The aim of this study was to determine the effect of different cooking processes 15 (microwaving, roasting, boiling grilling and frying) on enrofloxacin residues in 16 naturally incurred chicken muscle. Enrofloxacin and its metabolite ciprofloxacin were 17 analysed using a validated LC-MS method with limits of detection (LOD) and 18 quantification (LOQ) of respectively 2 and 5 ng g -1 of quinolones in muscle samples. 19

The method was demonstrated to be linear over the range of 5-500 ng g -1 . The mean 20 intra-day RSD at a concentration of 50 ng g -1 (n=6) was 6%; inter-day RSD was 12%.

21

A recovery study demonstrated that 65-101%, of the drug and metabolite could be 22 recovered from the tissue. The RSD with naturally incurred roasted chicken breast 23 was 9.18% at a concentration of 11 ±1,01 ng g -(n=6). In water, enrofloxacin 24 remained stable for 3 h when heated at 100 ºC. It was concluded that residue data 25 from raw tissues are valid for the estimation of the exposure of the consumers to this 26 drug as well as the ADI calculations because cooking procedures did not affect the 27 enrofloxacin residues which remained stable during heating. However, there was an 28 apparent decrease in quinolone concentration in the tissues because some was lost 29 by exudation or into the liquid used for cooking. Conversely, for a cooking procedure 30 with water loss, there was an apparent increase in residue concentration. 31 32 Keywords: Enrofloxacin, veterinary residues, chicken, cooking. 33 that are extensively used in human and veterinary medicine. These antibiotics have a 4 wide spectrum of action and high efficacy against infectious disease. They are highly 5 effective in treating against Gram positive, Gram negative and Micoplasma in 6 infection processes (Hooper et al., 2001a). The high level of use in animals and 7 humans, and to some degree of misuse in the sense of unnecessary use or use of 8 quinolones with poor activity in some developing countries, has been blamed for the 9 rapid development of bacterial resistance to these agents (Hooper, 2001b) which 10 means a hazard to human health. 11 12 Currently the levels of drug residues in raw food (meat and animals products) are 13 regulated. In this regard, the maximum residue limits (MRL) for the fluoroquinolone 14 enrofloxacin and its metabolite ciprofloxacin legally permitted in food under the laws 15 of the European Union (EEC, 1990) have been established for muscle, fat, liver, and 16 milk from several animal species, including chicken. 17 18 Since most of this type of food is cooked before consumption, more information 19 about the effect of cooking on residues is required to give a more accurate estimate 20 of consumer exposure to these chemicals and any breakdown products. However, 21 few studies have been carried out on the occurrence of the veterinary drug residues 22 in cooked foods, nor on the stability of these compounds following heat treatment 23 Raypa (Barcelona, Spain). Centrifuge Eppendorf 5415D (Hamburgo, Germany). 3

(

LC-MSMS System 4

The LC-MS-MS system consisted of an Agilent Technologies 1100 HPLC (Agilent 5

Technologies, Minnesota, USA) and a Q-Trap mass spectrometer with turbo ion 6 spray interface Applied Biosystems MSD Sciex (Toronto, Canada). The HPLC 7 column used was a Synergi Fusion-RP (50×2 mm) 4 µm, Phenomenex (Torrance, 8 CA, USA). The mobile phase was aqueous formic acid (0.1 %) mixed in gradient 9 mode (95 % to 65 % in 5 min) with acetonitrile. Flow-rate was 500 µL min -1 . 10 11 MS/MS detection was performed in mode MRM from the protonated molecular ion 12

[M+H] + of analyte to its product ion in positive ion mode. The product ion efficiency of 13 the [M+H] + was optimized by varying the spray, orifice and voltages. The collision 14 activated dissociation (CAD) of the [M+H] + of analytes was performed at quadrupole 15 using N 2 gas atmosphere. These CAD parameters were optimized by varying the 16 collision gas pressure and collision energy using a programme to obtain the 17 maximum intensity and reproducibility. The parameters are showed in the table 1. 18

"[insert Table 1 about here] 20 21

Animal treatment 22

Five chickens housed in individual cages were used. One of them were considered 23 as blank. The other four were dosed over 5 consecutive days with 15 mg/day of 24 enrofloxacin through the intramuscular route. The pharmacological speciality 25 Ganadexil (Enrofloxacin 5%), obtained from Industrial Veterinaria, S.A. (Esplugues 26 de Llobregat, Barcelona, Spain) was employed. 27 28

Cooking Procedure 29

Three samples (breast, whole leg and liver) from each chicken were subjected to five 30 different cooking treatments. Breast and whole leg were cooking using methods that 31 reflect the wide variation in domestic practices: microwaving, roasting, grilling, boiling 32 and frying. The liver was fried and roasted in an oven. All the pieces of tissue were 33 A 5 g portion of chicken tissue (breast, whole leg and liver) was fried for 10 min with 6 5 g of sunflower oil in a pan using an electric hob, turning occasionally. The cooked 7 muscle had a "well done" appearance on the outside. 8 9

Cooking by boiling. 10

A 5 g portion of chicken (breast and whole leg) was placed with 10 ml of water in a 11 vessel and boiled for 10 min, using an electric hob. 12 13 Cooking by microwaving. 14 A 5 g portion of chicken (breast and whole leg) with 1 g of sunflower oil was cooked 15 for 3.5 min at 800 W. 16 17 Cooking by roasting in an oven. 18 A 5 g portion of chicken (breast, whole leg and liver) with 1 g of sunflower oil was 19 cooked in a electric oven at 200 ºC for 10 min. 20 21

Cooking by grilling. 22

A 5 g portion of chicken (breast and whole leg) was grilled for 10 min. 23 24

Sample preparation 25

After the cooking treatments, the chicken muscle was homogenized using an Ultra-26 Turrax® blender. An accurately weighed sample (0.5 ± 0.05 g) was placed in an 27

Eppendorf tube and then 500 µL of a sodium hydroxide aqueous (10 mM) -28 acetonitrile (1:1) solution was added. The sample was sonicated for 10 min and 29 centrifuged at 13000 rpm for 20 min. Finally, 250 µL of the upper layer was placed in 30 an injection vial and 25 µL of internal standard (Ofloxacin, 1 µg mL -1 ) was added and 31 5 µL of this mixture were injected into the LC/MS/MS system. 32

Stability of enrofloxacin in heated water 1

Solutions containing 25 ngmL -1 enrofloxacin were prepared in water and dispensed 2 into a number of sealed vials. The vial solutions were placed in a thermostatic oven 3 and removed at intervals of 30 min during 3h. A 0.5 mL aliquot was used to analyse 4 enrofloxacin in the same way than tissue samples. The experiment was carried out at 5 100 ºC. 6 7 Validation 8

The analytical method was characterized for raw tissue with the repeated generation 9 of calibration graphs of raw tissue spiked prior to extraction with different amounts of 10 enrofloxacin and ciprofloxacin, using ofloxacin as the internal standard. Three 11 repetitions for each level were made each day over three days. Two analysts, using 12 different solutions each day, generated the data. The calibration graph was described 13 by the equation Y= mX+ b, where Y represents the response value of the analyte in 14 the solution (peak area), and X the number of nanograms of each quinolone on-15 column. To check the extraction methodology, we added 500 µL of a known 16 concentration mixture of enrofloxacin and ciprofloxacin in a sodium hydroxide 17 aqueous (10 mM) -acetonitrile (1:1) solution to 0.5 ± 0.05 g of muscle sample. The 18 spiked muscle (5, 50, 250 ng g -1 ) was them extracted as above. This was carried out 19 on three consecutive days three times each day. The intra-day assay was carried out 20 at a level of 50 ng g -1 repeated six times. To evaluate the method for cooked tissue, 21 the same breast piece naturally incurred and roasted in an oven at 200ºC was 22 analysed six times. 23 24

Results and Discussion 25

Method validation 26

Figure 1 shows a chromatogram of an extract of raw tissue spiked with 5 ng g The specificity of the method was assessed by testing a number of representative 1 blank muscle samples (n=10) to verify the absence of potential interfering 2 compounds. The LODs and LOQs, defined as the blank response (n= 10) plus three 3 or ten times the standard error of the blank response, was 10 pg and 25 pg 4 respectively, both injected on-column. These values correspond to 2 and 5 ng•g -1 of 5 quinolones in muscle samples. The chromatographic method was demonstrated to 6 be linear for the range studied (5-500 ng g -1 ) with P-value for lack-of-fit in the ANOVA 7 table greater or equal to 0.10 (calibration coefficient 0,9998 and 0,9996, for 8 ciprofloxacin and enrofloxacin, respectively). The mean intra-day RSD (n=6, c=50 ng 9 g -1 ) was 6%; inter-day assay gave a RSD of 12%. The extraction percentages were 10 within the established range 65-101%, for the drug and their metabolite. Appropriate 11 relative standard deviation was obtained with naturally incurred roasted breast of 12 9.18% (n=6, 11 ng g -1 ±1,01). Ciprofloxacin and enrofloxacin concentrations in the 13 samples were interpolated from the five-point calibration curves made with 0.5 g of 14 tissue as described above. All results quoted were corrected for analytical method 15 recovery daily obtained with a blank sample fortified at the LOQ (5 ng g -1 ). 16 17 There are no references in the literature to the effect of cooking on enrofloxacin 18 residues or related drugs in foods. Ciprofloxacin has been described as the main 19 metabolite of enrofloxacin, with extensive metabolism in chickens (Delaporte et al., 20 1994). In light of this, we analysed for both substances but, as only small amounts, or 21 undetectable levels of ciprofloxacin found were in all samples, only the enrofloxacin 22 residue levels are given in this paper. The stability of enrofloxacin in water at 100 ºC 23 was demonstrated over a period of 3 h (n=6, c=25 ng mL -1 RSD=14.3%). The 24 concentrations of enrofloxacin in each piece from each chicken used in the 25 experimental work are given in Table 2. The homogeneity of enrofloxacin residues 26 was demonstrated for the same piece of each chicken (see results from raw pieces). 27

Nevertheless, although the birds had been fed and treated in a similar way, 28 significant variations due to metabolic or other differences between individuals were 29 noted. 30

