetate by liquid chromatography tandem mass spectrometry. Otero et al. (2003) described method for determination of 14 fungicides in grapes with liquid-liquid extraction with dichloromethane-acetone mixture, clean-up with SPE and determination by liquid chromatography and diode array detection. HPLC with diode array detection was also used by Tiexeria (2004), after extraction with methanol and SPE clean-up. Juan-García et al. (2004) developed a LC/MS method based on SPE and stir-bar sorptive extraction (SBSE). Finally, Pang et al. (2006) described extraction with acetonitrile, 3-cartridge SPE and determination separately by GC/MS and LC/MS in grapes.

  concentration levels determined in grape samples and MRLs are presented in table 1.

  described a method for extraction with ethyl acetate-sodium sulphate,

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

  Pesticide residues found in wine grape samples from IPM were determined at harvest, in accordance with the Law on Plant Protection Products (Official Gazette of the Republic of

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	Slovenia no. 98/04) and Regulation on Residues of Plant Protection Products Found in and on
	Agricultural Commodities and Products (Official Gazette of the Republic of Slovenia, no.
	84/04).

Table 1 :

 1 Pesticides found in grape samples

		No. of samples Proportion	Range	MRL
	Pesticide		(%)	(mgkg -1 )	(mgkg -1 )
	Azoxystrobin	4	8.5	0.02-0.04	2
	Chlorothalonil	11	23.4	0.01-0.73	3
	Chlorpyriphos	9	19.1	0.02-0.13	0.5
	Cyprodinil	24	51.1	0.01-0.40	0.02*
	Dithiocarbamates	21	44.7	0.06-1.63	2
	Fludioxonil	2	4.3	0.02-0.03	0.02*
	Folpet	46	97.9	0.02-6.0	10
	Iprodione	2	4.3	0.01-0.30	10
	Kresoxim-methyl	1	2.1	0.01	1
	Metalaxyl	5	10.6	0.05-0.18	1
	Myclobutanil	1	2.1	0.02	1
	Phosalone	2	4.3	0.01-0.02	1
	Pyrimethanil	7	14.9	0.01-0.53	3*
	Trifloxystrobin	3	6.4	0.02-0.09	5

*national MRLs of the Republic of Slovenia, all others are EC MRLs Page 17 of 17 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants

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

Acknowledgements

The authors thank those who contributed to the work: Mateja Fortuna and the co-workers at the Central Laboratories of Agricultural Institute of Slovenia.


G F Hernández-Póveda

A Morales-Rubio

A. Pastor-García M De La Guardia

Extraction of polycyclic aromatic hydrocarbons from cookies: A comparative study of ultrasound and microwave assisted procedures

Keywords: Chromatography -HPLC, Extraction Additives/Contaminants: PAH PAHs, MAE, cookies, HPLC, fluorimetric detection

The chromatographic determination of 15 Polycyclic Aromatic Hydrocarbons (PAHs) in cookies has been improved in order to obtain a fast method with a low limit of detection through the combination of Microwave Assisted Extraction (MAE), oil saponification and solid phase extraction clean-up prior to the injection of purified extracts in a C18 201TP52 (5 µm, 250 mm x 2.1 mm) column. Using acetonitrile:water as mobile phase, with a 50 % to 95% w/w acetonitrile gradient for a fixed flow of 0.250 ml min -1 , 15 PAHs were separated in 45 min. The column temperature was maintained at 15ºC and fluorimetric detection was made at a fixed excitation wavelength of 264 nm and emission measurements at the best wavelength for each analyte, from 352 nm for 11H-benzo[b]fluorene to 500 nm for Indeno[1,2,3cd]pyrene. Recoveries for all 15 PAHs varied between 96 ± 4 and 105 ± 4 %, and the limits of detection ranged from 0.015 ng g -1 for chrysene to 0.7 ng g -1 for phenantrene. Results were compared with those obtained by conventional Soxhlet extraction during 8 hours refluxing with toluene, demonstrating that the methodology proposed is appropriate to quantify PAHs in cookies. Furthermore, the microwave assisted method was faster and used less solvent than the conventional and ultrasound assisted methods. The extraction time has been reduced to 9 min compared to the 8 hours required for Soxhlet extraction and 60 min required for ultrasound assisted treatment, and the solvent consumption has been reduced to 25 ml compared to the 155 ml and 90 ml required using Soxhlet and ultrasound respectively.

