Rosalia Ferracane

Alessio Tafuri

Antonio Logieco

Fabio , Galvano

Dolores Balzano

Alberto Ritieni

Simultaneous Determination of Aflatoxin B 1 and Ochratoxin A and Natural Occurrence in Mediterranean Virgin Olive Oil

Keywords: Mycotoxins, Mycotoxins -ochratoxin A, Mycotoxins -aflatoxins Ochratoxin A, aflatoxin B 1, olive oil, quality parameters

Olive oil, the most important dietary fat source of Mediterranean diet, can be contaminated by mycotoxins. An efficient analytical method for the simultaneous determination of aflatoxin B 1 (AFB 1 ) and ochratoxin A (OTA) in olive oil is reported. Thirty commercial samples of virgin olive oil, purchased in olive-press plants and supermarkets in southern Italy and North Africa, were analysed to verify the analytical procedure and monitor mycotoxin contamination. A simple, rapid and economic method was set up and tested for both the extractive step and the clean-up procedures for simultaneous AFB 1 and OTA determination in olive oil. Data obtained showed that OTA was detected with high frequency (80%) in samples from both geographical areas (up to 17.0 ng/g) while AFB 1 was found from three of four samples from North Africa (up to 2.4 ng/g). In addition, "not labelled" oil samples proved to be more contaminated by OTA then "labelled" samples (mean value of 2.47 and 0.66 ng/g, respectively). These findings indicate that olive oil can be significantly contaminated by mycotoxins and confirm that a scrupulous application of European Regulation 1019/2002, that prohibits the sale of not labelled olive oil, is strongly recommended. Conventional qualitative parameters such as peroxide number, spectrophotometric evaluation and acid values were not correlated with mycotoxin occurrence.

INTRODUCTION

Olive oil is one of the most important components of the Mediterranean diet and its physicalchemical, organoleptic and, particularly, nutritional properties have been studied in depth. As a result, the USA Food and Drug Administration recently announced the availability of a qualified health claim for monounsaturated fat from olive oil and reduced risk of coronary heart disease [US Food and Drug Administration]. Acid values, ∆K and peroxides number are widely considered nutritional and commercial parameters to define the commercial class of olive oils [Commission Regulation 1995]. These indices commonly used to assess the degree of oxidation of edible oils and fats were the peroxide value which measures primary oxidation, the specific absorption at 232 nm (K232), was related to the content of conjugate dienes, which were formed as a result of oxidation and during the bleaching phase in oil refinement [Official Journal of the European Communities, 1991]. In Italy olive oil consumption is 12-13 kg/person/year for a total of 750 thousand tons and a value of 3 thousand million euros [START_REF]91 11/7/1991 on the characteristics of olive oil and oliveresidue oil and on the relevant methods of analysis[END_REF]]. The regulation of European Community CE 656/95 [Commission Regulation 1995] established 28 analytical parameters to evaluate the quality of olive oil and fixed the minimal qualitative standards required for the various selling category. Indeed, olive oil to be commercialised as "extra virgin", "virgin" or lower and the lack of conformity of even just one of the analytical parameters causes the declassification of oil to inferior selling category. Some of the qualitative standards, i.e. free acidity, peroxides number, spectrophotometric indexes and panel test, are specific genuineness indices aimed to safeguarding consumers' health and to fight commercial frauds.

Mycotoxins are secondary bioactive metabolites produced by some aerobic microscopic fungi principally belonging to Aspergillus, Penicillium and Fusarium genera growing on plants during both field and storage time (CAST 2003). Aflatoxins (AFs) are produced by A. flavus and A. parasiticus and are very stable compounds soluble in polar organic solvents, in particular chlorurate solvents or in aromatic hydrocarbons. AFs are essentially found in cultivations and commodities made up of peanuts, plant oleaginous seeds, maize and by-products, almonds, walnuts, hazelnuts and dried figs [START_REF] Galvano | Mycotoxins in the human food chain[END_REF]. AF toxicity is a result of interactions with nucleic acids, determining deleterious effects on the protein synthesis, in particular on the expression of the gene p53, and integrity of nucleic acids with consequent damages to the organisms; for these reasons AFs are considered the most powerful natural hepatocarcinogenic agents. In addition, they are considered potent carcinogens, teratogens and mutagens and pose a severe hazard to animal and human health [START_REF] Chu | Mycotoxins: food contamination, mechanism, carcinogenic potential and preventive measures[END_REF][START_REF] Eaton | The Toxicology of aflatoxins, Human Health, Veterinary and Agricoltural Significance[END_REF][START_REF] Galvano | Mycotoxins in the human food chain[END_REF]. The risk of mycotoxins in olive oil has been reported by various authors [Gracian and Arevalo 1980;[START_REF] Tanoui-Elaraki | Conséquences de la contamination des olives par des Aspergillus toxinogènes sur la quantité et la qualité de l'huile de pression[END_REF][START_REF] Paster | Antimicrobial activity and inhibition of aflatoxins B 1 formation by olive plant tissue constituents[END_REF]Visconti et al. 1996;[START_REF] Mahjoub | A method for aflatoxin B 1 determination in olives[END_REF][START_REF] Yassa | Some factors affecting mould growth and aflatoxins production in olives[END_REF][START_REF] Eltem | Growth and aflatoxins B 1 production on olives and olive paste by molds isolated from Turkish-style natural black olives in brine[END_REF]. Olives are often stored for a long time in conditions that promote the growth of moulds, such as prolonged contact with the ground, in bags of jute, in little ventilated places all favouring the toxinogenic moulds colonization. [START_REF] Yassa | Aflatoxin B 1 production by moulds isolated from black table olives[END_REF] reported that a total of 40 mould strains were isolated from black table olives produced in Egypt. Among them, nine strains of A. flavus and five strains of A. parasiticus were found to produce aflatoxin B 1 (AFB 1 ) on culture media and olive paste. The refining step would partially remove AF from contaminated oil [Le [START_REF] Tutour | Simultaneous dection of aflatoxin B 1 and ochratoxin A in olive oil[END_REF][START_REF] Parker | Absence of aflatoxins from refined vegetable oils[END_REF]. However, pressed olive oil has been shown to contain 18-47% of the AFs originally present in the contaminated olive fruits [START_REF] Mahjoub | A method for aflatoxin B 1 determination in olives[END_REF].

Ochratoxins are a group of mycotoxins produced by various species of Aspergillus and Penicillium genera. They include nine different molecules, among which the better known and most toxic is ochratoxin A (OTA). OTA is soluble in organic polar solvents and in weakly basic water solutions and can be found as natural contaminant in various commodities as cereal, coffee, grape, cocoa almonds and figs [Majerus et al. 1993;[START_REF] Galvano | Mycotoxins in the human food chain[END_REF].

Although olive fruits can be affected by toxigenic fungi in pre-and post-harvest, there are few references in the literature concerning olive oil contamination by OTA [Le Tutour et al. 1983, Papachristou and[START_REF] Papachristou | Determination of ochratoxin A in virgin olive oils of Greek origin by immunoaffinity column clean-up and high-performance liquid chromatography[END_REF]. Our objectives in this study were a) to investigate efficient analytical method for simultaneous determination of AFB 1 and OTA in olive oil and b) to analyse commercial labelled and not-labelled oil samples to verify the analytical procedure and monitor the contamination in samples of virgin olive oil from two important olive oil productive area (southern Italy and North Africa).

MATERIALS AND METHODS

MATERIALS

Sampling

A total of 30 samples (two sub-samples for each lot) of virgin olive oil were purchased in olivepress plants and supermarkets in the South of Italy (Campania, Sicily and Puglia areas) and Morocco. Information about the origin of seven commercial samples were obtained from the bottle labels. All the samples were stored at 4 °C and in the dark prior to analysis by HPLC. Extraction and analytical performances were carried out analysing blank sample at the same time.

[Insert Table 1 about here] Table 1 provides a brief description of the samples which were collected. Two of samples are marked as organic while four samples were imported from Morocco and are classified as product of ecological and fair trade market.

Reagents

All reagents used were of analytical grade and for HPLC were purchased from Merck (Germany).

AFB 1 and OTA standards were purchased from Sigma (USA). Silica SPE cartridges of 500 mg/3mL were purchased from Applied Separation (USA). All the samples were filtered using regenerate cellulose filters RC 25 mm 0.20 µm (Chemtek Analytical, Italia) and 2 mL syringes (Plastipak, Italia).

Apparatus

A Rotary evaporator (Heidolph, Mod. W2000) and a centrifuge evaporator (Savant, Jouan, France). HPLC runs were performed an a Shimadzu (Japan) liquid chromatograph configured with a RF-10Ax1 fluorescence detector and pump LC-10AD and the control system SLC10 A. AFB 1 detection was carried out at λ ex = 360 nm and λ em = 440 nm while OTA detection was carried out at λ ex = 333 nm and λ em = 460 nm. Flow rate was 1 mL min-1 and the retention calculated times 6.4 min (RSD 1.5%,) for OTA [Insert figure 1 about here] (Figure 1) and 7.4 min (RSD 1.2%,) for AFB 1 [Insert figure 2 about here] (Figure 2) were obtained with ten consecutive injections of the same OTA and AFB 1 working solution within the same day. Final, mycotoxin identifications were performed by comparing retention times and UV spectra of the purified samples to pure OTA and AFB 1 standards. A further confirmation was performed by co-injecting samples together with OTA and AFB 1 standard solutions. AFB 1 calibration curve was build in the range 0.5-100 ng/g with a corresponding limit of detection of 0.25 ng/g while OTA calibration curve was build in the range 0.2-200 ng/g with a corresponding limit of detection of 0.1 ng/g [Insert figure 3 and 4 about here] All chromatographic results were statistically evaluated and the relative standard deviations (SD) were calculated.