31 As it can be observed in table 3, the change in enrofloxacin levels in the tissue is 1 dependant on the type of cooking. We noted a very different concentration of 2 quinolone in the tissues from different birds and, for that reason, we have presented 3 the data for the four chickens separately. The change in the concentration of 4 enrofloxacin residue induced by the different cooking procedures were presented as 5 a percentage increase or decrease. As shown in the table, extraction of residues took 6 place when we boiled or microwaved the chicken pieces. There was always a 7 reduction in the concentration, and the lost amount of analyte in the tissue was found 8 in the water or in the exudates. On the other hand, the amount of residue increased 9 in the case of roasting and grilling and this can be explained because the minor 10 moisture content of the treated piece caused the apparent concentration of the 11 quinolone residue. This takes to us to conclude that the cooking procedure did not 12 therefore affect the levels of quinolone. Thus we concluded that residue data from 13 raw tissues are valid for estimating consumer exposure and for making the ADI 14 (Acceptable Daily Intake) calculation because the cooking procedure did not affect 15 the enrofloxacin residues as they remained stable during the heating. 16 17

"[insert
The results reported here were consistent with these previously reported by other 18 authors that studied the fate of drug residues. As an example, streptomycin in eggs 19 subjected to boiling or frying did not change residue concentrations (Inglis and Katz, 20 1978;[START_REF] O'brien | Antibiotic residues in meat: 27 cooking and cold storage effects[END_REF], neomycin residues were not affected by cooking (Inglis 21 and Katz, 1978;[START_REF] Katz | Determination of neomycin residues in eggs and 20 stability of residues after cooking[END_REF], whereas oxytetracycline levels were 22 reduced to a monor extent by frying and roasting procedures [START_REF] O'brien | Antibiotic residues in meat: 27 cooking and cold storage effects[END_REF]. 23

Other veterinary drugs such as clenbuterol (Rose et al, 1995a), levamisole (Rose et 24 al, 1995b), sulphametazine (Rose et al, 1995c), oxytetracycline [START_REF] Rose | The effect of cooking 42 on veterinary drug residues in food[END_REF], 25 oxfendazole (Rose et al, 1997a), lasalocid (Rose et al, 1997b)

Figure1.Figure2.The effect of cooking on enrofloxacin residues in chicken tissues 1 2Fente*, C 4 5

 14 Figure1. Illustrative chromatogram of raw tissue spiked with 5 ngg-1 of ciprofloxacin (MRM: 332.3-245.3) and enrofloxacin (MRM: 360.4-316.4), using lomefloxacin (MRM: 362.3-261.2) as internal standard. 284x155mm (300 x 300 DPI)

2

  Enrofloxacin belongs to the group of potent antibiotics known as fluoroquinolones 3

  T25 homogenizer (Staufen,Germany). Ultrasounds bath UCI-200 2

4

  three times in the raw state to ensure the homogeneity of residue 1 distribution. In addition, the water used -or the exudates released-for the boiling or 2 microwave cooking were also analysed. 3Cooking by frying. 5

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Table 2

 2

	32	about here]
	33

Table 1 .

 1 Rose, M.D., Rowley, L., Shearer, G., Farrington, W.H.H. 1997 b. Effect of cooking on 1 veterinary drug residues in food: 6. Lasalocid. Journal of Agricultural and Food 2 Mass spectrometer parameters and MRM transition ions.

	or ivermectin (Rose et

Table 2 :

 2 Homogeneity data. Enrofloxacin residue levels (ng g -1 ) in raw tissues from the same piece (n=3) in different animals.

	Animal		Whole leg	Breast	Liver
	1	Mean RSD%	29,00 9,12	28,30 7,30	10,03 9,98
		Mean	10,45	5,25	29,63
	2	RSD%	11,85	13,33	10,11
		Mean	50,24	17,86	32,61
	3	RSD%	5,28	6,35	6,87
		Mean	25,52	15,05	65,71
	4	RSD%	10,79	13,46	3,87
		Mean	<LOQ	<LOQ	<LOQ
	Blank	RSD%	<LOQ	<LOQ	<LOQ

Table 3 :

 3 Effect of cooking on enrofloxacin residues (ng g -1 ) in chicken tissues. Illustrative chromatogram of raw tissue spiked with 5 ngg -1 of ciprofloxacin (MRM: 332.3-245.3) and enrofloxacin (MRM: 360.4-316.4), using lomefloxacin (MRM: 362.3-261.2) as internal standard.Figure2. Chromatogram of a breast portion cooking by microwaving from a chicken treated by injection, only peak corresponding enrofloxacin (MRM: 360.4-316.4) and internal standard lomefloxacin (MRM: 362.3-261.2) are presents.

			CHICKEN1 CHICKEN 2 CHICKEN 3 CHICKEN 4	a %
		Raw	29.00	10.45	50.24	25.52
		Boiled	16.00	<LQ	26.07	14.40	-48.11 to -43.57
		Fried	14.00	9.00	25.62	8.60	-66.30 to -13.88
	WHOLE LEG	Oven Microwave	41.80 17.00	22.78 <LQ	102.91 22.50	64.19 5.86	44.14 to 151.53 -77.04 to -41.38
		Grilled	42.13	14.80	75.00	51.50	41.63 to 101.80
		Water-Boiled	10.00	<LQ	53.60	22.46
		Water Microwave	14.54	<LQ	24.00	24.36
		Raw	28.30	5.25	17.86	15.05
		Boiled	12.30	<LQ	7.94	5.60	-62.79 to -55.54
		Fried	16.00	<LQ	12.22	5.28	-64.92 to -31.58
		Oven	45.01	12.46	32.06	61.69	59.05 to 309.90
		Microwave	10.74	<LQ	9.43	5.20	-65.45 to -47.20
	BREAST	Grilled	45.20	8.50	28.33	24.20	58.62 to 61.90
		Water-Boiled	38.00	<LQ	27.60	20.66
		Water Microwave	8.16	<LQ	5.20	14.38
		Raw	10.03	29.64	32.61	65.71
	LIVER	Fried	8.00	19.00	29.25	25.30	-61.50 to -10.30
		Oven	13.36	30.01	41.96	91.71	28.67 to 39.57

a %:Percentage of increase or decrease in enrofloxacin residue concentration. LQ:Limit of cuantification.

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Acknowledgements 29

Mr. Lolo acknowledges a scholarship from XUNTA DE GALICIA, Spain. We also


Moira ; Brett

Cphls

V Economou¹

C Papadopoulou

M Brett²

A Kansouzidou

Charalabopoulos

G Filioussis

K Seferiadis

Diarrheic shellfish poisoning due to toxic mussel consumption: first 1 recorded outbreak in Greece 2 3

Keywords: Diarrheic Shellfish Poisoning, Greece, mussels, algal toxins, Dinophysis, 17 outbreak

During the week 14-20 th January 2000, 120 people visited the Emergency Departments of 3 Thessaloniki (Northern Greece) hospitals complaining of acute gastrointestinal illness 4 after eating mussels (Mytilus galloprovincialis). The symptoms indicated diarrhoeic 5 shellfish poisoning and the toxicity of mussels harvested from Thermaikos Gulf in 6

Thessaloniki during the outbreak, was investigated using mouse bioassays. The 7 bioassays revealed toxicity to mice by the mussel samples, while high numbers of toxic 8 algae Dinophysis acuminata were identified in water samples from Thermaikos Gulf. 9

Harvesting of mussels was immediately suspended and a monitoring programme for algal 10 blooms was established from then onwards. During a follow-up of the mussels' toxicity 11 from January 2000 through January 2005, two more mussel samples were found positive 12 for DSP, one harvested in March 2001 from the area of the outbreak (Thermaikos Gulf) 13 and one harvested in January 2001 from North-western Greece (Amvrakikos Gulf). 14 However, no sporadic cases or outbreaks were reported during this period.

proven for OA, DTX-1 and DTX-3 [START_REF] Murata | Isolation and structural 3 elucidation of the causative toxin of diarrhetic shellfish poisoning[END_REF][START_REF] Rossini | Neoplastic activity of DSP toxins: The effects of okadaic acid and 17 related compounds on cell proliferation: Tumor promotion or induction of apoptosis? 18[END_REF]Vale and Sampayo 10 2002). DTXs are lipophilic and accumulate in the fatty tissue of shellfish. The toxin profiles 11 in shellfish display considerable seasonal and geographical variation (Yasumoto et al. 12 1985; [START_REF] Sechet | Causative species of diarrhetic 24 shellfish poisoning (DSP) in Norway[END_REF]). Ordinary cooking processes do not destroy DSP toxins (Gestal 13 -Otero 2000). 14 15 DSP symptoms include diarrhoea, nausea, vomiting, abdominal pain and chills, which 16 manifest within 30 min to a few hours after consumption of contaminated shellfish and 17 persist for 3 to 4 days [START_REF] Aune | Diarrhetic shellfish poisoning. Chapter 5[END_REF]. Although short-term effects are 18 relatively mild, long-term effects are more severe and chronic exposure may promote 19 tumour formation in the digestive system [START_REF] Hallegraef | Algal toxins in Australian shellfish[END_REF][START_REF] Rossini | Neoplastic activity of DSP toxins: The effects of okadaic acid and 17 related compounds on cell proliferation: Tumor promotion or induction of apoptosis? 18[END_REF]. OA and DTX1 20 are potent inhibitors of protein phosphatases and this mode of action may be linked to the 21 observed diarrhoea, degenerative changes in the absorptive epithelium of the small 22 intestine and tumour development [START_REF] Hallegraef | Algal toxins in Australian shellfish[END_REF] [START_REF] Rossebo | Etiologisk uklar matforgifning etter konsum av 15 blaskjell[END_REF]3 Underdal et al. 1985, Torgersen et al. 2005), Sweden [START_REF] Krogh | Outbreaks of diarrheic shellfish poisoning on the west 13 coast of Sweden[END_REF], Spain (Campos 4 et al. 1982;Fernández et al. 1996), Italy [START_REF] Viviani | ASP, 6 DSP, NSP and PSP monitoring in "mucilaginous aggregates" and in mussels in a 7 coastal area of the Northern Adriatic Sea facing Emilia -Romagna in 1988, 1989 8 and 1991[END_REF][START_REF] Ciminiello | Yessotoxin in mussels of the Northern Adriatic Sea[END_REF], 5 Portugal [START_REF] Vale | Esters of ocadaic acid and dinophysitoxin-2 in Portuguese 13 bivalves related to human poisonings[END_REF], Belgium (De [START_REF] Schrijver | An outbreak of diarrhoeic shellfish 12 poisoning in Antwerp, Belgium[END_REF] former USSR 6 (Konovalova 1993). There are also reports from Japan [START_REF] Yasumoto | Diarrhetic shellfish 17 poisoning[END_REF] New Zealand, Thailand, North and South America [START_REF] Hallegraef | Algal toxins in Australian shellfish[END_REF][START_REF] Scoging | Marine biotoxins[END_REF]Gestal 9 -Otero 2000). From the available literature it is concluded that DSP or the presence of 10 DSP toxins in mussels, is increasingly reported particularly in Europe (Table I). The 11 increased reporting may be the result of increasing knowledge and awareness about toxic 12 shellfish poisonings (DSP, ASP and PSP) and the implementation of 13 surveillance/monitoring programmes. Also, it has to be emphasized that toxin producing 14 algae and toxic mollusks are reported from new areas, but well documented cases in 15