Introduction

Polycyclic Aromatic Hydrocarbons (PAHs) are formed by the incomplete combustion of organic matter, at temperatures between 400 and 2000ºC, anthropogenic processes being the main sources of these compounds in the environment [START_REF] Swetman | Contamination of coconut oil by PAH[END_REF][START_REF] Mcintosh | Polycyclic aromatic hydrocarbons (PAH) concentration and composition determined in farmed blue mussels (Mytilus edulis) in a sea loch pre-and post-closure of an aluminium smelter[END_REF]). The carcinogenic characteristic of PAHs (Commission Regulation 2005), together with their lipophilic nature that favours accumulation in fats, justifies the careful analytical control of these compounds in human food [START_REF] Larsen | Toxicological Aspects of Food[END_REF]Poulsen 1987, European Union 2002).

PAHs are present in foods but especially in oils and fats, the possible contamination source being deposition from air, drying processes for vegetable materials or solvents employed during oil extraction (Welling andKaandorp 1986, Moret and[START_REF] Moret | Polycyclic aromatic hydrocarbons in edible fats and oils: occurrence and analytical methods[END_REF], and therefore it is necessary to get knowledge of different PAHs in food.

European legislation has established maximum limits for benzo [a]pyrene in several food categories (Commission Regulation 2005). A recent overview of European Union legislation on PAHs and analytical methods for official control of food and environmental samples concluded that it is necessary to have harmonization between legislation of different countries and development of well established and validated methods and standardization of procedures to establish databases for the PAH monitoring in food (Wenzl et al. 2006). The Spanish regulation limits to 2 ng g -1 the concentration of benzo[a]pyrene, benzo[e]pyrene, benz[a]anthracene, benzo [k]fluorantene, dibenz [a,h]anthracene, benzo[ghi]perylene, indeno [1,2,3cd]pyrene and benzo [b]fluorantene in the olive pomace oil, being limited the total concentration of these eight compounds to 5 ng g -1 (Spanish Official Bulletin 2001). The German Society for Fat Science has fixed a limit of 5 ng g -1 for the sum of heavy PAHs and 25 ng g -1 for the sum of heavy and light PAHs in oil (Speer et al 1990, Gertz and[START_REF] Gertz | Untersuchung und beurteilung von PAK in speisefetten und-ölen[END_REF].

Since many manufactured products contain oils or fats, it is necessary to improve the analytical methodologies for the fast, accurate and sensitive determination of PAHs in this type of products. GC and HPLC have been widely used for PAHs determination in edible fats and oils [START_REF] Lawrence | Polycyclic aromatic hydrocarbons in foods by HPLC with fluorescence detection[END_REF], Larsson et al 1987[START_REF] Speer | Determination and distribution of polycyclic aromatic hydrocarbons in native vegetable oils, smoked fish products, mussels and oysters, and bream from the river Elbe[END_REF][START_REF] Guillen | Polycyclic aromatic compounds: extraction and determination in food[END_REF][START_REF] Cejpek | Simplified extraction and cleanup procedure for the determination of PAHs in fatty and protein-rich matrices[END_REF][START_REF] Moret | Polycyclic aromatic hydrocarbons in edible fats and oils: occurrence and analytical methods[END_REF]. PAH extraction has been usually carried out by liquid-liquid partition with dimethylformamide-watercyclohexane [START_REF] Lawrence | Polycyclic aromatic hydrocarbons in foods by HPLC with fluorescence detection[END_REF][START_REF] Speer | Determination and distribution of polycyclic aromatic hydrocarbons in native vegetable oils, smoked fish products, mussels and oysters, and bream from the river Elbe[END_REF] or with dimethylsulfoxidecyclohexane [START_REF] Guillen | Polycyclic aromatic compounds: extraction and determination in food[END_REF] for GC analysis, or with acetonitrile for the quantitative extraction from pomace oil for HPLC analysis (Martinez-Lopez et al. 2005).

The main advantages offered by HPLC compared with GC are that: 1) it can provide additional selectivity and give separation of PAHs isomers, which are difficult to separate using GC; 2) provides an useful fractionation technique for the isolation of PAH for subsequent analysis by other techniques (de Boer and Law 2003); and 3) heat-sensitive compounds are not degraded during analysis (Cejpek et al 1995, de Boer and[START_REF] De Boer | Developments in the use of the chromatographic techniques in marine laboratories for the determination of halogenated contaminants and polycyclic aromatic hydrocarbons[END_REF].