HPLC column was

Validation parameters for extraction and analyses of mycotoxins

The validation parameters determined for extraction and analysis of the mycotoxins were: limit of detection (LOD), limit of quantification (LOQ), linearity range and recovery. For LOD and LOQ, recovery and linearity range 10 samples of olive oils per mycotoxin were selected on the basis of the absence of one of the mycotoxins. These samples were spiked with different amounts of standard of OTA or AFB 1 , then extracted and analysed. The limit of detection was calculated according to the formula: LOD = x + 3*SD, where x is the mean value of 10 analysed samples and SD is the calculated standard deviation.

LC-MS-MS analysis

LC-MS-MS analysis were performed using a system consisted of a series 200 binary micropump (Perkin Elmer, USA) and an API 3000 triple-quadrupole mass spectrometer (Applied Biosystem, Ontario, Canada).

The mass spectrometer was equipped with a Model 11 syringe pump (Harvard Apparatus, USA).

All the analysis were performed using a Turbo Ion Spray source, nebulizer temperature was set at 450°C for AFB 1 and 400°C for OTA; the collision-induced dissociation was performed using nitrogen as collision gas in the collision cell. AFB 1 analysis was performed on a Synergy column 4 MAX-RP 80 A, 50 x 2.00 mm (Phenomenex, USA). The solvent system was composed of 100% water (solvent A) and methanol-acetonitrile (90:10 v/v) (solvent B). The linear solvent gradient was 20-80% B in 10 min, 80-100 % B in 2 min, constant at 100% B for two min and finally 100-20% B in 2 min. All analyses were performed at a constant flow of 0.2 mL/min and were injected 5 µl of sample.

OTA analysis was performed by a direct sample injection to the MS/MS system, named flow injection MS/MS, using a mixture of methanol-water (70:30, v/v) at a flow rate of 0.2 mL/min as liquid carrier. The acquisition was carried out in MRM (Multiple Reaction Monitoring) in the negative-ion mode for OTA and positive-ion mode for AFB 1 . To promote ionization of the precursor ion, the voltage applied was respectively 5000 V for AFB 1 and -4500 V for OTA. Data acquisition and processing were performed using Analyst software 1.4

[Insert Table 2 about here]

In Table 2 the mass spectrometer parameters are listed for AFB 1 and OTA. Extraction and partition. Olive oil (20 g) and hexane (40 mL) were poured into a 250 mL separating funnel and extracted with three 40 mL portions of methanol/water extraction solvent (60/40 v/v) containing 4% NaCl. Methanol was evaporated under reduced pressure (35°C). Then, AFB 1 and OTA were extracted from the aqueous phase with three 40 mL portions of chloroform shaken for 2 min each time. The chloroform fractions were combined in a 250 mL flask and evaporated till dry in a rotary evaporator (35°C). The sample residue was dissolved in 5 mL of dichloromethane and the solutions were evaporated to dryness, under a gentle stream of nitrogen.

Clean-up

The silica Spe-ed SPE cartridge was pre-washed with 3 mL hexane and 3 mL dichloromethane.

Then, the sample residue was dissolved in 2 mL dichloromethane and loaded on the top of the column that was washed with 3 mL hexane, 3 mL anhydrous ethyl ether and 3 mL dichloromethane. AFB 1 was eluted with 4 mL chloroform/acetone (9/1 v/v) then evaporated to dryness in a centrifugal evaporator. At the end of AFB 1 extraction elution OTA was eluted with 4 mL methanol then evaporated in a centrifugal evaporator.

HPLC analyses

AFB 1 and OTA were eluted with 1 mL of mobile phase and 1 mL methanol, respectively, then filtered by regenerated cellulose filters and analysed under the HPLC conditions applied in the present study. The samples were evaporated to dryness under a gentle stream of nitrogen. The residues were dissolved in 1 mL of a methanol-water (70:30 v/v) to obtain the confirmation of molecular structure.

Confirmation by mass spectrometry

RESULTS AND DISCUSSION

With the aim to achieve an efficient analytical method for simultaneous AFB 1 and OTA determination in olive oil, a series of experimental tests were performed to improve the procedure for AFB 1 of [START_REF] Daradimos | Evaluation and validation of two fluorometric HPLC methods for the determination of Aflatoxin B 1 in olive oil[END_REF]. This latter method was modified to provide simpler, rapid and more economic both extractive steps and clean-up procedures. The extraction and the clean-up step were common for both AFB 1 and OTA and a single silica-gel column was used for the clean-up procedures.

AFB 1 and OTA were eluted in two different washing column solvents, then collected and separately analysed by HPLC. This clean-up procedure saved time, solvents and columns and without the need to use much more expensive immunoaffinity columns. To optimise the procedure different tests were carried out evaluating recovery of both singly and co-contaminated (AFB 1 and OTA) samples.

The artificial contaminations of olive oil was carried out using a methanol mycotoxin standard solution using a minimum quantity of solvent (10-20 µl). As methanol is not completely soluble in oil, n-hexane was added up to 1 mL solution to increase mycotoxin solubility. Contaminated olive oil was shaken for 5 min to achieve homogeneous contamination. During the extraction steps ethyl acetate was tested because it is more polar but its recovery factor was 30% therefore it was discarded.

The washing phase with diethyl ether was bypassed to improve speed of clean-up but in this way several compounds co-eluted with AFB 1 making peak identification difficult. The clean-up was performed by silica-gel column at low-pressure to speed up the procedure but this alternative was abandoned because the recovery factor for AFB 1 was really reduced.

The extraction step with n-hexane, included in the method of [START_REF] Daradimos | Evaluation and validation of two fluorometric HPLC methods for the determination of Aflatoxin B 1 in olive oil[END_REF], was eliminated simplifying the procedures. Recoveries were determined at three different concentrations and repeated three times on two consecutive days to estimate inter-day and intra-day variability. AFB 1 and OTA recovery factor were estimated to be 74.6% and 71.3%, respectively.

[Insert Table 3 about here] In a further test, olive oil was spiked with both mycotoxins at the same level (10 ng/g) and the recovery factors were estimated after three spiking experiments to be 71.8% for OTA and 73.3% for AFB 1 . OTA and AFB 1 were eluted by two different washing solvents from the same silica-gel column, thus unifying extractive and clean-up steps.

The main objective at the beginning of the study was to achieve a simple, reliable and efficient method for the determination of AFB 1 and OTA in olive oil. The secondary aim was to analyse commercial oil samples to verify the analytical procedure and monitor the contamination. The occurrence of AFB 1 and OTA, found in some of the samples and quantified by the HPLC method, was confirmed by mass spectrometry by flow injection for OTA (M1, M2, M3, S1, P1, P2, P3, P4, P6, P7, P8, P9, P12, L1, L2, L3, L4, L8, L6, L7, L9, L10, L11 samples) and HPLC/MS/MS for AFB 1 (C1, M1, M3 samples). Olive oil naturally contaminated from AFB 1 and OTA is showed in In order to further verify the HPLC results, all the samples were analysed by mass spectrometry (MS) identifying the molecules by their molecular weight. MS was economically disadvantageous but provided comparable analytical sensibility respect to HPLC.

Numerous samples did not provide information on origin and/or technological process because they were obtained from oil-mills in the South of Italy. In this part of Italy the "not labelled" olive oil is much more widely available than in the Centre and in the Northern provinces of the country, where the bottling industry is concentrated. The remaining samples came from both large and small supermarkets in order to estimate the quality of this source of supply. Most of these latter samples were of both notable and no notable brand, whereas the rest came from organic cultivation.

OTA and AFB 1 surveillance in olive oil was integrated from main quality parameters evaluation: acidity, peroxide number (PN) and spectrophotometric indices (K 232 , K 270 , ∆K).

[Insert Table 4 about here] Table 4 reports data of the analyses of virgin olive oil samples. Approximately 80% of the samples were contaminated with OTA in the range 0.1-17.0 with a mean value of 2.09 ng/g, 71.5% of "labelled" samples were contaminated in the range 0.1-1.8 ng/g with a mean value of 0.7 ng/g. Moreover, one of organic cultivation samples (P12) was contaminated with 1.80 ng/g of OTA.

In a recent study [START_REF] Papachristou | Determination of ochratoxin A in virgin olive oils of Greek origin by immunoaffinity column clean-up and high-performance liquid chromatography[END_REF], OTA was found in 44 (88%) of 50 olive oil samples (mean contamination 267 ng/kg ). It is opportune to underline that no regulatory limits exist of the OTA content in olive oils, whereas in cereals for human food the limit is 3 ng/g. Taken into consideration that the annual consumption of olive oil is 12-13 kg [START_REF]91 11/7/1991 on the characteristics of olive oil and oliveresidue oil and on the relevant methods of analysis[END_REF]] per person, the level of such mycotoxin in "not labelled" oil was estimated to be approximately 65 ng/day, equating to exposure of 1,09 ng/kg body weight/day while for the labelled olive oil the level was 18.3 ng/day equating to exposure of 0.3 ng/kg body weight/day (for a 60 kg person). The tolerable daily intake (TDI) for OTA was recently set at 0.2 ng/kg body weight/day [Commission Regulation 1998] therefore the samples of olive oil did not lead to exposure above the TDI although these samples clearly represent an important source of OTA. In addition, it is important to regard the effects of this contamination considering that olive oil is the main fat source in the Mediterranean diet. A greater hazard can be associated to consumption of "not labelled" olive oil whose OTA contamination levels were higher.