Europe are rare and this may be attributed to under-diagnosis and/or underreporting. The 16 shellfish most frequently implicated in DSP are mussels, but it has also been reported 17

Materials and methods 1

The DSP outbreak investigation 2

During the week 14-20 th January, 2000, one hundred and twenty people of both sexes, 3 aged from 8 to 70 years old, visited the Emergency Departments of the hospitals in the 4 city of Thessaloniki (Northern Greece), suffering from acute non-febrile gastro-enteritis 5 (Table II). The predominant symptoms reported by the patients were diarrhea, nausea, 6 vomiting, abdominal cramps and chills, which persisted for more than 24 h. All patients 7 reported onset of symptoms within 30 min to 10 h after consuming a mussel meal (deep 8

fried mussels, poached in cheese and tomato hot sauce, mussel pilaf and cooked mussel 9 salads) in various establishments (restaurants, taverns, homes) within the Greater 10 Thessaloniki area. All the hospitalized patients recovered completely within 12 to 36 h 11 after the administration (i.v) of saline solution and were completely asymptomatic upon 12 exit from the hospital. [Insert Table II about here] 13 14 Fecal samples from the hospitalised patients were examined for routine bacterial, viral 15 and parasitic agents causing non febrile gastroenteritis, but they were found negative. The 16 common history of the patients (reporting a mussels' meal), based on relative 17 questionnaires, combined with the clinical and laboratory findings were indicative of 18 diarrheic shellfish poisoning, thus the Veterinary Authorities were immediately notified 19 and consumption of mussels was banned until further notice. Mussel dishes are very 20 popular in the coastal areas of Greece, especially in the vicinity of Thessaloniki and 21 accordingly it was estimated that the actual number of people infected was actually much 22 larger than the 120 cases recorded at the hospitals' admissions desks. Hence, the 23 estimates were for a few hundreds of people infected, who either visited privately a 24 The patients who had prepared their meals at home reported purchase of fresh mussels 4 from seafood retails, supermarkets and open markets in the Thessaloniki greater area 5 and similar retail sources were reported by the restaurant and tavern owners. The fresh 6 mussels (Mytilus galloprovincialis) sold in the markets of Thessaloniki area and in most 7 markets in Northern and Northwestern Greece are harvested from aqua cultures located 8 into the Thermaikos Gulf (Figure 1). 9 10The Veterinary and Sea Biology authorities reported numbers of dinoflagellates ranging 11 from 3x10 4 to 5,4x10 4 cells/L of the toxic genus Dinophysis spp. in the seawater samples 12 from Thermaikos Gulf, and specifically of the species D. acuminata and D. sacculus 13 [START_REF] Koukaras | Intense DSP outbreak associated with a Dinophysis 11 bloom in Thermaikos Gulf (N.W. Aegean Sea, Greece)[END_REF]. No other episodes either of sporadic cases or outbreaks 14 associated with consumption of mussels exposed to toxin-producing Dinoflagellates of the 15 DSP type have been reported for the following four years, although the presence of toxic 16

Dinophysis acuminata was reported again in Thermaikos Gulf in March -May 2001 17 [START_REF] Koukaras | Intense DSP outbreak associated with a Dinophysis 11 bloom in Thermaikos Gulf (N.W. Aegean Sea, Greece)[END_REF]. However, suspension of harvesting and withdrawal of 18 mussels from the market are being applied sporadically in Thermaikos Gulf, whenever 19 algal blooms are detected during the phytoplankton monitoring program, which has been 20

established after the outbreak. An algal bloom is a rapid increase in the population of 21 phytoplankton algae in an aquatic system. The appearance of Dinophysis, even at low 22 densities such as 200 cells per litre, can cause already a toxification of shellfish that is 23 enough to affect humans (Van Egmond et al. 2004). According to the Greek Veterinary 24 The extraction of DSP toxins was performed following the method validated by the Marine 2 Laboratory, Aberdeen, UK, which is the EU designated UK National Reference Laboratory 3 (NRL) for Marine Biotoxins. The method is that of Yasumoto et al [START_REF] Yasumoto | Diarrhetic shellfish 17 poisoning[END_REF]) 4 with slight modifications. A portion of 100 g of homogenized mussel flesh was suspended 5 three times and thoroughly mixed with 100 ml of acetone (Scharlau, Analar grade) at 6 room temperature. The aliquots were centrifuged at 3000 rpm for 10 min in a Sorvall GLC-7 4 desktop centrifuge and the supernatants were collected and pooled. Acetone was 8 removed by rotary evaporation for 1h at 60 o C or until all trace of acetone had gone. An 9 Eyela type N rotary evaporator (Eyela Co, 3-3-4 Honcho Nihonbashi, Chuo-ku, Tokyo 10 103-0023, Japan) was used. The remaining aqueous-lipid extract was separated with 11 diethyl ether (Scharlau, Analar grade), backwashed with a small volume of water and 12 dried by rotary evaporation at 65 o C for 2 to 4 h. The remaining material was suspended in 13 4 ml of 1% Tween 60 (Merck) aqueous solution. 14 15

Mouse bioassay 16

BALB-C strain white mice weighting 19 to 21 g each were used for the mouse bioassay. 17

The mice were weighed prior to intra-peritoneal injection of 1ml of the mussel extract. 18 Three mice were used for each sample. All injected mice and controls were monitored for 19 a period of at least 24 h after injection. Initially the mice were observed constantly for the 20 first 6 h and from then every 1 h for the remaining 18 h. During the 24 h monitoring any 21 behavioral changes or symptoms were recorded in detail. When no typical symptoms of 22 DSP intoxication were observed after 24 h, the mice were monitored for an additional 24 h 23 period. The symptoms that were characterized as typical of DSP intoxication were initial 24 apathy, general weakness, difficulty in movement, spasms, contractions, bluish skin 25 death of two out of three mice within 24 hours after inoculation into each of them of an 2 extract equivalent to 5 g of hepatopancreas or 25 g whole body should be considered as a 3 positive result for the presence of one or more of the toxins mentioned, when a mouse 4 bioassay is utilized as a screening method. 5 6

Results and discussion 7

During this investigation positive mouse bioassay results were obtained in 4 out of 36 8 samples tested. Specifically, 3 samples out of 9 collected from the area of Halastra within 9 a two-year period from the onset of the DSP outbreak were found positive by the mouse 10 bioassay method. The two samples collected during the outbreak were toxic resulting in 11 death of mice within 85-90 min after the intra-peritoneal injection. The third positive 12 sample harvested from the area of Halastra was collected 15 months after the outbreak 13 when the mussel withdrawal was recalled. However, no sporadic cases or outbreak due to 14 DSP or similar gastrointestinal illness in humans were recorded during the period the DSP 15 positive sample was collected. 16 17 The fourth positive sample was harvested from the Amvrakikos Gulf in Northwestern 18 Greece, where no positive or suspect DSP cases have ever been recorded. Amvrakikos 19 Gulf is the nearest to Ioannina and like Thermaikos, it is shallow, calm, warm and thought 20 to be the most polluted gulf in the Ionian Sea, which is part of the Adriatic Sea, where 21 there are frequent reports for toxic blooms [START_REF] Boni | First cases of DSP in 15 the Northern Adriatic Sea[END_REF] There are a number of studies from different countries concerning DSP outbreaks, 5 sporadic cases or detection of toxins in a variety of seafood (Table I). The DSP outbreak 6 described in this paper is the first recorded in Greece and it was classified as a diarrheic 7 shellfish poisoning, based on clinical manifestations characteristic for DSP, the common 8 history of all patients reporting consumption of mussels, the absence of any known 9 pathogen in fecal and blood samples from all hospitalized patients, no detection of 10 antibodies to known pathogens, the subsequent analysis of mussels collected during the 11 outbreak and the detection of Dinophysis acuminata in the water of Thermaikos Gulf. The 12 actual number of the people affected cannot be precisely estimated, due to lack of 13 sufficient data. However, the number of 120 people needing urgent medical care within a 14 period of less than 7 days indicates a foodborne outbreak, of the group of seafood-borne 15 emerging diseases, the first recorded in Greece. . [Insert Table I about Chromatography -Mass Spectrometry, the presence of okadaic acid only (at levels 0.10 4 -0.20 µg/g) is confirmed. The hypersensitivity and the convulsions observed in mice 5 injected with the extract of the positive sample form Amvrakikos gulf, is indicative of the 6 simultaneous presence of an unknown lipophilic toxin along with okadaic acid and / or 7 DTX -2. Jumping before death, convulsions and short survival time is observed when 8 mice are injected i.p. with yessotoxin [START_REF] Ciminiello | Yessotoxin in mussels of the Northern Adriatic Sea[END_REF]. Although the presence of 9 yessotoxin in mussels has been reported in neighboring areas, it is quite improbable that 10 yessotoxins are the cause of these symptoms. Yessotoxins are poorly soluble in diethyl 11 ether and therefore probably could not co-elute with the other toxins (Ciminiello et al. 12 1997). Another possible explantation for the observed symptoms in this sample is that it 13 contained azaspiracid. After intraperitoneal injection of azaspiracid, mice exhibit 14 respiratory difficulties, spasms, paralysis of the limbs and death [START_REF] Ito | Multiple 12 organ damage caused by a new toxin azaspiracid, isolated from mussels produced 13 in[END_REF]. Still, the 15 presence of azaspiracid has not been proven by analytical methods in shellfish harvested 16 from the Mediterranean Sea [START_REF] James | Azaspiracid poisoning, the food-15 borne illness associated with shellfish consumption[END_REF]). 17 Microbiology Unit we were able to apply only the biological method (mouse bioassay). 13 The accumulation of algal toxins is one of the most critical problems in bivalve 14 aquaculture. Amongst the three most important categories of shellfish poisoning 15 (amnesic, diarrhoeic, paralytic), DSP is the least severe causing only gastrointestinal 16 symptoms. However, the accumulation of toxic algae in many bivalve producing areas has 17 a great economic impact as their appearance leads to the implementation of long term 18 bans on shellfish harvest, which is happening frequently at Thermaikos Gulf aquacultures 19 since the reported outbreak. The factors influencing the growth of the microalgal 20 All these factors are present in Thermaikos Gulf (North Aegean Sea), especially in the 1 Halastra site, where the constantly calm and warm waters favor mussel cultures. 2 However, the outfall of three large rivers in the gulf (Gallikos, Loudias and Axios), the 3 waste effluents of Greater Thessaloniki area (second largest city of Greece) and the 4 drainage of the plain of Central Macedonia (a plain with intensive agricultural activities), 5 make Thermaikos Gulf, ideal aquatic environment for the growth of microalgal population 6 and possible toxin production by dinoflagellates. Similar conditions exist at Amvrakikos 7 Gulf (North Ionian Sea), which is part of the South-central Adriatic Sea. Red tide blooms 8 are known to occur in the central Adriatic Sea since the 80's [START_REF] Boni | First cases of DSP in 15 the Northern Adriatic Sea[END_REF]). However, 9 toxic blooms associated with shellfish intoxication are becoming more frequent during the 10 last decade in this geographic area (Fattorusso et al. 1992