Traditional sample preparation procedures based on liquid-liquid or solid-liquid extraction (Moret andConte 1998, Toledo and[START_REF] Toledo | Benzo(a)pireno em óleos de milho produzidos e comercializados no Brasil[END_REF] or saponification (Gertz andKogelheide 1994, Arens and[START_REF] Arens | bestimmung von polyzyklischen aromatischen kohlenwasserstoffen in ölen und fetten[END_REF] without decomposition of PAHs and uses small solvent volumes, thus reducing sample contamination. Additionally, the use of closed reactors avoids losses of volatile compound [START_REF] Pastor | Efficiency of the microwave-assisted extraction of hydrocarbons and pesticides from sediments[END_REF].

In the present study microwave-assisted extraction (MAE), using different solvents has been applied to the determination in cookies of 15 PAHs of which 12 were highlighted by the US Environmental Protection Agency (EPA 1999), and 8 were identified by the Scientific Committee of Food as carcinogenic compounds (European Commission 2002). Results found were in good agreement with those obtained by conventional Soxhlet extraction with toluene and ultrasound-assisted extraction. So, one of the objectives of this study was to provide an appropriate tool for the quantification of PAHs in manufactured products suitable for consumers and inspection laboratories for which the separate ingredients of the cookies are not accessible.

Materials and Methods

Apparatus and material.

An Agilent 1100 Series HPLC system (Waldbronn, Germany) equipped with a manual injection valve 7725i Rheodyne (Rohnert Park, CA, USA) with 20 µl sample loop, a pump G1311A QUAT PUMP, a column oven G1316A COL COM, a solvent degassing system and a molecular fluorescence G1321A FLD detector, all those from Agilent, was used in this study. For PAHs separation and quantification a C18 I.

[Insert Table I about here] For sample preparation a IKA M20 Universe Mill (Staufen, Germany), a Büchi R 200 rotary evaporator with a water heating bath Büchi B 490 (Flawie, Switzerland), a Volter-velp shaker from Scientific (Milano, Italy), a Hermle Z 200 A Centrifuge (Wehingen, Germany), an ultrasound water bath from Selecta (Barcelona, Spain) and a microwave oven LG model MS-197H (Manchester, UK) were employed.

All the glassware material was cleaned with water and soap and stored for 8 hours with saturated K 2 Cr 2 O 7 in H 2 SO 4 solution, rinsed with distilled water and dried at 350ºC for 8 hours. Plastic syringes were cleaned with water and soap, rinsed with distilled water and stored for 5 hours with acetonitrile.

Reagents and standards.

50 µg g -1 stock solutions of PAHs were prepared, in toluene, from the corresponding solid compounds obtained from Fluka Chemika (Buchs, Switzerland) for Phenanthrene >97.0 % HPLC, Anthracene >99.0 % GC, Pyrene >99.0 % GC and and Indeno [1,2,3-cd]pyrene 98 %, making a special caution on the handle of solid products by using mask and gloves in order to avoid their hard toxic action on the operators.

Diluted multi-component standards, containing 1 µg g -1 of each one of the compounds considered, were prepared in acetonitrile from the stock solutions.

Finally, standard calibration solutions, from 1 to 20 ng g -1 , were prepared in acetonitrile from the multi-component solution. HPLC grade acetonitrile (d = 0.78 g ml -1 ) of 99.85 % purity, Toluene for pesticide residue analysis (d = 0.87 g ml -1 ) of 99.8 % purity and n-hexane for pesticide residue analysis (d = 0.66 g ml -1 ) were obtained from Scharlau (Barcelona, Spain). Ultrapure water was obtained with a resistivity of 18.2 MΩ cm with a Millipore MilliQ plus system (Paris, France).

Sample preparation.

To carry out the study of different parameters of the extraction from cookies for PAHs determination it was employed a sample pool (400 g), obtained from eight different trade mark cookies (50 g each) with fat content between 9.2 % and 21.0 %. Samples were stored shelter from humidity, light and heat sources.

Soxhlet extraction.

Five grams, accurately weighted in analytical balance (± 0.1 mg), of the pool sample were weighted and transferred inside the Soxhlet system. 155 ml of toluene were added and extraction process was carried out during eight hours by 48 cycles of 10 minutes. In the last cycle solvent volume was reduced to 25 ml and passed to a conical bottom test tube to evaporate it on the rotary evaporator. Results from this

Ultrasound assisted extraction.