Table 4 also shows data of AFB 1 contamination. Only 3 out of 30 samples were contaminated ranging from 0.54 to 2.50 ng/g and, in addition, the 66% of contaminated samples were not Italian and all were "not labelled". Along with data of Papachristou and Markaki 2004, who found the presence of AFB 1 (60 ng /kg) in only one sample out of 50, our data confirm that olives are a poor substrate for the biosynthesis of AFB 1 [START_REF] Leontopoulos | Black olives as substrate for Aspergillus parasiticus growth and aflatoxin B1 production[END_REF]]. As well as for OTA no regulatory limits exists for AFB 1 in olive oils.

In addition, the 10% of the samples were contaminated from both AFB 1 and OTA, and the two of these three samples have North African origin. As regard the quality indices of analysed oils (Table 4) it is interesting that OTA contamination was observed regardless the respect of the limits demanded for classify an olive oil as extra virgin oil. Indeed, OTA was present in M3 and P12 samples (classified as extra virgin oil), whereas P10, L12 and L5 samples (sold as extra virgin oil although not classifiable as such) where not contaminated by OTA and AFB 1 . The spectrophotometric indices, acid value and peroxides number reported in Table 4 were not related with AFB 1 and OTA occurrences resumed in the last two columns. This conclusion demonstrated that commercial class evaluation of olive oil and its safety level were not correlated; in fact the samples M3 was considered extra virgin olive oil but it was contaminated by both mycotoxins. On the other hand, many samples were mycotoxin free but commercially classified as virgin olive oil. Italian law [D.L. n.°155, 26/5/97] provides some systems of self-control for a correct management of the hygienic dispositions, assuring safe and healthy food. The self-control is the means through which every company can verify the effectiveness of the productive system, using monitoring procedure reported from HACCP (Hazard Analysis Critical Control Point) system [START_REF] Douglas | Minimising Risks Posed by Mycotoxins Utilising the HACCP Concept[END_REF].

HACCP system supplies the technical modalities to identify and to evaluate any risks associated to the various production steps, determining some corrective measures to prevent, eliminate or reduce the probability of a risks. Pre-requirement to develop a practical program HACCP system is the respect of corrected agricultural practices with respect of good manufacture roles [START_REF] Douglas | Minimising Risks Posed by Mycotoxins Utilising the HACCP Concept[END_REF]].

In Italy, despite its huge diffusion, oil-mills are dispensed by the application of HACCP. It is true that olive oil composition strongly hinders the microbial growth for many reasons, such as oxygen lack, reduced water content and presence of lipids as unique substrate; however this composition does not guarantee the absence of spore and toxins already present on the drupe during harvest and post-harvest storage.

Mycotoxins accumulation of olives on the plants depends on various climatic conditions, like temperature, rainfall, relative humidity. However, mycotoxins biosynthesis might take place also during some steps of olive fruit transformation such as collection, transport, storage and washing. Indeed, the main factor of biological risk for virgin olive oil production, is the growth of moulds on the olive fruits or on the residual olive cakes, that can contaminate insufficient cleaned pressing system [START_REF] Sacchi | L'HACCP in frantoio: guida all'autocontrollo dell'igiene in oleificio[END_REF]. The olives should have to be transformed within 12 hours from the harvest, because bad storage conditions affect the safety of olive oil increasing their acidity and conferring a mould and rancid taste. Therefore, the best way to prevent and control the mycotoxins contamination in olive oil is the prevention and the correct management of harvest, post-harvest and storage steps [START_REF] Park | Surveillance programmes for managing risks from naturally occurring toxicants[END_REF].

CONCLUSIONS

In conclusion "labelled" olive oils offers greater guarantees, such as quality indices of olive oil and for those examined, we confirmed what was reported on the label. On the contrary, "not labelled" oils offer no guarantees, because the declared extra virgin olive oils were not supported by analysis.

Moreover only olive oil quality indices does not provide assurance for the consumer about potential contamination with AFB 1 and OTA. Finally, oil samples bottled and labelled appeared to be safer because their OTA occurrence was on average four times less then "not labelled" samples (0.66 ng/g vs 2.47 ng/g). In addition, 13% of "not labelled" samples were contaminated with AFB 1 .

Evaluating conventional quality parameters such as peroxide number, ∆K and acid value this difference was confirmed. In fact, the PN value is 31.65 (S.D. 15.00) for "not labelled" oils vs.

16.14 (S.D. 3.18) of "labelled" oils and acid value is 2.24 (S.D. 1.51) for "not labelled" oils vs. 0.30

   4.6 x 150 mm) (Biorad, Belgium). Both the mobile phases for the AFB1 [water/methanol/acetonitrile (50/25/25 v/v/v)] and OTA [water(1% acid ) /acetonitrile (1% acetic acid) (1/1 v/v)] determinations, were filtered through Millipore HA VLP (0.45 µm) filters before use.

  number and spectrophotometric indices of oils were determined according to the analytical procedures described in the Reg. CEE 2568/91.Analysis of AFB 1 and OTAAFB 1 and OTA were determined according to the method of[START_REF] Daradimos | Evaluation and validation of two fluorometric HPLC methods for the determination of Aflatoxin B 1 in olive oil[END_REF] modified to provide a rapid, economical and reproducible analytical method.

Figure 5 [

 5 Figure 5

  (S.D. 0.15) of "labelled" oils. European Regulation n° 1019/2002 from November 2003 [Commission Regulation 2003] prohibits the sale of not labelled olive oil, guaranteeing the consumer a choice of the product.

  Figure legends

Table 1 . A brief description of the collected samples

 1

							Food Additives and Contaminants	Page 20 of 26
	Area
	1	0.2	0.3	0.4	0.5	0.6	0.7	0.8	0.9	1.0
				Amount (mg/kg)

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Table 2 . Spectrometer parameters to confirm AFB 1 and OTA by LC/MS/MS [MH]

 2

	AFB 1	313.1 [MH] +	285.0 269.0	56	36 42
			358.2		-24
	OTA	402.0 [MH] -	254.2 211.2	-55	-32 -37
			167.0		-45

+ : protonated molecule; [MH] -: deprotonated molecule; DP: declustering potential; CE: collision energy Compound Molecular ion Daughter ions (m/z) DP (V) CE (V)

Table 3 . Recovery of AFB 1 and OTA at different spiking levels

 3

	Page 25 of 26	Food Additives and Contaminants
	Spiking level	Calculated Recovery for OTA	Average	Calculated Recovery for AFB 1	Average
	A. 30 ppb B. 30 ppb	91.6% 90.6%	90,0%	87.5% 85.5%	86,5%
	A. 10 ppb B. 10 ppb	78.8% 90.6%	84.7%	74.0% 72.0%	70,1%
	A. 2.5 ppb B. 2.5 ppb	38.8% 39.6%	39,2%	65.5% 69.0%	67,3%

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Table 4 . Qualitative parameters and contamination of olive oil samples

 4

	Sample K 2 3 2 K 2 7 0	∆ ∆ ∆ ∆K	Acid value	Peroxides number	AFB 1 (ng/g)	OTA (ng/g)
	C1	2.36 0.357 0.00175	0.6	11	2.4	0.5
	L1	3.13 0.335 0.00025	4.2	24	ND	0.4
	L2	3.28 0.483 0.0060	4.2	21	ND	0.2
	L3	2.92 0.352 0.00175	3.8	64	ND	0.6
	L4	2.88 0.328 0.00275	3.9	25	ND	0.1
	L5	2.43 0.211 0.00025	1.7	26	ND	ND
	L6	3.30 0.512 0.00675	4.5	45	ND	0.7
	L7	3.09 0.289 0.00225	1.6	25	ND	6.0
	L8	3.30 0.513 0.00350	2.0	43	ND	0.4
	L9	3.18 0.309 -0.0020	2.8	32	ND	17.0
	L10	3.41 1.138 0.05220	4.7	25	ND	5.5
	L11	2.63 0.251 0.00170	0.5	24	ND	0.2
	L12	3.28 0.478 0.05100	3.9	51	ND	ND
	M1	2.13 0.325 0.0010	1.3	8	0.5	0.8
	M2	2.34 0.278 -0.0035	0.5	25	ND	0.6
	M3	2.21 0.268 -0.0035	0.9	10	1.0	1.5
	P1	3.18 0.295 -0.0020	1.2	49	ND	0.6
	P2	3.15 0.323 0.00075	1.1	32	ND	1.4
	P3	3.00 0.225 -0.0045	0.6	26	ND	9.2
	P4	2.87 0.205 -0.0300	0.5	21	ND	0.4
	P5	3.16 0.289 0.00175	2.0	37	ND	ND
	P6	2.68 0.197 -0.0025	0.4	14	ND	0.6
	P7	3.21 0.891 0.0860	0.1	17	ND	0.1
	P8	3.26 0.434 0.00325	3.6	45	ND	0.6
	P9	3.22 0.342 0.0010	1.5	22	ND	0.7
	P10	2.79 0.292 0.00175	0.4	58	ND	ND
	P11	2.59 0.201 -0.0035	0.3	11	ND	ND
	P12	2.69 0.195 -0.0037	0.3	15	ND	1.8
	P13	2.60 0.198 -0.0030	0.5	17	ND	ND
	S1	2.60 0.251 -0.0020	0.5	18	ND	0.4