Introduction 1 2

 2 Diarrheic shellfish poisoning (DSP) is a toxic syndrome caused by the ingestion of 3 shellfish contaminated with algal toxins produced by marine dinoflagellates belonging to 4 Dinophysis spp. (D. fortii, D. acuta, D. acuminata, D. caudate, D.hastate, D. mitra, D. 5 rotundata, D. tripos) (Hallegraef 1997; Yasumoto 2000) and Prorocentrum spp. (P. lima, 6 P. maculosum, P. concavum and P. hoffmianum) (Van Dolah 2000). The DSP toxins 7 belong to a group of fat-soluble polyether compounds, including Okadaic acid (OA) and 8 the closely related Dinophysistoxins (DTX). Gastrointestinal effects have only been 9

  from scallops, clams, cockles, oysters and crabs (Gestal -Otero 2000; Vale and 18 Sampayo 2002; Jørgensen et al. 2005; Torgersen et al. 2005). In the present paper the 19 first DSP outbreak in Greece is reported, together with the laboratory findings from mussel 20 samples collected during the outbreak and the subsequent four years after the outbreak.

2 3mussel toxicity investigation 4 Collection of mussel samples 5 FromFood

 245 authorities, seafood harvesting is suspended when toxic dinoflaggelate 1 counts are higher than 200 cells/L. The January 2000 to January 2005, 36 batches (500 g each) of fresh mussels were 6 examined for DSP toxins contamination. The mussel samples (species Mytilus 7 galloprovincialis) were randomly collected from local seafood markets. Twenty samples 8 were collected from Thermaikos Gulf (Halastra) in Thessaloniki (Northern Greece) and 9 sixteen samples from Amvrakikos Gulf (Preveza) (Northwestern Greece) (Figure1). 10 [Insert Figure 1 about here] Two out of the 36 samples were sent to the Food 11 Microbiology Unit of the Microbiology Department; University of Ioannina from colleagues 12 at the Hospital for Infectious Diseases in Thessaloniki during the DSP outbreak. These 13 samples were harvested from the Halastra area of Thermaikos gulf at the 20 January 14 2000. Samples after April 2001 until January 2005 were collected every 3 months. All 15 fresh mussel samples forwarded to our lab were deep-frozen (-30 o C) immediately after 16 collection and were transported by airplane (flight duration 40 min) in well insulated boxes 17 to the Food Microbiology Unit, within less than 24 hours after collection and upon arrival 18 were stored at -30 o C until testing. The samples were tested within 7 days time after their 19 arrival in our laboratory. The samples were defrosted at room temperature and were 20 thoroughly cleaned under a stream of tap water after external materials were brushed off 21 the shells. From each sample, 200 g of flesh was homogenized in a blending mixer and a 22 portion of 100 g was weighed using an Ohaus model 1500D balance (Ohaus Co, P.O. 23 Box 2033 19A Chapin Road Pine Brook, NJ 07058) prior to extraction. 24 25 Page 7 of 28 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Extraction of DSP toxins 1

Page 8 Food

 8 of 28 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk and respiratory distress and death. According to the Decision 2002/225/EC, 1

here] 16 17 19 (Food

 1619 The mouse bioassays performed during the outbreak are indicative of the presence of 18 lipophilic toxins. This finding combined with the increased concentration of D. acuminata Koukaras and Nikolaidis 2002), which is the most common DSP producing species in 20 Europe (Kumagai et al. 1996; Morono et al. 2002), indicates okadaic acid and / or DTX as 21 the potential cause of this outbreak. It should be noted that the third positive sample from 22 Thermaikos Gulf was collected in March 2001 when, according to other researchers 23 (Koukaras and Nikolaidis 2002) an increased number of Dinophysis spp. was observed. 24 Furthermore, the predominant DSP toxins detected in European mussels and especially 25 Page 10 of 28 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk of the Adriatic Sea are okadaic acid and DTX -2 (Draisci 1995; Pavela -1 Vranic et al. 2002). In a recent publication (Ciminiello et al. 2005) concerning 2 investigation of the toxin profile of Greek mussels M. galloprovinciallis by Liquid 3

18Food

  Detection of DSP is routinely based on mouse bioassays, HPLC methods and 19 immunoassays. It is generally acknowledged that biological tests suffer from considerable 20 variability and lack of sensitivity and because of increasing opposition to animal testing 21 many efforts have been made to detect DSP toxins by HPLC based methods. However, 22 the lack of availability of analytical standards and Certified Reference materials for all of 23 the DSP toxins currently precludes the sole use of chemical methods in routine monitoring 24 programmes. Additionally, the maximum level for DSP toxins set by regulatory authorities 25 Page 11 of 28 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk country to country. In general, levels of DSP toxins greater than 1 200 ng/g in shellfish are considered dangerous for human consumption (Hallegraef, 2 1997). The potential public health risks and the expanding geographical distribution of the 3 causative dinoflagellates have resulted in the issuing by the Commission of the European 4 Countries of the Decision 2002/225/EC of the 15 May 2002, which is an amendment of 5 the Council directive 91/492/EEC, which states that the maximum level of okadaic acid, 6 dinophysistoxins and pectenotoxins together, in bivalves intended for human consumption 7 (the whole body or any part edible separately) shall be 160 µg of okadaic acid 8 equivalents/kg. 9 10 To date, control measures to protect human health against DSP contaminated seafood 11 involve both biological and chemical procedures. At the time of the outbreak in the Food 12

Food

  population include: a) changes in water temperature favoring encysting or excysting of 21 dinoflagellates, b) decreased water salinity, c) increased concentrations of organic 22 substances, d) extended duration of sunlight and e) calm waters (Gestal -Otero 2000; 23 Caroppo et al. 2001). 24 25 Page 12 of 28 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

18 Page

 18 %) of patients with DSP symptoms seeking Hospital assistance 120 (100) Estimated No. of patients with DSP not seeking medical assistance >500 Laboratory tests No. of patients/no of faecal samples studied 120/120 No. of mussels' samples bought in the same venders as the cases during the onset of the outbreak 2 No. of unconsumed mussels (leftovers) tested None No. of mussels' samples bought in the same market as the cases after the end of the outbreak

  Yasumoto et al. 1980). Since then it has been recorded in several European 2 countries including France (van Egmond et al. 1993), Norway

	1985;
	F o r
	P
	e
	e r
	R
	e
	v i e
		w
		O n l
		y
	23	.
	24

  ; Yasumoto 7 et al. 1985; Yasumoto 2000; Kawabata 1989), India (Karunasagar et al. 1989), Australia, 8

  ; Fatorruso et al. 1992; Draisci 22 1995; Viviani et al. 1995; Ciminiello et al. 1997; 1998; Caroppo et al. 2001; Pavela -h prior to death, together with convulsions. The symptoms of 1 the mice following intraperitoneal injection are presented in Table III. [Insert Table III 2

	3	marked hypersensitivity 2 about here]
	4
		F o r
		P
		e
		e r
		R
		e
		v i e
		w
		O n l
		y
		10

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  ; Viviani et al. 1995; Draisci et 11 al. 1995; Ciminiello et al. 1997; Ciminiello et al. 1998; Draisci et al. 1999; Caroppo et al.