Five grams, accurately weighted in analytical balance (± 0.1 mg), of the pool sample were weighted on an erlenmeyer and between 10 to 35 ml of solvent were added.

The mixture was sonicated in the ultrasound water bath for 20 min and the solvent was passed to a conical bottom test tube to evaporate it on the rotary evaporator. This extraction-evaporation process was repeated three times for each sample using different portions of pure solvent, and extracts for each sample pooled.

Microwave assisted extraction.

Five grams, accurately weighted in analytical balance (± 0.1 mg), of the pool sample were weighted and transferred into a 115 ml PTFE reactor. 10 ml of toluene and 1 ml distilled water or 10 ml of an acetone:hexane (1:1) mixture were added to the sample and the reactor, hermetically closed, was irradiated at 700 w power during 3 to 15 minutes. Between the heating cycles, the reactor was maintained inside the microwave oven for 1 min without irradiation. After the extraction process, the reactor was cooled in an ice bath during 5 min, then, it was opened and 8 g of anhydrous Na 2 SO 4 were added to retain water. The solvent was passed to a conical bottom test tube to evaporate it on the rotary evaporator. Three aliquots of 5 ml solvent were added to the reactor after extraction, mixed with sample and transferred to a centrifuge test tube to collect all oil that could be on the reactor wall and sample surface and the solvent was evaporated on the rotary evaporator. For each extraction method analytical blanks were prepared for pure water and solvents following the same treatment than that employed for samples.

Blank preparation.

Extracts clean-up through liquid extraction and solid phase purification.

Cookies extracts obtained by the different ways indicated above, that contain approximately 1 g of oil, were mixed with 10 ml acetonitrile to extract the PAHs from the fatty matrix. The mixture was shaken rigorously for 3 min in a volter shaker and allowed to stand for 5 min in an ice bath. After that, it was centrifuged at 3500 rpm for 10 min. The upper layer was separated and further evaporated. Ten ml acetonitrile were added to the remaining oil, and the liquid extraction was repeated. After 3 extraction steps, the mixture of acetonitrile extracts was evaporated to dryness. The residue obtained after acetonitrile evaporation was dissolved in 1 ml hexane and to remove any rest of oil, it was saponified with 100 mg solid NaOH. Solid phase extraction, using a Sep Pack Silica Plus cartridge from Waters (Milford, MA, USA), was employed to clean the hexane layer solution. To do it, the cartridge was conditioned by passing 1 ml methanol and after that hexane extracts were loaded at a flow rate of 1 ml min -1 , being cleaned the cartridges with 1 ml of hexane. After that, hexane was evaporated and the residue dissolved in 1 ml acetonitrile, being filtered through a 0.22 µm nylon filter. A volume of 20 µl of the acetonitrile solution was injected in the chromatographic system for PAHs determination.

Chromatographic method.

An acetonitrile (ACN) gradient of 0 min 50 % ACN, 7 min 50 % ACN, 20 min 80 % ACN, 25 min 80 % ACN and 30 min 95 % ACN with a 0.250 ml min-1 mobile phase flow and 15 C column temperature was used to perform the chromatographic

Results and discussion.

Soxhlet extraction.

Table II shows the concentration, in ng (PAH)/g (cookie), of the 15 PAHs studied in the pooled sample. Results obtained are the average of 4 independent analyses and have been taken as a reference, to evaluate microwave and ultrasound assisted extractions.

[Insert Table II about here]

Ultrasound assisted extraction.

Preliminary experiments using ultrasound extraction were carried out to choose the optimum solvent, evaluating 30 ml of toluene and 30 ml acetone-hexane (1:1) mixture. Recoveries with toluene were between 43 % and 80 % as compared with Soxhlet extraction, being better than those obtained by using acetone-hexane with recoveries ranging from 86 % to 105 %.

Using acetone-hexane the effect of the solvent volume on the recovery of PAHs maintaining constant a total extraction time of 1 hour (3 times 20 min). As can be seen, Table III shows that the recovery of PAHs increases with increased solvent volume, but no differences in mean recovery were found between 30 and 35 ml.

Furthermore, for six of the PAHs, recoveries lower than 90 % were achieved and because of that, on spite of the reduction of solvent consume and extraction time as [Insert Table III about here] Microwave extraction.

Table IV shows the recovery data of PAHs from the pooled cookies employing different irradiation times. As can be seen, 2 steps of 7.5 min were necessary to achieve the quantitative recovery of all considered compounds, as compared with those obtained by Soxhlet, when 10 ml toluene and 1 ml water were employed.

