Vito Simeone
email: simeone@iamb.it

Nuray Baser

Donato Perrelli

Gianluigi Cesari

Hamid El Bilali

Patrizia Natale

& P Natale

-Mai B ) -Organic

Residues of rotenone, azadirachtin, pyrethrins, and copper used to control Bactrocera oleae (Gmel.) in organic olives and oil

Keywords: Extraction, HPLC, Stripping Voltammetry Additives/Contaminants: Copper, Pesticide residues organic olive growing, rotenone, azadirachtin, pyrethrins, copper, residues, olives, oil

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

Introduction

Given the increasing concern about food safety, it is necessary to develop a control system of the production chain, which still displays some weaknesses, particularly for organic products. A knowledge of the post-treatment decay process for the active ingredients applied in organic agriculture on different substrates and in the various growing conditions to control pathogens is an important starting point to define proper application methods.

In the Mediterranean basin, olive growing and olive oil production play a major role in organic agriculture. However, the fungal infection by Bactrocera oleae (Gmelin) can seriously affect the quality of olives and therefore the quality of olive oil. Given the high incidence of infection, especially in some areas and on some varieties, appropriate control strategies are required, based also on the use of natural predators and/or products of plant origin (botanicals). The latter generally display a rapid degradation and a low toxicity risk for non-target organisms and mammals. However, their insecticidal action is sometimes of short duration (from a few hours to a few days), their cost is high and their availability on the market is limited. Furthermore, as it occurs with any plant protection product, they can entail risks for the operators and the environment. Among these natural substances, pyrethrins, rotenone, azadirachtin, quassine, nicotine ryanodine, sabadilla and some essential oils are the most used [START_REF] Bagnoli | Problematiche relative all'utilizzo di prodotti di origine vegetale per il controllo della mosca delle olive: i prodotti di origine vegetale (botanicals) nella lotta ai fitofagi[END_REF]. As regards azadirachtin, pyrethrum and rotenone, little is known about their residual activity in olives and olive oil.

Azadirachtin A and its isomers act on the feeding behaviour or as growth regulators [START_REF] Schmutterer | Properties and potential of natural pesticides from the neem tree, Azadirachta indica[END_REF]). Natural pyrethrins act, mainly by contact or ingestion, on the central and peripheral nervous system, thus causing immediately the insect paralysis [START_REF] Casida | Pyrethrum; a benefit to human welfare[END_REF]. Pyrethrins are used against B. oleae as adulticide with a two-day pre-harvest interval. Rotenone is a broad-spectrum bio-insecticide [START_REF] Copping | The BioPesticide Manual[END_REF]) which acts by contact and ingestion, inhibiting the respiratory processes. Recent studies have included rotenone among the causes of Parkinson's disease [START_REF] Sherer | Mechanism of toxicity in rotenone models of Parkinson's disease[END_REF].

Copper derivatives can be used in organic agriculture in accordance with the limitations set by Commission Regulation (EC) No 473/2002. The activity is exerted by the copper ion, which is mainly a fungicide, but also a repellent, an anti-oviposition elicitor and is active against the first larval stages of B. oleae [START_REF] Girolami | Aspetti della simbiosi batterica di Dacus olaee Gmelin in natura e negli allevamenti di laboratorio[END_REF][START_REF] Nuzzaci | Nuove metodologie di lotta antidacica[END_REF][START_REF] Tzanakakis | Considerations on the possible usefulness of olive fruit fly symbionticides in integrated control in olive groves[END_REF].

In the present work, the post-treatment decay curves of pyrethrins, azadirachtin, rotenone and copper were studied as these substances were applied on olives in different control programmes against B. oleae, to assess their persistence and their transfer into the olive oil. I. The first sampling was carried out before the applications and subsequent samplings after the applications, at intervals reported in Table II.

Materials and Methods

Trials in fields and preparation of samples

In order to evaluate the residues on the olives, each sample consisted of about 1 kg of olives collected from the whole canopy; in contrast, to assess the residues in the oil, about 20 kg of olives, harvested following the post-harvest interval for each active ingredient, were milled on the harvest day with an electric grinder and the oil was extracted from the paste by centrifugation (15-16% average oil content) at room temperature (12-16 °C).