		F o r F o r F o r F o r F o r F o r
		P P P P P P
		e e e e e e
	12	e r e r e r e r e r e r
	13	2001; Pavela -Vranic et al. 2002).	R R R R R R
	14 15 16 17	e e e e e e DSP intoxication is still an under reported illness, which is attracting interest after notification of outbreaks (Gestal -Otero 2000; Yasumoto 1985; Vale and Sampayo 2002). v i e v i e v i e v i e v i e v i e Therefore knowledge of its existence along with the recently increasing reports of red tide w w w w w w
	18 19 20	blooms spreading from Europe to America to Australia should trigger a constant awareness for this emerging seafood zoonotic disease. O n l O n l O n l O n l O n l O n l y y y y y y

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	Page 21 of 28	Food Additives and Contaminants Food Additives and Contaminants
	Table I u Ciminiello et al. 2005 Table II. 1997 February-June Croatia k NR Number 2004 April Chile x NR v Wu et al. 2005 Characteristic	Mussels Mytilus chilensis	OA: 0.133 µg/g mg/kg DTX-2: 0.1 µg/g DTX-1:19 µg/100 g Values
	Year/month 1984 October w Madigan et al. 2005 Demographics 1997 March-November Norway l NR Country of cases Sweden a Several NR=Not Reported, ND=Not Determined, OA= Okadaic acid, DTX = Dinophysitoxin, Seafood Toxins determined Mussels >170 MU/kg OA/DST-1: 349 µg Blue mussels x Garcia et al. 2004. Male No. (%) 73 (60.8)
	hundred Several Norway b ~ 400 DSTs=Diarrhetic Shellfish Toxins 1984 -1985 1998 Female No. (%) Slovenia m hundred Mean age in years (range)	Blue mussels Blue mussels	ND -	47 (39.2) 35
	F o r P e e r F o r P e e r 1990 August Canada b 16 1990 France c 415 1990 Mexico Gulf d NR F o r P 1998 September-November Spain n NR 2000 January F o r F o r F o r Greece o >120 P P P e e r 2001 June-July Portugal p 7 e e e e r e r e r	Mussels Mussels imported from Denmark Oysters (Crassostrea virginica) Mussels Mussels (M. galloprovincialis) Clams, Razor clams, Green crabs	DTX-1: 100µg/100g OA: 170 µg/100 g OA: 18-1010 µg /100g ND OA: 0.162 µg/100g OA: 32 µg/100 g
	1991 January 2002 February	R e R e 120 Mussels R e Belgium q 403 Chile b Blue mussels R R R imported from e e e	OA DTX -1 OA: 22.9 µg/100g
	1991-1994 2002 Summer	v i e w v i e w Several hundred Mussels (Mytilus edulis) v i e w Denmark Brown crabs v i e v i e v i e Norway r ~200 Ireland e (Cancer pagurus) w w w	OA: <0.7 µg/g DTX-2: 6.3 µg/g OA: 29 µg/100g DTX-2: 2 µg/100g
	1993 Summer-1994 Summer 1994 February 2002 September 2002 August-2004 March	Italy f Portugal g 18 NR Portugal s 32 Denmark t NR	O n l y O n l y Mussels (M. galloprovincialis) OA: 32-151 µg/100g DTX-1: 0-0.32 µg/100g Donax clams OA: 130 µg /100 g O n l y Blue mussels OA: 1328-1862 µg/100g Surf clams -Blue OA: 4.3-163.1 O n l O n l O n l mussels µg/100g y y y
	1994 June 2003 February	2 Greece u NR UK h	(Donax trunculus) Mussels galloprovincialis) Mussels (M.	OA OA: 0.10-0.20 µg/g
	1997 March 2003 May-October	UK i China v	49 NR	Mussels Clams, Scallops	OA: 25.3-36.7 OA: 3.2-17.5 µg/100g
				Gastropods	µg/100 g
	1997 June 2003 December	UK j Australia w NR 55	Mussels Oysters	-DSTs total: 0.253
			1 2 3 4 5

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk a Krogh et al. 1985. b Aune and Yndestad 1993. c Van Egmond et al. 2004. d Dickey et al. 1992. e Carmody et al. 1996 f Draisci et al. 1995. g Vale and Sampayo 1999. h Anon. 1994. i Scoging and Bahl 1998. j Anon. 1997. k Pavela -Vrančič et al. 2002. l Ramstad et al. 2001. m Mozetic and Bozic 2001. n Moroño et al. 2003. o Papadopoulou et al. (present study) p Vale and Sampayo 2002. q De Schrijver et al. 2002. r Torgersen et al. 2005 s Vale et al. 2003 t Jorgensen et al. 2005. Page 23 of 28 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

TABLE III .

 III

			Food Additives and Contaminants	Page 26 of 28
		Amvrakikos gulf
	Sampling Date 15/4/2002 Outbreak 15/1/2000 Outbreak 19/1/2000 15/4/2000 6/12/2000 19/1/2001 22/2/2001 23/3/2001 10/9/2001 20/7/2002-20/7/2005 20/12/2001	Place (one sample/place) Thermaikos Gulf Thermaikos Gulf Thermaikos Gulf Thermaikos Gulf Thermaikos Gulf Amvrakikos gulf Thermaikos Gulf Amvrakikos gulf Thermaikos Gulf Thermaikos Gulf Amvrakikos gulf F o r P F o r P e e r R e v i e w Symptoms observed No symptoms Apathy, weakness, difficulty in movement, respiratory distress, spastic contractions just before death Apathy, weakness, difficulty in movement, respiratory distress, spastic contractions just before death No symptoms No symptoms Apathy, weakness, difficulty in movement, hypersensitivity, respiratory distress No symptoms Apathy, weakness, difficulty in movement, respiratory distress No symptoms e e r R O n l y > 48 h Median Mouse survival Time (0/3) (number of mice dead/number of mice tested) 1 h 25 min (3/3) 1 h 30 min (3/3) > 48 h (0/3) > 48 h (0/3) 9 h (3/3) > 48 h (0/3) 5 h (3/3) > 48 h (0/3) e Thermaikos Gulf Amvrakikos gulf No symptoms > 48 h (0/3) Thermaikos Gulf No symptoms > 48 h (0/3) v i e	Negative DSP toxins Result Positive Positive Negative Negative Positive Negative Positive (0/3) Negative Negative
			w
	FIGURE 1.	7 l O n
			y

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

Vranic et al. 2002). Also, it should be noted that the mice injected with the extract of the

homoyessotoxin in dinoflagellates of the Adriatic Sea. Toxicon 37:1187 -1193.

Fattorusso E, Ciminiello P, Constantino V, Magno S, Mangoni A, Poletti R, Pompei M,

Viviani R. 1992. Okadaic acid in mussels of Adriatic Sea. Marine Pollution Bulletin

24: 234-237.

Page 15 of 28 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

along the coast of Karnataka state (India). In: Okaichi T, Anderson DM, Nemoto T,

editors. Red Tides: Biology, Environmental Science and Toxicology. Elsevier,

Amsterdam. p. 61 -64

Van Egmond HP, Aune T, Lassus P, Speijers GJA, Waldock M. 1993. Paralytic and

Journal Natural Toxins 2: 41-83.

Tetrahedron 41: 1019 -1025.

Yasumoto T, Oshima Y, Sugawara W, Fukuyo Y, Oguri H, Igarishi T, Fujita N. 1980.

poisoning. Bulletin of the Japanese Society of Sciences Fish. 46: 1405 -1411.

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

Acknowledgments 21

The present study was carried out during a bilateral joint research project on Marine

the joint project was financially supported by grants from the


Cristina Blasco

G Font

Y Picó

Solid-phase microextraction-liquid chromatography -mass spectrometry applied to the analysis of insecticides in honey*

Keywords: Solid-Phase Micro Extraction, Liquid Chromatography-Mass Spectrometry, Organophosphorus insecticides, Carbamate insecticides, Honey, Food Analysis

Solid-phase microextraction-liquid chromatography-mass spectrometry applied to the analysis of insecticides in honey

Introduction

Honey is produced when the nectar and sweet deposits from plants are gathered, modified and stored in the honeycomb by honey bees. The definition of honey stipulates a pure product absent of residues, which include, but are not limited to pesticide residues. However, the legislation is complex and is not always clear and consistent [START_REF] Herrera | Determination of pesticides and PCBs in honey by solid-phase extraction cleanup followed by gas chromatography with electron-capture and nitrogenphosphorus detection[END_REF]. The European Union (EU) and United States (USA) have a very similar position, up to now, only three Maximum Residue Limits (MRLs) have been defined for acaricides amitraz, coumaphos, and cymiazole , which are directly applied within the beehive to control the parasitic mite Varroa jacobsoni (Commission Regulation (EC) No.2377/90 1990; US Environmental Protection Agency (EPA) 2005).

There is no legislation relating to other pesticide residue limits in honey but countries such as Germany, The Netherlands and Italy have established those MRLs. In some cases, these tolerances are specific to particular substances and in other cases, there are a general limit of 10 µg kg -1 or 50 µg kg -1 for any of these residues [START_REF] Piro | The EU legislation for honey residue control[END_REF]. Consumers are extremely sensitive to the issue of pesticide residues in food, particularly, for a product such as honey that is regarded as natural and pure should be as free from residues as possible. In addition, according to the Directive 96/23/EC, which contains guidelines for residues control in animals and in their products (honey included), there is a defined set of substance categories that must be monitored comprising, among others, organophosphorus compounds (Council Directive 96/23/EC 1996). In this way, honey and other bee products can concentrate pesticide residues used in the area close to the beehives because bees travel long distances to collect nectar and pollen, constituting excellent bioindicators of the environmental contamination in the surrounding area [START_REF] Ghini | Occurrence and distribution of pesticides in the provence of Bolognia, Italy, using honeybees as bioindicators[END_REF]. The determination of pesticides is generally performed by mass spectrometry (MS) coupled to gas (GC-MS) or liquid chromatography (LC-MS) because it offers the possibility of confirming the identity of their residues (Fernandez et al. 2002a;[START_REF] Mastovska | Practical approaches to fast gas chromatography-mass spectrometry[END_REF][START_REF] Hogendoorn | Recent and future developments of liquid chromatography in pesticide trace analysis[END_REF][START_REF] Van Der Hoff | Trace analysis of pesticides by gas chromatography[END_REF] Zoonen 1999; [START_REF] Pico | Environmental and food applications of LC-tandem mass spectrometry in pesticide-residue analysis: An overview[END_REF]). The development of chromatographic (gas and/or liquid) methods implies optimization of a wide variety of preliminary steps, e.g. isolation of the analytes and interfering compounds removal (clean up/extraction) prior the separation. Liquid-solid extraction (LSE) (Blasco et al. 2004b;[START_REF] Jimenez | Capillary gas chromatography with mass spectrometric and atomic emission detection for characterization and monitoring chlordimeform degradation in honey[END_REF], supercritical fluid extraction (SFE) [START_REF] Rissato | Supercritical fluid extraction for pesticide multiresidue analysis in honey: determination by gas chromatography with electron-capture and mass spectrometry detection[END_REF]), solid-phase extraction (SPE) [START_REF] Herrera | Determination of pesticides and PCBs in honey by solid-phase extraction cleanup followed by gas chromatography with electron-capture and nitrogenphosphorus detection[END_REF][START_REF] Albero | Analysis of pesticides in honey by solid-phase extraction and gas chromatography -Mass spectrometry[END_REF][START_REF] Cossu | Monitoring of pyrethroid residues in Sardinian honey by solid phase extraction and high-performance liquid chromatography[END_REF][START_REF] Blasco | Assessment of pesticide residues in honey samples from Portugal and Spain[END_REF]Fernandez et al. 2002b) matrix solid-phase dispersion (MSPD) [START_REF] Albero | Multiresidue determination of pesticides in honey by matrix solid-phase dispersion and gas chromatography with electroncapture detection[END_REF], solid-phase microextraction (SPME) [START_REF] Volante | A SPME-GC-MS approach for antivarroa and pesticide residues analysis in honey[END_REF][START_REF] Yu | Development of new solid-phase microextraction fibers by sol-gel technology for the determination of organophosphorus pesticide multiresidues in food[END_REF]Blasco et al. 2004a) and stir-bar sorptive extraction (SBSE) (Blasco et al. 2004a) are the most common techniques used to obtain suitable extracts for analysis.