[Insert Table IV about here]

In order to reduce the microwave irradiation time, a polar solvent should be used.

Because of that, additionally than the toluene:water mixture, an acetone-hexane (1:1) mixture was used. As can be seen in table IV quantitative recoveries of PAHs were achieved in only 9 min irradiation at 700 w. In these experiments, three x 3 min irradiation time steps were employed in order to avoid overpressure because the efficiency of heating of the acetone:hexane was higher than that obtained for toluene:water.

Figures of merits of the PAHs determination in cookies.

Results obtained for the 15 PAHs provided in all cases sensitive and linear regression graphs from 0.5 ng g -1 to 20 ng g -1 using 5 standards and being found regression coefficient higher than 0.9997. For the three procedures indicated in the experimental section, the limit of detection for each one of the 15 PAHs studied were Data provided evidence that all the extraction methods employed provided comparable limit of detection values and data found (see table V) varied from 0.015 ng g -1 for Chrysene to 0.9 ng g -1 for Phenanthrene.

[Insert Table V about here]

Comparison of the extraction procedures.

Ultrasound assisted extraction gave comparable recovery results (see table III

Analysis of samples by the recommended extraction procedure.

Table VI shows concentration of PAHs in two types of commercial cookies obtained by using the recommended procedure, microwave irradiation of samples with 10 ml acetone-hexane mixture for 3 steps of 3 min at 700 W power. Data reported are the average of three replicate analysis of each sample.

[Insert PAHs. However, acetone:hexane provided quantitative recoveries in 9 min compared to the 15 min required using toluene:water. Solvent consumption is another advantage of the proposed method, compared to Soxhlet and ultrasound assisted procedures. It has been reduced to 10 ml, plus additional 15 ml for cleaning the reactor, compared to the 155 ml and 90 ml employed in Soxhlet and Ultrasound procedures respectively. The limits of detection obtained by the three procedures assayed are of the same order of magnitude for all a5 PAHs.

Concerning the PAH content on the marked samples, none of the eight PAHs regulated by law was found at concentrations higher than 2 ng g -1 and the sum of the concentrations of the 8 heavy PAHs was lower than 5 ng g -1 and the sum of heavy and light PAHs was lower than 25 ng g -1 . Results shown as mean ± standard deviation of three independent analyses 1: Limit of detection in cookies.

  , are time and solvent consuming, and are inadequate for a rapid and environmentally friendly determination of PAHs. Microwave assisted treatment reduces the extraction time

201TP52 ( 5

 5 µm, 250 mm x 2.1 mm) Vydac (Columbia, MD, USA) column, a guard column 201TP (5 µm, 2.1 mm) from Vydac and a signal processor Chem Station HP (Waldbronn, Germany) were employed. The experimental conditions employed for PAHs separation and detection are indicated in Table

  Benz[a]anthracene >98 % GC-HPLC; Aldrich Chemie (Milwaukke, USA) for Fluoranthene 99 %, 11H-benzo[b]fluorene >98.0 % HPLC, Chrysene 95 %, Benzo[b]fluoranthene 98 %, Dibenz[a,c]anthracene 97.0 % and Benzo[a]pyrene >96.0 % HPLC; Sigma Chemical Company (St.Louis, MO, USA) for Dibenz[a,h]anthracene 97 % and AccuStandard (New Haven, CT, USA) for Benzo[k]fluoranthene 98 %, Benzo[e]pyrene 99 %, Benzo[ghi]perylene >98.0 % GC

  considered to establish the reference values of fat and PAHs in this pool.

  under study. By using a fixed excitation wavelength of 264 nm the best emission wavelength, indicated in table I, were employed for quantification.

  extraction, this procedure cannot be considered as an efficient alternative.

  interpolation, in the corresponding calibration line, of the smallest chromatographic peak obtained at each retention time of the PAHs considering the criterion of 3 times background noise, and taken into consideration the original sample (ng PAH/g cookie).