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Abdellah ; Zinedine

Jose Miguel Soriano
email: jose.soriano@uv.es

Cristina Juan

Brahim Mojemmi

Juan Carlos Molto

Abdelaziz Bouklouze

Yahia Cherrah

Larbi Idrissi

Rajae Elaouad

Jordi Manes

J C Moltó

R El Aouad

& J Mañes

Incidence of ochratoxin A in rice and dried fruits from Rabat and Salé

Keywords: Ochratoxin A, rice, dried fruits, Morocco

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Introduction

Ochratoxin A (OTA), chemically known as N-{[(3R)-5-chloro-8-hydroxy-3-methyl-1oxo-7-isochromanyl]-carbonyl}-3-phenyl-L-alanine, is a mycotoxin described the first time by [START_REF] Van Der Merve | Mycotoxins. Part II. The constitution of ochratoxins A, B and C, metabolites of Aspergillus ochraceus Wilhm[END_REF]. OTA is a ubiquitous secondary fungal metabolite primarily produced by the genera of Aspergillus (e.g. A. ochraceus) and Penicillium (e.g. P. verrucosum). Several other Aspergillus species have been described as producers of this toxin, including strains of A. alliaceus, A. ostianus, A. sclerotiorum, A. sulphureus, A. melleus, A. petrakii, A. glaucus, A. niger, A. awamori, A. foetidus, A. carbonarius, A. albertensis, A. auricomus and A. wentii [START_REF] Varga | Ochratoxin production by Aspergillus species[END_REF]. In colder climates, Penicillium species were found to be responsible for OTA contamination of several agricultural products. OTA has been widely detected in cereals (barley, wheat, maize, oat) (Speijers & Van Egmond, 1993;[START_REF] Trucksess | Determination and survey of ochratoxin A in wheat, barley and coffee-1997[END_REF], green coffee [START_REF] Leoni | Ochratoxin A in Brazilian roasted and instant coffees[END_REF], grape juice [START_REF] Zimmerli | Determination of ochratoxin A at the ppt level in human blood, serum, milk and some foodstuffs by high-performance liquid chromatography with enhanced fluorescence detection and immunoaffinity column cleanup: methodology and Swiss data[END_REF], and wine (Blesa et al., 2004a;Miraglia & Brera, 2002). OTA contamination of dried fruits was found to be due to the action of black aspergilli in Europe including Spain [START_REF] Abarca | Aspergillus carbonarius as the main source of ochratoxin A contamination in dried vine fruits from the Spanish market[END_REF], France [START_REF] Sage | Fungal microflora and ochratoxin A risk in french vineyards[END_REF], the Czech Republic [START_REF] Ostry | MYKOMONmonitoring project of toxigenic fungi in food in years 1999-2001[END_REF] and in other parts of the world such as Argentina [START_REF] Romero | Toxigenic fungi isolated from dried vine fruits in Argentina[END_REF] and Australia [START_REF] Leong | Occurrence of fruit rot fungi (Aspergillus section Nigri) on some drying varieties of irrigated grapes[END_REF].

OTA is receiving increasing attention for its toxic effects and high incidence in a wide range of food commodities. OTA has been shown to be nephrotoxic, carcinogenic, immunotoxic, genotoxic and teratogenic to all animal species tested. The genotoxicity of OTA has been postulated in vivo and in vitro [START_REF] Creppy | Update of survey, regulation and toxic effects of mycotoxins in Europe[END_REF]. Genotoxic effects such as DNA strand breaks, sister chromatid exchanges, chromosomal aberrations and induction of micronuclei have been observed in some mammalian cell systems in response to OTA exposure [START_REF] Mally | DNA adduct formation by ochratoxin A: Review of the available evidence[END_REF]. The presence of OTA in blood from healthy humans confirms a continuous and widespread exposure. A positive correlation among human nephropathies and dietary OTA exposure or plasma concentrations arises from several epidemiological studies. OTA has been implicated in a human disease of kidney referred to as Balkan endemic nephropathy, characterized by tubule interstitial nephritis and associated with high incidence of kidney, pelvis, ureter and urinary bladder tumors in some Eastern European countries in particular Bulgaria, Romania, Serbia, Croatia, Bosnia and Hertzegovinia, Slovenia and Macedonia (Pfohl-Leszkowicz et al., 2002). Consumption of food contaminated with OTA during pregnancy and/or childhood is suspected to induce lesions in testicular DNA that puberty could promote testicular cancer [START_REF] Monaci | Determination of ochratoxin A in foods: State of the art and analytical challenges[END_REF]. Sufficient experimental evidence for carcinogenicity in animal studies has led to the classification of OTA as a possible human carcinogen (group 2B) by the International Agency for Research on Cancer (IARC 1993). The Joint Committee FAO/WHO of Experts on Food Additives (JECFA) has established the provisional tolerable weekly intake (PTWI) of OTA at 100 ng/kg of body weight corresponding to approx. 14 ng/kg b.w./day [START_REF] Jecfa | Safety evaluation of certain mycotoxins in food[END_REF].

In North African countries, the most suspected food susceptible to be contaminated by OTA are domestic and imported cereals like wheat and sorghum, olives, poultry products and spices [START_REF] Grosso | New data on the occurrence of ochratoxin A in human sera from patients affected or not by renal diseases in Tunisia[END_REF]. Data published suggested the evidence association of elevated exposure to OTA with cases of human nephropathies in Tunisia [START_REF] Abid | Ochratoxin A and human chronic nephropathy in Tunisia: is the situation endemic?[END_REF]. In Morocco, a North African country surrounded by the Atlantic and Mediterranean seas, there is a lack of investigations related to OTA occurrence in food and feed, even though climatic conditions, crop production and handling practices are favourable to mycotoxin contamination. In a recent report, the natural occurrence of OTA in barley, corn and wheat from Morocco was reported and the co-occurrence of OTA with Fusarium toxins (Zearalenone and Fumonisin B 1 ) has been also confirmed in corn samples by [START_REF] Zinedine | Natural occurrence of mycotoxins in cereals and spices commercialized in Morocco[END_REF]. Although cereals (corn, wheat, barley) were studied for mycotoxin occurrence, rice has never been investigated. The Moroccan population consumes huge amounts of dried fruits directly or as ingredient included in popular foods such as "Chebbakia", "Sellou/Sefouf", "Zammita" and "Mkharka" especially during the "Ramadan" fasting month and festival days. Almost all nuts such as pistachio, walnuts and peanuts consumed in Morocco are imported and little is known about their quality. Consequently, there are no legal parameters concerning the presence of mycotoxins, especially OTA, that control their entry to the country and, up to now, there is a lack of information in the literature about the occurrence of OTA in these products. The purpose of this survey is to investigate the presence of OTA in rice and dried fruits available in the area of Rabat and Salé in Morocco.

Material and methods

Sampling

Chemical and reagents

Acetonitrile, cyclohexane, formic acid, acetic acid and LC grade methanol was supplied by Merck (Darmstadt, Germany). LC-grade water was obtained by filtering deionised water through a 0.45-µm filter with a Waters-Millipore (Milford, MA, USA) system.

Solvents and water were degassed for 20 min using a Branson 5200 ultrasonic bath (Branson Ultrasonic Corp., CT, USA). Phosphate-buffered saline (PBS) was bought from Sigma (St. Louis, MO, USA).

OTA crystalline material was purchased from Sigma (St. Louis, MO, USA). Stock standard solution with concentration of 500 µg/ml was prepared in methanol, kept in a closed container at -20 °C, wrapped in aluminium foil, and held for less than 3 months.

Standard working solutions were prepared by appropriate dilution in the same solvent and stored in glass-stopped tubes at -20 °C.

Extraction procedure -OTA in rice

The method used for OTA in rice was developed in the laboratory and reported by Blesa et al. (2004b). Briefly, samples of rice (200 g) were prepared using a food processor and mixed thoroughly. An aliquot (2.5 g) of the sample was placed into a mortar (50 ml capacity) and was gently blended with 1.5 g of the solid phase C 8 (50 µm, Análisis Vínicos, Tomelloso, Spain) for 5 min using a pestle, to obtain a homogeneous mixture. This mixture was introduced into a 100 mm x 9 mm i.d. glass chromatographic column with a coarse frit (no. 2) and covered with a plug of silanized glass wool at the top of the column. OTA was eluted with 20 ml methanol-formic acid (99:1, v/v) with a vacuum manifold. The eluate was evaporated to 3 ml with a gentle stream of N 2 at 45 °C, and then, it was filtered through a nylon acrodisk (0.45 µm) and centrifuged at 5000 rpm for 10 min (Heraeus, Germany). The extract was filtered again and evaporated to dryness with N 2 at 55 °C and reconstituted in 500 µl of the mobile phase for LC analysis.

-OTA in dried fruits

The method of [START_REF] Stroka | Immunoaffinity column cleanup with liquid chromatography using post-column bromination for determination of aflatoxins in peanut butter, pistachio paste, fig paste, and paprika powder: collaborative study[END_REF] slightly modified was used. Briefly, 10 g of samples of pistachio, peanut and walnut added with 1g of NaCl were blended with 40 ml of methanol-water (80:20) and 20 ml of cyclohexane for 3 minutes. After separation of the two phases, cyclohexane was eliminated. For figs and raisins, samples were extracted only with 40 ml of methanol-water (80:20). The extracts were filtrated on Whatman filter paper No. 4. An aliquot of 10 ml was diluted with 60 ml of PBS buffer (pH 7.4).

An immunoaffinity column (IAC OchraTest TM , Vicam, USA) for ochratoxin analysis was conditioned with 10 ml of PBS buffer by gentle syringe pressure at a flow rate of 5 ml/min. Then, the mixture of the filtrate diluted extract (70 ml) was applied to the IAC column (1-2 drops per second), followed by a washing with 20 ml of LC grade water and then dried with air. OTA was then slowly eluted from the IAC with 2 ml methanol into a glass vial; the eluate was evaporated to dryness with a gentle stream of N 2 at 55 ºC and reconstituted in 500 µl of the mobile phase for LC analysis.