SPME is frequently chosen by many analysts as an efficient technique for sample preparation because it is ideally suited for coupling with MS, constituting one of the tools that helps to improve the usefulness of mass spectrometric techniques for screening and quantification of pesticides in food samples [START_REF] Zambonin | Coupling solid-phase microextraction to liquid chromatography. A review[END_REF][START_REF] Rissato | Supercritical fluid extraction for pesticide multiresidue analysis in honey: determination by gas chromatography with electron-capture and mass spectrometry detection[END_REF][START_REF] Vas | Solid-phase microextraction: a powerful sample preparation tool prior to mass spectrometric analysis[END_REF]. Most SPME procedures to determine pesticides in honey have been performed in combination with gas chromatography (GC) where, after the extraction step, the analytes are thermally desorbed into the injector of the chromatograph [START_REF] Volante | A SPME-GC-MS approach for antivarroa and pesticide residues analysis in honey[END_REF][START_REF] Yu | Development of new solid-phase microextraction fibers by sol-gel technology for the determination of organophosphorus pesticide multiresidues in food[END_REF]). The SPME of antivarroa residues using 100 µm thickness polydimethylsiloxane (PDMS) fibers was discussed (Volante et ) demonstrating that it can be a useful tool for the rapid screening of residues in honey. Organophosphorus insecticide residues were extracted using five fiber coatings (30 µm thickness PDMS, 85 µm thickness polyacrilate (PA), 65 µm polydimethylsiloxane divinylbencene (PDMS/DVB) and sol-gel-derived bisbenzocrown ether/hydroxyl-terminated silicone oil (OH-TSO)) and found that the OH-TSO showed excellent extraction characteristic for organophosphorus [START_REF] Yu | Development of new solid-phase microextraction fibers by sol-gel technology for the determination of organophosphorus pesticide multiresidues in food[END_REF]. SPME-GC is, however, generally limited to the analysis of volatile and thermally stable compounds [START_REF] Piro | The EU legislation for honey residue control[END_REF]Council Directive 96/23/EC 1996). A typical alternative to overcome these limits is interfaced SPME with liquid chromatography (LC) [START_REF] Vas | Solid-phase microextraction: a powerful sample preparation tool prior to mass spectrometric analysis[END_REF]. The desorption step marks the difference between SPME-GC and SPME-LC because for the latter it is carried out by liquid removal. SPME and SBSE in combination with LC-MS were evaluated to determine six OPPs in honey, desorbing the compounds in 1 mL of methanol and injecting them in the LC. SBSE showed higher concentration capability and greater accuracy and sensitivity (Blasco et al. 2004a). However, SPME could present two advantages that have not been exploited to determine residues in honey yet. One would be the use of a more proper fiber coating (could markedly increase the method efficiency) and the other would be the capability of desorbing the analytes directly in the LC system (could improve sensitivity more than 100 hundred times).

The present study was aimed at checking the reliability of these two possibilities. For this purpose, an SPME-LC-MS method for the simultaneous determination of eleven organophosphorus and one carbamate pesticides (bromophos ethyl, chlorpyrifos methyl, chlorpyrifos ethyl, diazinon, fenoxycarb, fonofos, phenthoate, phosalone, pirimiphos methyl, profenofos, pyrazophos, and temephos) in honey was developed using SPME were optimized. Finally, the potential of the method was demonstrated by the analysis of honey samples. As target analytes, organophosphorus and carbamate insecticides were selected instead of the acaricides directly applied on beehives because the formers were used widely in the agricultural crops. The presence of these insecticides establishes how much honey is affected by environmental contamination. The selection of the particular organophosphorus (and one carbamate) was carried out in basis of their application to the surrounding area of the beehives. Their intense application was established after a survey among cooperatives, farmers and pesticides vendors of the geographical area.

Experimental

Chemicals

Pesticide standards (bromophos ethyl, chlorpyrifos methyl, chlorpyrifos ethyl, diazinon, fenoxycarb, fonofos, phenthoate, phosalone, pirimiphos methyl, profenofos, pyrazophos, and temephos) were purchased from Sigma-Aldrich (Madrid, Spain). HPLC-grade methanol was purchased from Merck (Darmstadt, Germany) and sodium chloride (analysis grade) was supplied by Scharlau (Barcelona, Spain). The individual stock solutions were prepared in methanol at a concentration of 1000 mg L -1 and stored at 4ºC. Standard working solutions at various concentrations were daily prepared in ultrapure water obtained from Milli-Q SP Reagent Water System (Millipore, Bedford, MA, USA).

Chromatographic and detection conditions

The mobile phase was a methanol/water gradient at a flow-rate of 0.7 mL min -1 . The gradient was 70% methanol from 0 to 15 min, followed by a linear gradient to 90% from 15 to 20 min, then increased again linearly to 95 % from 20 min to 25 min, and finally, maintained at 95% methanol from 25 to 30 min and re-equilibrates to the initial conditions in 10 min.

Optimum operating parameters of the APCI interface in NI mode were: vaporizer temperature, 450 ºC; nebulizer gas, nitrogen at a pressure of 60 psi; drying gas, also nitrogen, at a flow rate of 4 L min -1 and temperature of 350 ºC; capillary voltage, 3500 V; and corona current 25 µA. The chromatograms were recorded in full-scan and selected-ion monitoring (SIM) modes. Full scan conditions were m/z ranged from 50 to I.

Solid-phase microextraction (SPME)

A SPME holder assembly for automated sampling and several replaceable fibers were obtained from Supelco (Bellefonte, PA, USA). Silica fibers with four different coatings, 85 µm thick polyacrilate (PA) film, 7, 30 and 100 µm polydimethylsiloxane (PDMS), 60 µm polydimethylsiloxane/divinylbenzene (PDMS/DVB) and 50 µm carbowax/templated resins (CW/TPR) were employed for comparative studies. The new fibers were conditioned in methanol by stirring for 30 min and the used ones were cleaned in methanol by stirring for 15 min before extraction. The SPME device has been described elsewhere [START_REF] Zambonin | Coupling solid-phase microextraction to liquid chromatography. A review[END_REF][START_REF] Wardencki | A review of theoretical and practical aspects of solid-phase microextraction in food analysis[END_REF][START_REF] Vas | Solid-phase microextraction: a powerful sample preparation tool prior to mass spectrometric analysis[END_REF].

Spiked samples were prepared by adding 1.5 g of honey with different volumes of the working solutions into 6 mL clear vials (Análisis Vinicos, Tomelloso, Spain) and by keeping for 1 h at room temperature to ensure the appropriate distribution of the insecticides in honey. Then, the honey was added with 5 mL of water and stirring for 10 min to appropriately dissolve the honey. The extraction was carried out for 120 min under magnetic stirring in order to improve mass transfer to the aqueous sample into the fiber coating. Desorption was performed in static mode placing the fiber into the desorption chamber, which has been previously filled with methanol-water (70:30 v/v) for 15 min. To avoid possible memory effects, the fiber was left continuously exposed to the mobile phase flow during the analysis. The injection was recorder programming the autosampler for a blank run, and using the handle of the SPME interface as external start.

Results and discussion

Organophosphorus Analysis

The twelve organophosphorus insecticides studied can be separated and simultaneously detected by liquid chromatography-mass spectrometry using atmospheric pressure chemical ionization (APCI) and negative ionization mode (NI). In many application dealing with pesticide residue analysis, electrospray in positive ionization mode was used [START_REF] Alder | Residue analysis of 500 high priority pesticides: Better by GC or LC-MS/MS?[END_REF][START_REF] Pico | Environmental and food applications of LC-tandem mass spectrometry in pesticide-residue analysis: An overview[END_REF]). However, the literature shows that the best sensitivity is obtained either by electrospray or APCI, depending on the make of the instrument and design of the ionization source [START_REF] Pico | Environmental and food applications of LC-tandem mass spectrometry in pesticide-residue analysis: An overview[END_REF]. Different studies that compares the behaviour of different pesticides, including organophosphorus in both ionization source, using the same design as that applied in the present study show that organophosphorus pesticides are more sensitive by APCI than ES (Fernandez et al. 2002b;[START_REF] Blasco | Assessment of pesticide residues in honey samples from Portugal and Spain[END_REF][START_REF] Fernandez | Determination of carbamate residues in fruits and vegetables by matrix solid-phase dispersion and liquid chromatography-mass spectrometry[END_REF][START_REF] Fernandez | Simultaneous determination of carbamate and organophosphorus pesticides in honeybees by liquid chromatography-mass spectrometry[END_REF][START_REF] Thurman | Choosing between atmospheric pressure chemical ionization and electrospray ionization interfaces for the HPLC/MS analysis of pesticides[END_REF]. The same studies compared sensitivity in positive and/or negative ionization modes showing that carbamates were not detected with the ESI source in the NI mode at acceptable levels [START_REF] Fernandez | Determination of carbamate residues in fruits and vegetables by matrix solid-phase dispersion and liquid chromatography-mass spectrometry[END_REF][START_REF] Fernandez | Simultaneous determination of carbamate and organophosphorus pesticides in honeybees by liquid chromatography-mass spectrometry[END_REF]. Organophosphorus insecticides ionized in both PI and NI. The most sensitive ionization mode depends on the structure of each particular compound (Fernandez et al. 2002b;[START_REF] Blasco | Assessment of pesticide residues in honey samples from Portugal and Spain[END_REF] [START_REF] Thurman | Choosing between atmospheric pressure chemical ionization and electrospray ionization interfaces for the HPLC/MS analysis of pesticides[END_REF]). These previous works cover the pesticides selected in the present study and clearly shows that best agreement to determine simultaneously all these compounds with proper sensitivity is the negative ionization mode (Fernandez et al. 2002b;[START_REF] Blasco | Assessment of pesticide residues in honey samples from Portugal and Spain[END_REF][START_REF] Fernandez | Determination of carbamate residues in fruits and vegetables by matrix solid-phase dispersion and liquid chromatography-mass spectrometry[END_REF][START_REF] Fernandez | Simultaneous determination of carbamate and organophosphorus pesticides in honeybees by liquid chromatography-mass spectrometry[END_REF].