  ) to those obtained by Soxhlet for Benz[a]anthracene, Chrysene, Benzo[b]fluoranthene, Dibenz[a,c]anthracene, Benzo[k]fluoranthene, Dibenz[a,h]anthracene and Indeno[1,2,3-cd]pyrene. However, the microwave assisted treatment using both, 15 min with toluene:water or 9 min with acetone-hexane (see table IV), gave quantitative results for all the PAHs studied. Taken in consideration the microwave assisted extraction time, the recommended procedure is that based on the use of acetonehexane mixture (1:1). The regression between data obtained by the microwave assisted extraction using acetone-hexane (y) and the Soxhlet procedure (x) provided an equation y = 1.0144 x -0.0065, with a regression coefficient R 2 of 0.9999, thus evidencing the good comparability of results and confirming the validity of the microwave assisted extraction procedure in front of the classical Soxhlet extraction. The paired Student's t test for both groups of data was lower than the theoretical t value (1.771) for a 95% probability level and 13 degrees of freedom. So, it can be concluded that the developed procedure is comparable to that found by using classical Soxhlet extraction. Validation of the recommended extraction procedure. From our results the recommended extraction procedure was based on the use of microwave irradiation and the acetone:hexane mixture (1:1). Recovery studies on a pooled sample were performed to show evidence of the absence of losses or contamination during the determination of PAHs. Data found for the pooled sample spiked (n = 3) with known concentration of PAHs (0.5 ng g -1 of each PAHs), showed quantitative recoveries for the 15 PAHs studied. The recommended procedure provided recoveries that ranged from 86% in the case of Benz[a]anthracene, Benzo[b]fluoranthene and Benzo[ghi]perylene to 110% for Phenanthrene. For the other PAHs studied the recoveries were from 95 % to 105 %.

  Kaandorp B. 1986. Determination of polycyclic aromatic hydrocarbons (PAH) in edible vegetable oils by liquid chromatography and programmed fluorescence detection. Comparison of caffeine complexation and XAD-2 chromatography sample clean-up. Zeitschrift fur Lebensmittel Untersuchung und-Forschung 183:111-115. Wenzl T, Simon R, Kleiner J, Anklam E. 2006. Analytical methods for polycyclic aromatic hydrocarbons (PAHs) in food and the environment needed for new food legislation in the European Union. Trends in Analytical Chemistry 25

  :water were both appropriate to achieve the quantitative extraction of all 15

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Table VI about here] Conclusion The microwave-assisted procedure developed for the extraction of 15 PAHs in cookies provided results in good agreement with those obtained by the conventional Soxhlet extraction refluxing with toluene for 8 hours. Acetone:hexane and Page 13 of 24 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk toluene

Table I

 I Experimental conditions employed for the separation and detection of PAHs by

	Page 21 of 24
	Table II Table III
	Concentration of PAHs in a cookies pool sample obtained from Soxhlet extraction Effect of acetone-hexane (1:1) mixture volume on the ultrasound assisted extraction
	HPLC with toluene. of PAHs.
	Conditions for HPLC fluorimetric determination Compound C ± s (ng g -1 ) Compound Solvent volume (ml) used in each step*
	Compound Phenanthrene Phenanthrene Anthracene Anthracene Fluoranthene Pyrene 11H-benzo[b]fluorene 10 F o r Fluoranthene Pyrene 11H-benzo[b]fluorene Benz[a]anthracene F o r Phenanthrene 64 ± 1 Anthracene 40 ± 1 Fluoranthene 58 ± 2 Pyrene -F o r Benz[a]anthracene Chrysene Benzo[e]pyrene Benzo[b]fluoranthene Dibenz[a,c]anthracene P e e r Chrysene Benzo[e]pyrene Benzo[b]fluoranthene Dibenz[a,c]anthracene Benzo[k]fluoranthene P e e r 11H-benzo[b]fluorene 18 ± 9 Benz[a]anthracene 50 ± 4 P Chrysene 34 ± 1 e Benzo[e]pyrene 41 ± 2 Benzo[b]fluoranthene 52 ± 2 e r Benzo[k]fluoranthene R λ λ λ 20 λ em (nm)* t R (min) H 9.463 ± 0.001 25 30 364 0.350 ± 0.008 (R ± s) % H 18.2 402 20.1 444 22.0 384 23.4 352 1.94 ± 0.08 < LOD 0.123 ± 0.003 0.80 ± 0.03 78 ± 9 85 ± 3 88 ± 1 62 ± 2 84 ± 2 87 ± 2 78 ± 9 86 ± 5 89 ± 5 ---26.2 396 27.0 368 29.3 384 32.2 440 34.3 380 0.219 ± 0.005 0.24 ± 0.03 0.22 ± 0.02 0.288 ± 0.009 0.126 ± 0.004 56 ± 1 78 ± 2 86 ± 2 75 ± 4 91 ± 5 99 ± 1 76 ± 4 100 ± 7 104 ± 4 65 ± 4 90 ± 3 95 ± 4 74 ± 9 98 ± 3 105 ± 2 35.6 450 Benzo[a]pyrene 0.226 ± 0.004 R Dibenz[a,c]anthracene 55 ± 9 80 ± 4 95 ± 8 98 ± 2 R 36.3 Benzo[a]pyrene 404 37.4 Dibenz[a,h]anthracene 400 41.3 Benzo[ghi]perylene 416 42.4 Indeno[1,2,3-cd]pyrene 500 e v i e Dibenz[a,h]anthracene 0.34 ± 0.02 Benzo[ghi]perylene e Benzo[k]fluoranthene 22 ± 4 69 ± 5 104 ± 5 103 ± 6 e 0.114 ± 0.001 Indeno[1,2,3-cd]pyrene 0.093 ± 0.009 v i e Benzo[a]pyrene 9 ± 7 45 ± 4 71 ± 3 87 ± 3 Dibenz[a,h]anthracene 15 ± 1 36 ± 9 76 ± 3 95 ± 3 Benzo[ghi]perylene -32 ± 3 73 ± 3 86 ± 2 v i e 45.1 w Average ± standard deviation (n = 4) w Indeno[1,2,3-cd]pyrene -37 ± 8 78 ± 9 93 ± 6 w	35 88 ± 3 89 ± 1 89 ± 1 -89 ± 1 94 ± 8 106 ± 6 97 ± 3 106 ± 3 97 ± 7 102 ± 4 87 ± 3 97 ± 2 86 ± 7 95 ± 7
	*: An excitation wavelength of 264 nm was employed in all cases H : The standard deviation of the retention time for 8 independent analyses in different work sessions was in all cases of the order of ± 0.1 min. O n l O n l Mean 38 ± 4 61 ± 6 86 ± 5 94 ± 3 94 ± 4 *:Total extraction time was 1 hour (3 steps of 20 minutes) O n l
	y y :Values expressed are the recovery percentages compared with Soxhlet extraction H y
	± the standard deviation of 3 independent assays.
		21