LC analysis

The chromatographic system consisted of a Shimadzu (Kyoto, Japan) SCL-6A system

Confirmation

The identity of OTA in positive samples was confirmed by methyl ester formation according to the method of [START_REF] Zimmerli | Determination of ochratoxin A at the ppt level in human blood, serum, milk and some foodstuffs by high-performance liquid chromatography with enhanced fluorescence detection and immunoaffinity column cleanup: methodology and Swiss data[END_REF]. Briefly 200 µl of the extract was diluted to 2.5 ml methanol and 0.1 ml concentrated HCl was added. The solution was left standing overnight at room temperature. Thereafter, the methanol was evaporated and the residue was taken up in 200 µl of mobile phase. Then 20 µl were injected to the LC system. 90% of the OTA was methylated by this method. The resulting methyl ester has a quite different retention time than OTA, this allows the confirmation of OTA identity by an almost complete disappearance of the first OTA peak and the presence of a new one.

Results and discussion

Recoveries for OTA on samples spiked at a level of 10 ng/g for dried raisins and dried figs and at a level of 5 ng/g for pistachio, peanut, walnut and rice are reported in Table I. The limit of quantification (LOQ) (S/N, 10:1) of OTA was 0.02 ng/g in rice, 0.03 ng/g in pistachio, peanut and walnut and 0.03 ng/g in dried raisins and dried figs.

OTA in rice

Results of occurrence of OTA in rice samples showed that OTA was present in 18 out of 20 analyzed samples, the incidence is 90%. Figure 1 shows a chromatogram of a rice positive sample. Levels of contamination in positive samples ranged between 0.02±0.01 and 32.4±2.10 ng/g, where the average of OTA in positive rice samples is 4.15±1.45 ng/g. Our findings showed high incidence of OTA in Moroccan rice comparing with data described for non-organic and organic rice from Spain where the incidence of occurrence of OTA were 7.8 and 30 % respectively [START_REF] González | Occurrence and daily intake of ochratoxin A of organic and non-organic rice and rice products[END_REF]. Worldwide contamination of rice with OTA was reported in UK [START_REF] Scudamore | Surveillance of stored grain from the 1997 harvest in the United Kingdom for ochratoxin A[END_REF] in Vietnam [START_REF] Trung | Fungal contamination of rice from South Vietnam, mycotoxigenesis of selected strains and residues in rice[END_REF], in rice germs and rice germ cake from Egypt [START_REF] Abdelhamid | Occurrence of some mycotoxins (aflatoxins, ochratoxin A, citrinin, zearalenone and vomitoxin) in various Egyptian feeds[END_REF]), in Portugal [START_REF] Pena | Determination of ochratoxin A in Portuguese rice samples by high performance liquid chromatography with fluorescence detection[END_REF], in France [START_REF] Leblanc | Estimated dietary exposure to principal food mycotoxins from The First French Total Diet Study[END_REF] and in polished rice from Korea [START_REF] Park | Fungal mycoflora and mycotoxins in Korean polished rice destined for humans[END_REF].

[Insert Figure 1 about here]

In Morocco, rice cultivation fluctuates vastly depending especially on climatic conditions. On a potential of 25 000 ha in the Gharb plain, the harvested area varies from 500 ha to 13000 ha. One average, the Moroccan population consumes 60 000 tons each year (2 kg/person/year). Due to drought the country has endured during the last two decades, rice yield production decreased dramatically from 44 000 tons in 1993 to 2500 tons in 1995 leading to extensive importation from other countries. Rice (Oryza ) is an important food crop worldwide along with wheat and corn, and has been major food in several countries. [START_REF] Park | Fungal mycoflora and mycotoxins in Korean polished rice destined for humans[END_REF] has been reported that rice is naturally contaminated with A. ochraceus. Rice is an aquatic plant and is usually harvested at very high moisture levels (35-50 %). Therefore, mycotoxin-producing moulds could contaminate the grain and produce important quantities of OTA during storage. Furthermore, rice is a better substrate for the characterization of OTA-producing A. ochraceus strains.

Up to now, no Moroccan government regulations for the maximum permitted levels of mycotoxins in foods and feeds are still in force. However, a project prepared by the Inter-ministerial Committee for Food Control and Frauds Repression (Comité Interministériel pour le Contrôle Alimentaire et la Répression des Fraudes, CIPCARF) and the Moroccan Service of Industrial Normalization (Service de Normalisation Industrielle Marocaine, SNIMA) to protect both human and animal health is under discussion. The maximum limit for OTA in cereals set by the Moroccan project is 30 ng/g (FAO 2004). This value is higher than maximum residue level (MRL) fixed by European regulations for OTA in cereals and cereal products at levels that cannot be greater than 5 and 3 ng/g, respectively (European Commission 2005). The maximum limit of OTA established by the codex in cereals and cereal products is about 5 ng/g (JECFA 2001). Our findings showed that 15% of analyzed samples exceeded the maximum permitted level of OTA set by the European Union in cereals.

OTA in dried fruits

The incidence of OTA in dried raisins, dried figs, walnuts, and peanuts were 30, 65, 35, and 25% respectively (Figure 2). In pistachio samples, OTA was below the to 2.36±0.75 in peanut, from 0.03±0.01 to 1.42±0.45 in dried figs, from 0.05±0.02 to 4.95±0.02 in dried raisins and from 0.04±0.01 to 0.23±0.05 in walnut. The average of OTA in positive samples of peanut, dried figs, dried raisins and walnut are 0.68±0.15, 0.33±0.12, 0.96±0.25 and 0.11±0.06 respectively. All positive samples of dried fruits were below the maximum tolerable level of OTA (10 ng/g) set by European regulations in dried vine fruits (FAO 2004). However, no maximum limit is set by the Moroccan project of mycotoxin regulations for OTA in dried fruits.

In comparison, previous UK surveys reported an incidence of OTA in raisins of 85% [START_REF] Macdonald | Ochratoxin A in dried fruit: method development and survey[END_REF] and 97% (MAFF 1999), while a German survey [START_REF] Engel | Ochratoxin A in sweets, oil seeds and dairy products[END_REF] reported a 95% overall incidence of OTA in raisins and currants. Data from Finland and France have indicated incidences of 71 and 46%, respectively (Miraglia & Brera, 2002).

Recent data from Canada reported that OTA was present in 79% of samples of raisins [START_REF] Lombaert | Ochratoxin A in dried vine fruits on the Canadian retail market[END_REF]. OTA was present in 95% of dried fig samples from Brazil and 26% contained levels higher than 5 ng/g, and samples were infected particularly with A. niger [START_REF] Imanaka | Incidence of toxigenic fungi and ochratoxin A in dried fruits sold in Brazil[END_REF]. In Turkey, [START_REF] Senyuva | Survey for co-occurrence of ochratoxin A and aflatoxin B1 in dried figs in Turkey by using a single laboratoryvalidated alkaline extraction method for ochratoxin A[END_REF] In Morocco, traditional techniques for the transformation and conservation of fruits are still used. Theses practices are very optimal conditions (especially temperature, humidity and fruits damages) for mould growth and mycotoxin production. The natural drying, which may consists in direct exposition of the fruit to the sun, is widely used especially in rural area. Fruits (raisins, figs etc.) having reached a sufficient degree of maturity are gathered and transported to drying places such as the terrace of house or a piece of ground fenced-off to prevent access of animals. These surfaces of drying are in general exposed to a maximum sunning and are papered with herbs to avoid the contact with the ground. Fruit are spread out over these surfaces without preliminary treatment.

After drying, fruit are collected and stored. During the process of fruit drying, the sugar is concentrated as the moisture content decreases resulting in an almost selective medium for xerotolerant moulds such as A. niger section nigri species. Among black aspergilli, A. carbonarius is the most important as OTA producing isolate observed more frequently [START_REF] Abarca | Aspergillus carbonarius as the main source of ochratoxin A contamination in dried vine fruits from the Spanish market[END_REF][START_REF] Sage | Fungal microflora and ochratoxin A risk in french vineyards[END_REF]. Other black aspergilli including the A. niger aggregate and A. aculeatus have also been found to produce OTA on grapes [START_REF] Battilani | Epidemiology of toxin-producing fungi and ochratoxin A occurrence in grape[END_REF]. Recently, OTA production in A. tubingensis isolates originating from grapes was observed by [START_REF] Medina | Study of Spanish grape mycobiota and ochratoxin A production by isolates of Aspergillus tubingensis and other members of Aspergillus section nigri[END_REF].

Conclusion

As far as we know, this is the first report that has shown the contamination of rice and dried fruits available in Morocco by OTA. The acceptable level of OTA which are fixed by European regulations was exceeded in 15% of analyzed samples of rice. It can be concluded that the occurrence of OTA in some dried fruits and rice samples is due to the fact that may be some food safety and quality standards (good agricultural practices (GAPs), good manufacturing practices (GMPs), and the hazard analysis and critical control point (HACCP) system) need to be applied and performed in most of Moroccan food units to control growth of moulds and mycotoxin production during harvesting, distribution and storage periods. A survey of a large number of prepared food samples during theses periods needs to be investigated.

  One hundred samples of dried fruits (20 raisins, 20 walnuts, 20 peanuts, 20 dried figs and 20 pistachios) and twenty samples of rice were purchased in July -August (2005) from retail shops of Rabat and Salé in Morocco, stored in plastic bags at 4ºC, taken to the Laboratory of Food Chemistry and Toxicology of the University of Valencia of Spain and immediately analyzed for OTA.