Few publications involving SPME for the purpose considered here have been found in the literature [START_REF] Yu | Development of new solid-phase microextraction fibers by sol-gel technology for the determination of organophosphorus pesticide multiresidues in food[END_REF][START_REF] Volante | A SPME-GC-MS approach for antivarroa and pesticide residues analysis in honey[END_REF][START_REF] Capillo | Solid-phase microextraction and gas chromatography with atomic emission detection for multiresidue determination of pesticides in honey[END_REF], despite the fact that honey is an obvious subject for using SPME because its solubility in water. None of the above articles used LC-MS, even through the last reviews published on the subject clearly demonstrate that LC is currently competing with GC for the status of reference analytical technique to determine pesticide residues. LC has been found be able to determine more pesticides and at much higher sensitivity than GC. This comment is also valid for organophosphorus pesticides, which are most often analyzed by GC methods up to now [START_REF] Alder | Residue analysis of 500 high priority pesticides: Better by GC or LC-MS/MS?[END_REF].

The results obtained are in agreement with other LC-MS studies of organophosphorus pesticides, which demonstrated that despite the organophosphorus pesticides are compounds that can be determined by gas chromatography, they can be also identified and quantified by LC-MS with almost the same sensitivity [START_REF] Hogendoorn | Recent and future developments of liquid chromatography in pesticide trace analysis[END_REF][START_REF] Pico | Environmental and food applications of LC-tandem mass spectrometry in pesticide-residue analysis: An overview[END_REF][START_REF] Cossu | Monitoring of pyrethroid residues in Sardinian honey by solid phase extraction and high-performance liquid chromatography[END_REF]Fernandez et al. 2002b;Blasco et al. 2004a).

The ions used in selected ion monitoring (SIM) have been reported in Table I. The most intense ion was used for quantification and the second and third ion for confirmation, to meet the criteria established by the EU to confirm the identity of a pesticide (European directly desorbed from the fiber displayed an abundance of the ions signals within the reported criterion. However, it should be noted that differences, although within the confirmation criteria, were more accentuated for the confirmation ions with low relative abundance.

Optimization of the SPME sample treatment

Conditions for the SPME absorption of the pesticides were optimized in honey samples spiked at 0.1 µg g -1 of each studied pesticide and desorbed off-line. Initial conditions were 1 g of honey samples was dissolve in 5 mL of water by stirring for 10 min. The water volume was the only condition that was maintained through the experiments, because it is the minimum volume to be able to dissolve properly the sample. Then, the fiber was immersed in the sample for 90 min. The SPME-LC off-line desorption of pesticides in honey samples was achieved by exposing the fiber to 1 mL of methanol for 15 min in an ultrasonic bath. This volume enables the fiber coating to be immersed completely in the solvent.

Extraction efficiencies of three PDMS of different thick coatings, PA, CW/TPR, and PDMS/DVB fibers, were evaluated and compared in order to select the best fiber. The showing that organophosphorus insecticides were extracted to a different extend depending on the nature of the polymeric coating. The PDMS/DVB fiber was the most effective in concentrating pesticides, and PA coating was the less effective. The addition of saturate NaCl (up to 350 mg mL -1 ) to the extraction solution, which changes the ionic strength of the aqueous solution and may change the partitioning of the analytes into the fiber, was evaluated for the six fibers. There was no measurable effect on the recoveries, except a modest improvement of the extraction efficiency of PA for profenofos. The PDMS/DVD coating was selected for further experiments because it gave satisfactory extraction efficiencies for all the pesticides which were studied and the adjustment of the ionic strength was not further considered.

Suspended matter as well as dissolved compounds could be responsible for interference on sample extraction by SPME by the absorption of analytes, forming micelles and/or interfering with diffusion (hindering to analytes to reach the fiber). The results of recovery test using different amounts of sample are shown in Fig. 2. The recovery was much better using low amounts of samples, but acceptable values (between 19 and 85 %) are achieved using 1.5 g of sample. This was a good compromise between recovery and amount of sample to be processed, which should be great for a trace analysis to obtain appropriate limits of detection. partitioning has been correlated with octanol-water distribution coefficients (Kow); log Kow values ranged from 3.3 (diazinon) to 6.5 (bromophos ethyl). However, honey matrix decreases the extraction recovery, which indicates a disturbance of the extraction process. In this way, the experimental results obtained for extraction recovery do not correspond with log Kow values (i.e. diazinon vs bromophos-ethyl). One reported explanation [START_REF] Yu | Development of new solid-phase microextraction fibers by sol-gel technology for the determination of organophosphorus pesticide multiresidues in food[END_REF]) is that compared with water, diffusion coefficients are smaller in the more viscous sugar solutions. However, the lower diffusion coefficients would affect the rate for reaching equilibrium but not the distribution between sample and fiber. Another more realistic explanation is that suspended matter and dissolved compound from the matrix block the analytes by different mechanisms decreasing the recoveries, as has been discussed in the optimization of the amount of sample [START_REF] Albero | Analysis of pesticides in honey by solid-phase extraction and gas chromatography -Mass spectrometry[END_REF][START_REF] Albero | Multiresidue determination of pesticides in honey by matrix solid-phase dispersion and gas chromatography with electroncapture detection[END_REF]Blasco et al. 2004a).

The desorption conditions by the SPME-LC commercial interface were optimized to obtain the best possible detection limits. The optimization of the desorption conditions is the most critical step in SPME-LC. Dynamic desorption mode, which ensures quantitative recoveries, causes a significant increase of peak width and peak asymmetry compared to conventional loop injection (20 µL), deteriorating the chromatographic efficiency and resolution. Thus, a static desorption technique was evaluated as a possible alternative. Desorption was performed with different methanol water mixtures. 15 composition of the mobile phase containing the optimal proportion of methanol. The fiber was soaked in the static mobile phase contained in the desorption chamber (60 µL volume) for a variable period of time before injection into the LC column. The best conditions were reached after 15 min of static desorption followed by injection into the mobile phase stream. The peak shape observed for conventional loop injection was now quite well preserved.

Performance of the SPME-LC-MS method

Recovery, repeatability, and detection and quantification limits using fortified honey samples are reported in Table II. Recoveries of the 12 organophosphorus insecticides were in the range of 19-92 % (n=5). The intraday precision was better than 19 % at the LOQ levels and between 6-14 % at 10 LOQ levels. Limits of detection (LODs) were between 0.001 and 0.1 µg g -1 and limits of quantification (LOQs) between 0.005 and 0.5 µg g -1 .LODs and LOQs have been dictated by the signal intensity of the less abundant ion to ensure a proper confirmation of the pesticide identity prior to quantify it.

The whole analytical procedure using SPME combined with LC-MS was tested for linearity in the range of LOQs to 100 times LOQs. As shown in Table III, a linear relationship was obtained for each pesticide in this range (six points calibration) from water and honey by SPME-LC. Great differences in the regression equations between water and honey were found, which is reasonable since the differences in the recovery between water and honey are also important.

The proposed method showed appropriate linearity, precision (relative standard deviation (RSDs) less than 19 %), and high sensitivity, in-line with the international SPME of pesticides in honey samples showed, in most cases, low recoveries that can be explained by the complexity of the matrix and the interactions between analytes and matrix components, as it has previously been commented. SPME of organophosphorus pesticides in fruit and fruit juices without clarify gave small recoveries (<10 %) [START_REF] Yu | Development of new solid-phase microextraction fibers by sol-gel technology for the determination of organophosphorus pesticide multiresidues in food[END_REF]Blasco et al. 2004a). Low recovery does not necessarily imply insufficient precision of the method. However, the matrix variability forces to construct the calibration curve by using blank honey samples (free from the analytes to be quantified) spiked with analytical standards of the target pesticides at different concentrations.

The optimized procedure was applied to the analysis of several real samples of different origin. The samples were analyzed in triplicate and each of the replicates was injected twice. Table IV shows the pesticides and the concentrations found. Of the 26 samples analyzed, pesticide residues were detected in six samples (25 %). Diazinon was the most frequently detected pesticide. The detection of several of the investigated compounds in real samples confirms the need for monitoring these residues in honey and other bee products. These finding are in concordance with the most recent studies of pesticide residues content in Spanish honey samples (Blasco et al. 2004b;[START_REF] Jimenez | Capillary gas chromatography with mass spectrometric and atomic emission detection for characterization and monitoring chlordimeform degradation in honey[END_REF][START_REF] Herrera | Determination of pesticides and PCBs in honey by solid-phase extraction cleanup followed by gas chromatography with electron-capture and nitrogenphosphorus detection[END_REF][START_REF] Albero | Analysis of pesticides in honey by solid-phase extraction and gas chromatography -Mass spectrometry[END_REF].

Comparison of methods

For analyzing pesticide residues in honey, SPME has been already tested in a few studies with recoveries comparable to those obtained in the present study [START_REF] Capillo | Solid-phase microextraction and gas chromatography with atomic emission detection for multiresidue determination of pesticides in honey[END_REF][START_REF] Volante | A SPME-GC-MS approach for antivarroa and pesticide residues analysis in honey[END_REF][START_REF] Yu | Development of new solid-phase microextraction fibers by sol-gel technology for the determination of organophosphorus pesticide multiresidues in food[END_REF]). The significantly lower recoveries in honey solution compared to water were also noted in these studies [START_REF] Capillo | Solid-phase microextraction and gas chromatography with atomic emission detection for multiresidue determination of pesticides in honey[END_REF][START_REF] Volante | A SPME-GC-MS approach for antivarroa and pesticide residues analysis in honey[END_REF][START_REF] Yu | Development of new solid-phase microextraction fibers by sol-gel technology for the determination of organophosphorus pesticide multiresidues in food[END_REF]). Traditional sample treatment methods are almost invariably based on solvent and solid-phase extraction followed, sometimes, by various cleanup steps. These method provided higher recoveries [START_REF] Blasco | Assessment of pesticide residues in honey samples from Portugal and Spain[END_REF]Fernandez et al. 2002b;Fernandez et al. 2002a) but SPME is a relatively easy and fast technique for the analysis of pesticides present in trace amounts and presents the advantage of avoiding the use of organic solvents [START_REF] Capillo | Solid-phase microextraction and gas chromatography with atomic emission detection for multiresidue determination of pesticides in honey[END_REF][START_REF] Volante | A SPME-GC-MS approach for antivarroa and pesticide residues analysis in honey[END_REF][START_REF] Yu | Development of new solid-phase microextraction fibers by sol-gel technology for the determination of organophosphorus pesticide multiresidues in food[END_REF]).