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uk Food Additives and Contaminants F o r P e e r R e v i e w O n l y 22 Table IV

  Effect of irradiation time on the microwave extraction of PAHs by using toluene-water and acetone-hexane mixtures.

	Compound	Toluene-water				acetone-hexane mixture
		Irradiation time (min)*				Irradiation time (min)*
		1.5+1.5	3+3	4.5+4.5	6+6	7.5+7.5	3+3	3+3+3	3+3+3+3
				(R ± s) %	H			(R ± s) %	H
	Phenanthrene	24 ± 1	49 ± 1	99 ± 1	100 ± 4	100 ± 1	62 ± 9	102 ± 1	104 ± 1
	Anthracene	56 ± 1	60 ± 3	76 ± 3	91 ± 4	104 ± 4	71 ± 4	105 ± 3	103 ± 2
	Fluoranthene Pyrene 11H-benzo[b]fluorene Benz[a]anthracene F o 39 ± 5 -21 ± 2 65 ± 4 r	63 ± 4 -26 ± 1 75 ± 1	87 ± 3 -68 ± 8 85 ± 2	91 ± 4 -90 ± 4 92 ± 5	100 ± 5 -102 ± 1 100 ± 2	58 ± 3 -30 ± 1 74 ± 3	99 ± 1 -101 ± 1 105 ± 4	96 ± 1 -100 ± 1 101 ± 2
	Chrysene Benzo[e]pyrene Benzo[b]fluoranthene Dibenz[a,c]anthracene Benzo[k]fluoranthene	51 ± 2 36 ± 2 P 49 ± 2 e 15 ± 1 32 ± 1 e 60 ± 2 45 ± 4 57 ± 3 40 ± 4 42 ± 6 r	83 ± 7 73 ± 3 85 ± 3 70 ± 4 71 ± 4	101 ± 1 86 ± 3 91 ± 5 91 ± 1 91 ± 3	105 ± 4 99 ± 4 98 ± 7 101 ± 2 98 ± 5	64 ± 2 61 ± 2 66 ± 9 80 ± 1 71 ± 3	103 ± 4 99 ± 3 103 ± 3 105 ± 3 96 ± 4	100 ± 2 100 ± 2 98 ± 3 99 ± 4 100 ± 1
	Benzo[a]pyrene Dibenz[a,h]anthracene Benzo[ghi]perylene	12 ± 3 4 ± 4 -	44 ± 3 14 ± 3 R 13 ± 2 e 81 ± 4 48 ± 5 19 ± 2	92 ± 4 81 ± 7 60 ± 3	104 ± 5 96 ± 1 98 ± 6	62 ± 3 16 ± 1 53 ± 1	101 ± 3 97 ± 9 100 ± 2	99 ± 1 98 ± 9 102 ± 1
	Indeno[1,2,3-cd]pyrene Mean *Time in minutes correspond to each step assayed. -24 ± 1 34 ± 2 44 ± 2 v 26 ± 1 69 ± 4 i e w 65 ± 3 88 ± 4	106 ± 4 101 ± 4	75 ± 6 60 ± 5	100 ± 1 101 ± 3	99 ± 1 100 ± 2
					O n l
					y