  Separation was carried out on a C18 column Phenomenex (150×4.6 mm I.D, 5 µm., Scharlau, Barcelona, Spain). A 20-µl aliquot of the sample was injected onto the column. The mobile phase consisted of acetonitrile/water/acetic acid (49.5/49.5/1, v/v/v) pumped at a flow rate of 0.8 ml/min. Determination of OTA was performed at 333 and 464 nm as excitation and emission wavelengths, respectively.

  As shown, the limit of quantification (LOQ) allows OTA determination at the maximum levels indicated in the European legislation. Results of the study reflected that the analysis and recoveries were considered as valid for analyzing residues of OTA in cereals and dried fruits according with European specification(European commission 2002) [Insert TableI about here]

  Analytical results showed that OTA levels ranged from 0.10±0.05

  reported the contamination of fig samples from the 2003 and 2004 crops with both OTA and aflatoxins. Bayman et al. (2002) reported high levels of OTA (up to 1850 ng/g) in figs grown in California. In the European Commission's 2002 Scientific Co-operation (SCOOP) project report, the analysis of 20 samples of fig imported to The Netherlands reported an incidence of 10% with a maximum concentration of 0.8 ng/g (Miraglia & Brera, 2002). [Insert Figure 2 about here]

Figure 1 .

 1 Figure caption

Figure 2 :

 2 Figure 2: Percent distribution of OTA in different analyzed matrixes.

  Figure 1

Table I .

 I Recoveries and limit of quantification (LOQ) for OTA in different analyzed

	Page 19 of 21		Food Additives and Contaminants
	matrixes
	Food commodity		Recovery (%)	LOQ (ng/g)
	Rice Peanut	F o r		83.0 93.6	0.021 0.027
	Pistachio Walnut	P e	86.8 72.1	0.027 0.027
	Dried figs Dried raisins	82.4 75.5 r e	0.030 0.030
				R
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http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

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

Acknowledgments

This research has been supported by the Spanish Agency for International Cooperation "AECI" (Programme Mixte Inter-Universitaire Maroco-Espagnol 2004, Project No. 128/P/M/04) and the Spanish Ministry of Education and Science (AGL-2003-01407).

The first author is most grateful to the Presidency of Mohamed V University Rabat-Agdal for their collaboration. C. Juan thanks the grant given by Spanish Ministry of Education and Science.


R J Mccracken

D G Kennedy

Dr Glenn Kennedy
email: glenn.kennedy@afbini.gov.uk

Keywords: nitrofurans, eggs, LC-MSMS, eggshell, yolk, albumen

The detection, accumulation and distribution of he detection, accumulation and distribution of he detection, accumulation and distribution of he detection, accumulation and distribution of nitrofuran residues in egg yolk, albumen and shell nitrofuran residues in egg yolk, albumen and shell nitrofuran residues in egg yolk, albumen and shell nitrofuran residues in egg yolk, albumen and shell

INTRODUCTION INTRODUCTION INTRODUCTION INTRODUCTION

Nitrofuran antibiotics were prohibited for use in food-producing animals in the European Union (EU) and other countries, in response to evidence that these compounds are both carcinogenic and mutagenic (van [START_REF] Van Koten-Vermeulen | Toxicological evaluation of certain veterinary drug residues in food[END_REF].

In 2002 the EU had to impose restrictions on the import of poultry products from Thailand (Commission Decision 2002/251/EC) and Brazil (Commission Decision 2002/794/EC), countries whose food exports to the EU were found to contain nitrofuran residues. Since May 2003, more than150 Rapid Alerts have been issued by the EU through its Rapid Alert System for Food and Feeds (http://europa.eu.int/comm/food/fs/sfp/ras_index_en.html) regarding the detection of nitrofurans in food from 29 countries. The presence of such residues in food, either through illegal use of the drugs or through inadvertent contamination, has made it essential for monitoring authorities to have appropriate detection methods in place to prevent such residues reaching the consumer.

An analytical method using LC-MSMS was developed under the auspices of FoodBRAND (www.afsni.ac.uk/foodbrand), an EU funded project (Cooper et al. 2005a). This method and others, based on Solid-Phase Extraction [START_REF] Leitner | Determination of the metabolites of nitrofuran antibiotics in animal tissue by high-performance liquid chromatographytandem mass spectrometry[END_REF]Connely et al. 2003), are now used throughout the world to detect the marker residues of the nitrofuran drugs. Previous reports have shown that the tissue-bound side chain metabolites are the most suitable markers for nitrofuran detection in animal tissues, because they are more stable in vivo than the parent compounds and persist in tissues for many weeks (Cooper et al. 2005a). However, in the case of nitrofurazone (NFZ), whose side-chain metabolite is semicarbazide (SEM), this practice has proved unreliable, because SEM has been attributed to other sources of contamination in food [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]. Following a spate of positive SEM detection in coated chicken products, the source of SEM contamination was attributed to the presence of azodicarbonamide, a flour treatment agent, used in the coating material. It did not therefore indicate the use of nitrofurazone in chicken production.

Consequently, attention has focused upon finding alternatives to SEM as a marker for the detection of nitrofurazone abuse. Recently, it was reported that intact nitrofurazone accumulates in whole eyes of chickens treated with the drug (Cooper et al. 2005b). The detection of the parent drug in retina is therefore a realistic alternative to SEM to monitor for abuse in primary production. This, of course, is not possible for the analysis of processed meat products. Arising from the crisis in 2002 of nitrofuran contamination in imported poultry and shellfish, and the subsequent doubts over the use of SEM as a marker residue is the need for further information on the accumulation of nitrofurans in food products. This requirement goes hand-in-hand with the need for sensitive confirmatory methods for detecting appropriate residues, as required under Commission Decision 2002/657/EC.

As part of a continuing investigation into the most appropriate markers for nitrofuran detection and the development of confirmatory methods, an evaluation of the residues for the four main nitrofurans in chicken eggs was undertaken. It has been known for some time that furazolidone (FZD) accumulates as intact parent compound in chicken eggs [START_REF] Botsoglou | Determination of furazolidone in eggs by high-performance liquid chromatography[END_REF][START_REF] Mccracken | Evaluation of the residues of furazolidone and its metabolite, 3-amino-2-oxazolidinone (AOZ), in eggs[END_REF]. Levels of the drug reached a plateau of approximately 300 µg kg -1 after the fourth day of treatment, and parent compound could still be detected up to the ninth day after withdrawal from medication. Two analytical methods were required to monitor the accumulation of residues in eggs. Firstly, the existing LC-MSMS method for nitrofuran metabolites in tissues was applied to the analysis of eggs. Secondly, a new LC-MSMS method for the detection of the intact parent compounds was developed. This new method is presented for the analysis of whole egg, with validation data obtained using the guidelines contained in Commission Decision 2002/657/EC. The accumulation of parent compound and side-chain metabolites in yolk, albumen and shell is presented in order to illustrate, that for poultry eggs, the analysis of intact parent compound residues is feasible as part of a residue monitoring programme. The procedure is less complicated than the detection of metabolites, and samples are processed rapidly, thus providing a satisfactory alternative to metabolite testing. This is particularly important in the case of nitrofurazone, because the parent compound can be determined instead of semicarbazide, which has been shown to be insufficiently specific as an indicator for nitrofurazone use.

The study of nitrofuran accumulation in chicken eggs was extended to include the analysis of the drugs and metabolites in eggshell. There are several varied applications for the utilisation of eggshell waste from poultry production. In the US, scientists have produced dietary phosphorous supplements for poultry, pigs and pets [START_REF] Miller | Using the old egg (shell)[END_REF]. Eggshell has been used to replace limestone as a source of calcium, and has been combined with phosphoric acid to produce mono-and di-calcium phosphate. The use of eggshell powder as a source of calcium has also been evaluated for human nutrition [START_REF] Schaafsma | Mineral, amino acid and hormonal composition of chicken eggshell powder and the evaluation of its use in human nutrition[END_REF]. It has been demonstrated that eggshell matrix proteins enhance calcium transport in human intestinal epithelial cells [START_REF] Daengprok | Hen eggshell matrix proteins enhance calcium transport in the human intestinal epithelial cells[END_REF]. Collagen, derived from eggshell membrane, is used in cosmetics, wound dressings, surgical implants and intrusive device coatings. Gelatine is used in the food and confectionery industries, and sialic acid is a precursor for use in anti-inflammatory drugs. The utilisation of eggshell waste is therefore another route potentially for nitrofuran contamination to reach the consumer.

The work presented therefore has three important implications. Firstly, that confirmation of nitrofuran intact parent compounds in eggs is possible using LC-MS/MS. Secondly, that intact parent compounds accumulate in eggs and are appropriate markers for monitoring nitrofuran abuse in laying chickens. Thirdly, that nitrofurans accumulate in eggshell and because of the utilisation of eggshell waste there exists the potential for nitrofurans to reach the consumer by this route.