Most SPME were carried out in combination with GC with different detectors [START_REF] Capillo | Solid-phase microextraction and gas chromatography with atomic emission detection for multiresidue determination of pesticides in honey[END_REF][START_REF] Yu | Development of new solid-phase microextraction fibers by sol-gel technology for the determination of organophosphorus pesticide multiresidues in food[END_REF][START_REF] Volante | A SPME-GC-MS approach for antivarroa and pesticide residues analysis in honey[END_REF]). However, the method described here is based on separation and determination by LC-MS, which allows to increase the number of pesticides analyzable because LC-MS is able to determine any compound independently of its volatility and thermolability [START_REF] Pico | Environmental and food applications of LC-tandem mass spectrometry in pesticide-residue analysis: An overview[END_REF]).

There is one previous study that compares SBSE and SPME for determining six organophosphorus pesticide residues. In that study, SPME enrichment was performed 18 using a poly(dimethylsiloxane) (PDMS) fiber to compare both techniques in the same conditions (Blasco et al. 2004a). However, PDMS is not the optimum coating to adsorb organophosphorus pesticides because recoveries are between 3.2 to 7.6 %. In the present work, the fiber coating selected was tested and the method extracts substantially large amount of the analytes from honey (from 19 to 92 %). In addition, the previous study desorbs the analytes from the fiber in 1 mL of methanol and 5 µL of this extract were injected in the LC-MS systems, attaining LOQs between 0.8 and 2 µg g -1 . The present study directly desorbs all the analytes adsorbed in the fiber in the LC systems by means of a commercial six port-valve device. This system allows to obtain LOQs from 0.005 to 0.5 µg g -1 , which is around hundred times lower for most of the studied compounds. These low LOQs document the effectiveness of this method in measuring trace levels of pesticide residues.

Conclusions

A method combining SPME with LC-MS that directly desorbs the analytes from the fiber to the LC system by means of a commercial device has been developed for first time.A PDMS/DVB coated fiber ensures good extraction efficiency, whereas the static desorption mode ensures the best compromise in terms of analyte transfer efficiency and preservation of chromatographic efficiency. When SPME and analysis by LC-MS are combined, a highly sensitive and selective analytical procedure is obtained for multiresidue screening of pesticides in complex food samples such as honey. Additional advantages, derived from easy removal of sugar and extraction solvents providing enhanced protection to the LC-MS system, are also characteristics of the proposed procedure.

  -MS by means of the commercially standard six port LC injector, with a special fiber-desorption chamber. The SPME conditions (extraction and desorption)

  (Supelco, Bellefonte, PA, USA) consisted of a standard six-port reodyne valve equipped with a desorption chamber (total volume = 60 µL), installed between the autosampler of the LC and the analytical column. The LC system, a Hewlett Packard (Palo Alto, CA, USA) HP-1100 Series LC-MSD system, consisted of a HP-1100 autosampler with 200 µL loop, a G1312 binary pump, a Luna C 18 column (250 x 4.6 mm I.D, particle size 5 µm) protected by a Securityguard cartridge C 18 (4 x 2 mm I.D), both from Phenomenex (Madrid, Spain), and diode array UV-Vis detector that can be coupled in series to a single quadrupole MS analyzer with an APCI interface usable in either positive ionization (PI) or negative ionization (NI) modes, controlled by HP Chemstation software version A.06.01.

  a scan time of 0.75s. Time-scheduled SIM using five windows was developed, as it is shown in Table

  samples were taken from local markets of Valencia, those samples were from different floral origins, thyme, multi flowers, rosemary, heather, lavender, orange blossom, lemon, acorn and eucalyptus. Honeys were locally produced. These samples were stored in their original containers (always glass jars) at room temperature in a dark place.

  Health and Consumer Protection 2004). According to this criterion, the presence of a target pesticide in a given matrix is demonstrated if, compared with a reference standard analyzed in the same experimental conditions, (i) the LC retention time agrees within 2 %, and (ii) the abundance of signals of quantification ion plus two confirmation ions agree within 20 %. Fenoxycarb and temephos gave an in-source collision induced dissociation spectra (CID) with only one confirmation ion. The in-source CID spectra of pesticides extracted from honey and

  of the target analytes on each fiber are reported in Fig.1,

Fig. 3

 3 Fig.3shows the extraction efficiency profiles obtained by plotting the peak area versus

Fig. 4

 4 Fig. 4 displays the dynamic and static desorption as well as the effect of methanol

  (European Commission Directorate General Health and Consumer Protection 2004; European Commission Directorate General Health and Consumer Protection 2000).

  Fig. 5 illustrates a typical chromatogram of an unspiked honey sample, a spiked honey

  Figure captions.

Figure 1 .

 1 Figure 1. Relative extraction efficiencies (recovery %) of the studied insecticides by

Figure 2 .

 2 Figure 2. Relative extraction efficiencies (recovery %) of the studied insecticides using

Figure 3 .

 3 Figure 3. Variation of the peak areas with the extraction time using the PDMS/DVB

Figure 4 .

 4 Figure 4. Chromatograms obtained from different desorption modes and mixtures. (A)

Figure 5 .

 5 Figure 5. Chromatograms of (A) honey spiked at LOQ levels, (B) unspiked honey that does not contain any of the studied pesticides, and (C) sample that contains 17.2 ng g -1

FFigure 1 .

 1 Figure1. Relative extraction efficiencies (recovery %) of the studied insecticides by each fiber(1 g honey/5 mL water).

FoodFigure 2 .Figure 3 .Figure 4 .Figure 5 .

 2345 Figure 2. Relative extraction efficiencies (recovery %) of the studied insecticides by different amount of honey using the PDMS/DVB fiber.

  ;

	F o r
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Table I .

 I Time scheduled SIM conditions for monitoring the studied insecticides.

			Food Additives and Contaminants
	Pesticide	Time	Compound	Quantification	Confirmation		Fragmentor	Dwell
		window	retention		ion, m/z			time
		(min)	time (min)		(relative	ions,	m/z	(V)	(ms)
				abundance)
						(relative
						abundance)
	Fenoxycarb Phenthoate Fonofos	F o r 0-18 16.154 16.796 0-22 19.016 F o r		185 (100) 319 (100) 153 (100)	300 (15) 110 (70), 157 (12) 137 (52), 109	40 60	199 199 98
	Diazinon Phosalone Pyrazophos		P e e r 19.016 P 20.317 e 21.215 e r	275 (100) 338 (100) 372 (100)	(68) 159 (40), 151 (40) 185 (80), 142 (40) 157 (24), 125 (62)	98 98 98
	Chlorpyrifos methyl Profenofos Pirimiphos ethyl Temephos Bromophos ethyl Chlorpyrifos ethyl	22-24 24-25 25-30	22.735 24.559 25.589 26.050 26.873 27.228	R e v i e w 302 (100) 304 (66), 141 (70) 207 (100) 343 (50), 345 (63) R 304 (100) 180 (50), 169 (10) e 451 (100) 217 (17) 351 (100) 363 (30), 239 v i e (30) 330 (100) 332 (66), 169 (73 ) w	60 40 70	400 400 98 98 98 98
						O n l O n l
						y y
								31 32

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Table II .

 II Performance of the SPME-LC-MS method for the studied insecticides in honey samples LODs (S/N=3) were calculated considering the ion of those reported in TableIgiving the worst S/N for each analyte. 2 LOQs (lower amount of pesticide that provided acceptable recoveries and repeatabilities (< 20 %)) were calculated for the lowest of the confirmation ions

	mpound	LOD (µg/g) 1	LOQ (µg/g) 2	Recovery (%) (X ± RSD, n=5)	10 LOQ (µg/g)	Recovery (%) (X ± RSD, n= 5)
	noxycarb	0.03	0.1	44 ± 12	1	47 ± 9
	enthoate	0.007	0.02	56 ± 10	0.2	59 ± 8
	noFos	0.003	0.01	50 ± 14	0.1	56 ± 10
	azinon	0.004	0.01	70 ± 8	0.2	69 ± 6
	osalone razophos lorpyrifos methyl fenofos imiphos ethyl mephos omophos ethyl lorpyrifos ethyl 1 F 0.07 0.001 0.1 0.02 0.003 0.001 0.003 0.003 o r P 0.2 0.005 0.5 0.05 0.01 0.005 0.01 0.01 e F o r P e e r e r	89 ± 6 43 ± 10 38 ± 15 41 ± 13 48 ± 12 19 ± 19 28 ± 17 37 ± 16	2 0.05 5 0.5 0.1 0.05 0.1 0.1	92 ± 6 45 ± 8 40 ± 12 43 ± 10 50 ± 8 21 ± 14 34 ± 11 43 ± 11
			R R
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				w w
				O n l O n l
					y y
						34

Table IV .

 IV Pesticide levels found in honey samples

	Sample	Pesticide	Level (ng g -1 ) (X ± SD, n=3)
	Orange blosom 1	Temephos	7.2 ± 0.9
	Orange blosom 2	Fonofos	15 ± 2.2
		Pyrazophos	6.2 ± 1.2
	Rosemary	Diazinon	17.2 ± 2.8
	Lemon 1	Diazinon	12.4 ± 1.1
	Lemon 2	Pirimiphos ethyl	19 ± 3.0
	Lavander	Bromophos ethyl	11.5 ± 1.2

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Acknowledgements

This work was financially supported by the Spanish Ministry of Science and Technology and the European Regional Development Funds (ERDF) (Project AGL2003-01407) and to the Generalitat Valenciana (GRUP 03/164).

Data obtained in real samples indicate the importance of analytical control methods for honey and the need of further studies of contaminant sources in relation to food safety, the protection of the environment, and the management of agricultural resources.

**The data were subjected to linear regression analysis of peak area (y) of the compound against the spiked concentration (x). For the equations, 6 plots (each point represents the mean of duplicate determinations) with different concentrations for each compound were used.