Table V

 V Limit of detection calculated for each of the tested procedures.

						LOD a
						(ng g -1 )
	Compound		Soxhlet	Ultrasound	Microwave	Microwave
						(Toluene)	(Ac:Hx) b
	Phenanthrene Anthracene Fluoranthene Pyrene F o r	0.6 0.09 0.4 0.4			0.9 0.11 0.3 0.5	0.4 0.12 0.5 0.4	0.7 0.19 0.3 0.5
	11H-benzo[b]fluorene Benz[a]anthracene P 0.015 0.2			0.018 0.3	0.019 0.3	0.017 0.11
	Chrysene	0.03 e			0.02	0.03	0.015
	Benzo[e]pyrene Benzo[b]fluoranthene		0.18 0.06 e r	0.3 0.09	0.2 0.03	0.18 0.03
	Dibenz[a,c]anthracene Benzo[k]fluoranthene		0.10 0.07	0.13 0.011 R	0.10 0.015	0.19 0.03
	Benzo[a]pyrene Dibenz[a,h]anthracene Benzo[ghi]perylene Indeno[1,2,3-cd]pyrene		0.05 0.03 0.03 0.02		0.019 0.013 e 0.02 0.018 v i e	0.03 0.07 0.019 0.016	0.03 0.06 0.02 0.02
						w
	Results shown as mean ± standard deviation of three independent analyses a: Limit of detection in cookies ng PAHs/g cookies b: Acetone + hexane mixture O n l
						y

Table VI

 VI Analysis of commercial cookies by the recommended procedure.

			Concentration (ng g -1 )
	Compound	LOD 1	Sample 1	Sample 2	Pool
		(ng g -1 )	(18.5 % oil)	(21.0 % oil)	(16.0 % oil)
	Phenanthrene	0.7	8 ± 2	3 ± 1	9.6 ± 0.1
	Anthracene	0.19	0.5 ± 0.1	< LOD	0.37 ± 0.02
	Fluoranthene	0.3	0.70 ± 0.07	0.90 ± 0.01	1.92 ± 0.04
	Pyrene	0.5	< LOD	< LOD	< LOD
	11H-benzo[b]fluorene	0.017	1.3 ± 0.1	0.025 ± 0.001 0.124 ± 0.003
	Benz[a]anthracene	0.11	0.98 ± 0.06	1.5 ± 0.1	0.81 ± 0.05
	Chrysene	0.015	0.22 ± 0.05	0.10 ± 0.01	0.23 ± 0.02
	Benzo[e]pyrene	0.18	< LOD	0.2 ± 0.2	0.2 ± 0.2
	Benzo[b]fluoranthene	0.03	0.031 ± 0.004	0.10 ± 0.01	0.22 ± 0.01
	Dibenz[a,c]anthracene	0.19	0.2 ± 0.3	1.8 ± 0.5	0.30 ± 0.02
	Benzo[k]fluoranthene	0.03	0.03 ± 0.02	0.077 ± 0.002 0.122 ± 0.009
	Benzo[a]pyrene	0.03	0.04 ± 0.02	< LOD	0.23 ± 0.01
	Dibenz[a,h]anthracene	0.06	< LOD	< LOD	0.33 ± 0.07
	Benzo[ghi]perylene	0.02	0.658 ± 0.003 0.124 ± 0.005 0.114 ± 0.005
	Indeno[1,2,3-cd]pyrene	0.02	0.07 ± 0.03	0.208 ± 0.006 0.092 ± 0.002

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Acknowledgements

Authors acknowledge the financial support of the Laboratorio de Higiene Laboral y Ambiental of the University of Valencia.