EXPERIMENTAL EXPERIMENTAL EXPERIMENTAL EXPERIMENTAL

Materials

Furazolidone (FZD), nitrofurazone (NFZ), furaltadone (FTD) and nitrofurantoin (NFT) were obtained from Sigma-Aldrich (Poole, UK). The metabolite compounds: 3-amino-2-oxazolidinone (AOZ) and semicarbazide (SEM) hydrochloride were obtained from Sigma-Aldrich; 1-amino-hydantoin (AHD) hydrochloride and 3-amino-5-morpholinomethyl-1,3-oxazolidin-2-one (AMOZ) were obtained from Chemical Synthesis Services (Craigavon, UK). D 4 -Furazolidone, D 5 -furaltadone, 13 C 15 N 2 -nitrofurazone, D 4 -AOZ, and D 5 -AMOZ were also obtained from Chemical Synthesis Services. 13 C 15 N 2 -SEM and 13 C 3 -AHD were obtained from Witega (Berlin, Germany). All other reagents and solvents were analytical and HPLC grade, respectively. Samples of whole egg, yolk, albumen (5 g) or shell (0.5 g) were placed in 30 ml glass centrifuge tubes. Internal standard solution (50 µl of 1 µg ml -1 solutions in methanol) containing D 4 -FZD, D 5 -FTD and 13 C 15 N 2 -NFZ was added to each tube. Ethyl acetate (12 ml) was added, and samples were homogenised using a Silverson highspeed emulsifier for 1 min. Following centrifugation at 2200 rpm for 15 min., supernatants were transferred to clean tubes and evaporated to dryness under nitrogen at 50 o C. Acetonitrile (5 ml) was added to the dry residue, and hexane (3 ml) was added while vortexing. Following centrifugation, the hexane layer was discarded and the acetonitrile evaporated under nitrogen at 50 o C. The dry residue was redissolved in 500 µl of methanol-water (1:1, v/v), transferred to Eppendorf vials and centrifuged at 10 000 rpm for 10 min. Aliquots (140 µl) were transferred to autosampler vials prior to analysis by LC-MS/MS.

LC-MS/MS analysis of parent nitrofurans.

An Agilent 1100 Series HPLC system (Agilent Technologies, USA) coupled to a Quattro Ultima ® Platinum tandem mass detector (Micromass, UK) incorporating MassLynx ® software were used for nitrofuran analysis. The mass spectrometer was operated in electrospray positive mode for the analysis of FZD and FTD, and in electrospray negative mode for NFT and NFZ. Data acquisition was performed in multiple reaction monitoring (MRM) mode. The time-sectored ion transitions, dwell times, cone voltages and collision energies are shown in Table 1. The source settings were as follows: source and desolvation temperatures were 120 and 350°C, respectively, cone and desolvation gas flows were 99 and 800 L hr -1 , respectively, The HPLC analytical column was a Columbus C18 (2) 5µm, 2.0 x 150 mm (Phenomenex, UK). A binary gradient mobile phase was used at a flow rate of 0.2 ml min -1 . Solvent A consisted of 0.5 mM ammonium acetate and methanol (80:20, v/v), and solvent B was methanol. The gradient ran from 10 % solvent B initially to a maximum of 80% after 8 min, was held until 15 min, returning to 10 % after 15.5 min. The sample injection volume was 5 µl. Nitrofuran concentrations in samples were calculated by comparing the ratio of the nitrofuran base peak to the internal standard peak with the same ratio in the calibration curve standards.

Validation of the method for parent nitrofurans in eggs.

The method for the determination of parent nitrofuran compounds was validated for whole egg homogenates in accordance with the guidelines stated in Commission Decision 2002/657/EC. Whole egg homogenate, fortified with each of the nitrofurans (FZD, FTD, NFT and NFZ) at concentrations of 2, 3 and 4 µg kg -1 were analysed on three separate occasions (n=7). Samples were quantified against standard calibration curves in the range, 0, 1, 2, 5, 10 and 20 µg kg -1 .

Determination of nitrofuran side-chain metabolites in eggs using LC-MS/MS.

The protein-bound, side-chain metabolites for each of the nitrofurans were determined using the extraction procedure reported previously for the determination of AOZ in eggs [START_REF] Mccracken | Evaluation of the residues of furazolidone and its metabolite, 3-amino-2-oxazolidinone (AOZ), in eggs[END_REF]). Identification and quantification was performed using liquid chromatography tandem mass spectrometry.

The mass spectrometric

Treatment of laying hens with nitrofurans.

Four groups of six laying hens were held in wire-floored cages. They were allowed ad libitum access to fresh water at all times. Each group was fed a standard layers diet for three days prior to commencement of the experiment. After this period one group was fed a layer feed medicated at 300 mg kg -1 with furazolidone. In the same way the other groups were fed with furaltadone, nitrofurantoin or nitrofurazone. After feeding for 1 week and with no withdrawal from treatment, eggs were collected for 2 days from each cage. They were separated into yolk, albumen and shell, and analysed for the presence of bound residues of AOZ, AMOZ, SEM and AHD, and for parent compounds. The analysis for parent compounds was performed on the day of sampling.

RESULTS AND DISCUSSI RESULTS AND DISCUSSI RESULTS AND DISCUSSI RESULTS AND DISCUSSION ON ON ON

Method validation results for the analysis of parent nitrofurans are shown in Tables 2 and3. Calibration curves for standard solutions were linear in the range 1-20 µg kg -1 (Table 2). Decision Limits (CCα) for the parents were <1 µg kg -1 for all four compounds (Table 3). Detection Capabilities (CCß) were lower for furazolidone and furaltadone by comparison with nitrofurazone and nitrofurantoin. Nevertheless, confirmation of residues was possible at 2 µg kg -1 for all four compounds in all of the replicates analysed. Intra and inter run CVs are well within the acceptable values predicted by the Horwitz equation (Commission Decision 2002/657/EC).

Concentrations for furazolidone, furaltadone and nitrofurazone were calculated using the appropriate internal standard. An internal standard was not available for nitrofurantoin, however concentrations were calculated using the D 4 furazolidone.

For this reason, recovery values for nitrofurantoin were lower than the other 3 nitrofurans.

There are numerous methods reported for the determination of nitrofuran residues in eggs. [START_REF] Kumar | Determination of nitrofuran residues in poultry muscle tissues and eggs by liquid chromatography[END_REF] reported an LC method with UV detection. The limits of detection were 1 µg kg -1 for nitrofurazone and furazolidone; and 2 µg kg -1 for furaltadone. This procedure, however, would not meet present EU criteria as a confirmatory method. [START_REF] Draisci | Determination of nitrofuran residues in avian eggs by liquid chromatography-UV photodiode array detection and confirmation by liquid chromatographyionspray mass spectrometry[END_REF] using LC-ion spray mass spectrometry with an atmospheric pressure ionisation interface. Limits of detection were 3.2, 1.6 and 1.0 µg kg -1 for nitrofurazone, furazolidone and furaltadone, respectively. Only single protonated-molecular ions were monitored, and again this would not meet EU criteria as a confirmatory method.

Since 2002, and the start of the crisis related to nitrofuran contamination in poultry and shellfish, most residues monitoring programmes have focussed upon the detection of the side-chain metabolites. This was justified on the basis of the stability in vivo of metabolite residues compared to the parent compounds. However, this is not necessarily the case when considering other matrices such as eggs. [START_REF] Botsoglou | Determination of furazolidone in eggs by high-performance liquid chromatography[END_REF] and is 1.1 days [START_REF] Cooper | A study of the kinetics of semicarbazide and nitrofurazone in chicken eggs and egg powders[END_REF]. Parent nitrofurazone was detected in eggs up to 3 days post-withdrawal when chickens were fed at only 1% of the therapeutic dose (3 mg kg -1 ). In eggs pasteurised for 4 min. at 64-66 o C, followed by spray drying at 180-230 o C, parent nitrofurazone depleted by 65%. It is possible, however that albumen powder containing over 5 µg kg -1 , SEM may contain detectable quantities of parent nitrofurazone.

The distributions of parent compound and side chain metabolite residues in yolk, albumen and shell for each nitrofuran are shown in Table 4. For all four drugs, residues of parent compounds accumulated in yolk, albumen and shell. The chemical basis for the detection of AOZ, AMOZ, AHD and SEM means that any parent compound present in the sample will also be measured as its corresponding metabolite together with any other moiety containing acid-releasable AOZ, AMOZ, AHD or SEM. In this study, where there was no withdrawal from treatment before eggs were collected, it was expected that most of the residues occurring in the yolk and albumen might be present as intact parent compound. This was not the case. In terms of the number of moles detected, the concentrations of side-chain metabolites greatly exceeded that of the parent compound for all four nitrofurans. This suggests that over the 6-day treatment period, and despite zero withdrawal, most of the drug was present not as the parent -but as other moieties which contain the intact side-chain. [START_REF] Mccracken | Evaluation of the residues of furazolidone and its metabolite, 3-amino-2-oxazolidinone (AOZ), in eggs[END_REF] reported that the concentration of furazolidone in egg homogenates stored at -20°C fell by 44% after 55 days. In the present study however, eggs were analysed immediately after collection for parent compound residues. Any For furazolidone the albumen/yolk ratio of parent residues was 0.7 compared to 1.02 of AOZ. Similar values were found for furaltadone: 0.82 of parent and 1.06 for AMOZ; and for nitrofurantoin: 0.83 parent compared to 1.01 for AHD. Residue concentrations of nitrofurantoin were lower than the other nitrofurans, once again reflecting the poor absorption of this drug in chickens [START_REF] Mccracken | Transfer of nitrofuran residues from parent broiler breeder chickens to broiler progeny[END_REF]. The concentration of residues and their distribution in yolk and albumen was quite different in the case of nitrofurazone. Higher concentrations of parent compound were detected in yolk (73%) compared to albumen (23%), giving an albumen/yolk ratio of 0.31. The albumen/yolk ratio for SEM was 0.55. Reasons for the difference in distribution between compounds may be attributable to the physiochemical characteristics of the drugs and to the physiology of the hen and the formation of the egg. Kan and Petz (2000) compiled a database from the literature on drug residues in egg yolk and white. White/yolk ratio values for the four nitrofurans compare favourably with those reported here, but the authors concluded that there was no single factor apparent that determines the distribution of drug between yolk and albumen. The difference between the yolk/albumen ratio for nitrofurazone and the other nitrofurans therefore may not be significant. Contrary to this, Kan and Petz also discussed the striking difference in distribution between yolk and white of narasin and salinomycin (white/yolk ratios of 0.31 and <0.007, respectively). These large molecules (molecular weights: 765 and 751 for narasin and salinomycin, respectively) differ only by one methyl group. Similarly, the differences in distribution of the nitrofurans may in part be due to the molecular structure of the compounds. The most striking difference in residue distribution of the nitrofurans however, was that 50% of the total SEM residues found in the egg were detected in the shell. A mean concentration of 24.2 nmol g -1 of SEM was found in shell compared to only0.24 nmol g -1 of parent nitrofurazone. Eggshells used as negative controls were free of SEM, which eliminates the possibility of SEM occurring by any means other than nitrofurazone treatment of the laying birds. This concentration is significantly higher than for the side-chain metabolites of the other nitrofurans and illustrates the potential for SEM contamination to occur in eggshell products. The concentration of residues in shell were slightly higher for furazolidone compared to furaltadone but did not amount to more than 10% of the total residues in the egg. The result reported for NFT in shell represents detection of the drug in only 1 of the six samples tested.

The utilisation of eggshell waste is a growing industry. Eggshell is mainly composed of calcium carbonate (94%), but also contains small amounts of magnesium carbonate, calcium phosphate and organic matter including protein. AOZ, AMOZ, AHD and SEM have been considered as part of the side-chain metabolites of the nitrofurans that are bound to protein in the tissues of the animal. It is not known whether all of the metabolites detected in the eggshell are bound to the eggshell proteins, but it remains a possibility. However, it is clear that nitrofuran residues accumulate in eggshell and therefore pose a threat to health, where eggshell products are consumed.

In conclusion, residues of nitrofurans are deposited throughout the stages of egg production: in the development of yolk, in the albumen and during the shelling process. These residues, at least with zero withdrawal from treatment, can be detected as intact parent compound. The method presented is suitable as a confirmatory

  , RF lens 5.0. Argon was used as collision gas and the multiplier was operated at 650 V.

  (1 g) or shell (0.5 g) was mixed in a 50 ml centrifuge tube with internal standard solution containing D 4 -AOZ, D 5 -AMOZ, 13 C 15 N 2 -SEM and 13 C 3 -AHD (50µl of 100 ng ml -1 ), 4 ml of water, 0.5 ml of 1 M hydrochloric acid and 150 µl of 2-nitrobenzaldehyde (50 mM in dimethylsulphoxide). The tube was stoppered and the mixture incubated in a water bath at 37 o C for 16 hours. Nitrofuran side-chain metabolites (AOZ, AMOZ, SEM and AHD) present in the egg sample were derivatised by the 2-nitrobenzaldehyde to produce the corresponding nitrophenyl derivative. Following the incubation, the pH of the mixture was adjusted to approximately 7.4 by the addition of 5 ml of 0.1 M dipotassium hydrogen orthophosphate and 0.4 ml of 1M sodium hydroxide. The nitrophenyl derivatives were extracted by shaking twice with 5 ml of ethyl acetate. The solvent was removed under nitrogen at 50°C and the residue redissolved in 0.5 ml of methanol:water (1:1, v/v). The sample was transferred to a centrifuge tube and centrifuged at 10 000 rpm for 10 min. An aliquot of the supernatant was transferred to an autosampler vial for LC-MS/MS analysis.

  [START_REF] Mccracken | Evaluation of the residues of furazolidone and its metabolite, 3-amino-2-oxazolidinone (AOZ), in eggs[END_REF] demonstrated that intact furazolidone residues accumulate in eggs and may still be detected as such up to 9 days after withdrawal of medicated feed. A Minimum Required Performance Limit (MRPL) of 1.0 µg kg -1 has been set for all four side-chain metabolites in poultry meat (Commission Decision 2003/181/EC). Subsequently, Commission Decision 2005/34/EC extended the MRPL definition to cover all matrices. No MRPL has been established for the nitrofuran parent drugs in eggs, or in any other tissue. The developed method may find two potential applications. Firstly, it may be used as the primary test used to detect nitrofuran residues in eggs. Secondly, it is quicker and simpler than methods for the determination of the side chain metabolites. It may also be used to back up the results obtained using the more widely used method for the detection of the side chain metabolites. Specifically, it might be used to confirm the presence of nitrofurazone following the finding of semicarbazide in eggs. This would provide unambiguous evidence for the administration of nitrofurazone to the chickens and would remove any possible ambiguity associated with the use of semicarbazide as a marker. Stability experiments performed at this laboratory and reported elsewhere indicate that while

  occurred during the period of treatment, and much of it prior to deposition in the egg.

  Figure 1a.

  Figure 1c

Figure 1a .

 1a Figure 1a. Chromatograms (un-smoothed data) of MRM in electrospray negative mode of a blank egg extract spiked with nitrofurazone at 2 µg kg -1 , and similar chromatograms of a standard solution equivalent to 2 µg kg -1 . Two transitions are shown for nitrofurazone: 197.2>150.2 and 197.2>124.2; and 200.2>153.2 corresponding to the internal standard 13 C 15 N 2 -nitrofurazone.

Figure 1b .

 1b Figure 1b. Chromatograms (un-smoothed data) of MRM in electrospray positive mode of a blank egg extract spiked with furazolidone at 2 µg kg -1 , and similar chromatograms of a standard solution equivalent to 2 µg kg -1 . Two transitions are shown for furazolidone: 226.2>209.3 and 226.2>139.2; and 230.2>139.2 corresponding to the internal standard D 4 -furazolidone.

Figure 1c .

 1c Figure 1c. Chromatograms (un-smoothed data) of MRM in electrospray positive mode of a blank egg extract spiked with furaltadone at 2 µg kg -1 , and similar chromatograms of a standard solution equivalent to 2 µg kg -1 . Two transitions are shown for furaltadone: 325.3>281.3 and 325>252; and 330.3>286.3 corresponding to the internal standard D 5 -furaltadone.

Figure 1d .

 1d Figure 1d. Chromatograms (un-smoothed data) of MRM in electrospray negative mode of a blank egg extract spiked with nitrofurantoin at 2 µg kg -1 , and similar chromatograms of a standard solution equivalent to 2 µg kg -1 . Two transitions are shown for nitrofurantoin: 237.1>194.2 and 237.1>152.2.

Table 1 . Data acquisition parameters for the Multiple Reaction Monitoring (MRM) of parent nitrofurans.

 1 procedure and can be used together with the established method for the metabolites to form an effective residue control programme.

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Table 2 . Typical values for calibration of parent nitrofuran standard solutions in the

 2

	range 1-20 µ µ µ µg kg	-1
		Typical equation of curve	Correlation coefficient (r)
	Nitrofurazone	y = 0.7872x -0.033	0.9989
	Furazolidone	y = 0.8322x + 0.194	0.9971
	Furaltadone	y = 0.9698x + 0.206	0.9993
	Nitrofurantoin	y = 0.0781x + 0.011	0.9977

Table 3 . Summary of validation results for the determination of parent nitrofurans in whole egg homogenate.

 3

		Fortification Level (µ µ µ µg kg -1 )	Overall Mean (µ µ µ µg kg -1 ) n=21	Overall recovery (%)	Within Day CV	Between Day CV
	Furazolidone	2.0	2.2		110	6.4	7.4
		3.0	3.3		110	7.0	6.8
		4.0	4.5		113	5.7	9.0
		CCα α α α	0.7 µ µ µ µg kg	-1
		CCß	1.2 µ µ µ µg kg	-1
	Furaltadone	2.0	2.3		115	6.7	6.2
		3.0	3.3		110	4.8	4.9
		4.0	4.6		115	4.5	7.6
		CCα α α α	0.5 µ µ µ µg kg	-1
		CCß	0.8 µ µ µ µg kg	-1
	Nitrofurazone	2.0	2.3		115	9.6	11.8
		3.0	3.3		110	10.1	12.9
		4.0	4.6		115	7.0	12.1
		CCα α α α	1.0 µ µ µ µg kg	-1
		CCß	1.7 µ µ µ µg kg	-1
	Nitrofurantoin	2.0	1.5		75	10.0	23.5
		3.0	2.3		77	7.3	6.9
		4.0	2.8		70	7.3	8.4
		CCα α α α	1.0 µ µ µ µg kg	-1
		CCß	1.7 µ µ µ µg kg	-1

Table 4 . The distribution of nitrofuran parent compound and side chain metabolite residues in yolk, albumen and shell.

 4

			Parent compounds		Side-chain metabolites
			(Mean ± ± ± ± SE, n=6)		(Mean ± ± ± ± SE, n=6)
		Yolk	Albumen	Shell	Yolk	Albumen	Shell
	Furazolidone
	nmol g -1	1.82 ± 0.30 1.29 ± 0.16 0.23 ± 0.03	4.68 ± 0.57	4.81 ± 0.66 1.12 ± 0.38
	Furaltadone
	nmol g -1	1.83 ± 0.08 1.50 ± 0.10 0.11 ± 0.03	2.93 ± 0.23	3.10 ± 0.14 0.28 ± 0.07
	Nitrofurantoin
	nmol g -1	0.28 ± 0.02 0.23 ± 0.03	0.15	1.47 ± 0.12	1.49 ± 0.11 0.17 ± 0.03
	Nitrofurazone
	nmol g -1	4.18 ± 0.59 1.30 ± 0.13 0.24 ± 0.04 15.13 ± 0.81 8.44 ± 1.19 24.2 ± 2.97

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