Extraction of the chemical residues from olives and oil

Protocols by [START_REF] Caboni | Persistence of azadirachtin residues on olives after field treatment[END_REF] and [START_REF] Cabras | Rotenone residues on olives and in olive oil[END_REF] were applied to extract azadirachtin, rotenone and pyrethrins from the olives: 25 g of olives and 10 g of Na 2 SO 4 were added to 25 mL of acetonitrile, stirred for 10 min, and then sonicated for 15 min. The particulate was removed by filtering.

Extracts were kept at 4°C until testing. To detect the same substances in the oil, the extraction protocol by [START_REF] Cabras | Rotenone residues on olives and in olive oil[END_REF] was applied. Acetonitrile (10 mL) and 100 µL of oxalic acid (10% aqueous solution) were added to 1 g of oil aliquots. The mix, stirred for 30 min, was then allowed to stand for 15 min for the phase separation; the organic fraction was filtered onto regenerated cellulose microfilters, before being tested by HPLC.

To determine copper on the olives, olive aliquots (20 g) were placed in 100 mL of a 10% HCl aqueous solution. After 30 min stirring, the aqueous phase was recovered and then filtered prior to testing. To assess the presence of copper in the oil, 50 mL of n-hexane and 15 mL of 37% HCL were added to 20 g of oil. The mix was brought to light boil for 30 min and then allowed to cool to room temperature before the quantitative recovery of the lower hydrochloric acid phase. The extraction flask was washed 3 times with 10 mL of distilled water, making the mix boil lightly for 5 min; the aqueous phase was recovered and added to the hydrochloric acid phase collected earlier. The

HPLC analysis of chemical residues

The analytical determination of azadirachtin, pyrethrins and rotenone was carried out by a Thermoquest SpectraSystem ® Mod. 1000 (Thermo Finnigan, USA) liquid chromatograph, provided with a UV-DAD detector, model UV6000LP (TSP UV 6000 diode array detector) and a Waters X-Terra RP18 reporting the concentrations of increasing standard solutions as a function of the area of the relative chromatographic peaks. High correlation coefficients (0.995-1.000) were obtained between 0.08 and 5 mg kg -1 for azadirachtin, 0.005 and 5.0 mg kg -1 for rotenone, 0.02 and 5.0 mg kg -1 for pyrethrin II and pyrethrin I. The limit of quantification (LOQ) was 0.08 mg kg -1 for azadirachtin, 0.005 mg kg -1 for rotenone and 0.02 mg kg -1 for pyrethrins. The recovery of the active ingredient added to untreated matrices (samples were fortified with 0.5-5 mg kg -1 azadirachtin, 0.02-2 mg kg -1 rotenone, 0.1-2 mg kg -1 pyrethrins) was between 93 and 102%. Retention times were: 18.6 min for azadirachtin, 24.6 min for rotenone, 25.7 min for pyrethrin II and 27.3 min for pyrethrin I. Copper was determined by the standard addition method by chemical stripping by the equipment ION3 (Steroglass s.r.l, San Martino in Campo, Italy).

Copper determination was carried out at a potential range of -800/-100 mV, setting the electro deposition potential at -950 mV and introducing 20 ml of 1 N HCl into the chamber, including 1 ml of Hg(II) and 1 g l -1 of the extract to test. The integration interval was 370/-125 mV. For the quantitative analysis 100 µL of 5 mg kg -1 standard copper were added twice and three addition measurement cycles were performed. The linearity interval was determined between 0 and 1000 µg L -1 (R 2 >0.995 ). When olives or oil were fortified with 0.1-1.0 mg/kg copper, the recovery ranged between 90 and 100%.

Data management

Each evaluation of residue concentration is given as the average of the values coming from three different sample extractions ± standard deviation. Such values were converted into their natural logarithms and plotted as a function of time (in days) after applications, by first-order rate equations. Half-time decay values (DT 50 ) were obtained by these plots as ln2/k, where k represents the rate constant, and reported in the tables together with correlation coefficients (R 2 ). .

Results

In both testing years and in the experimental conditions applied, azadirachtin was not detected in any olive or extracted olive oil, even when three applications were carried out and sampling was performed immediately after olive drying. Probably, the levels of azadirachtin residues on the olives were so low as not to be detected by HPLC analysis, as a result of the low sensitivity of the method (0.08 mg kg -1 ) and the high decay rate of this chemical. Rotenone decay and half-life in olives given one to three treatments are indicated in Tables III-V. For olive samples collected for each trial 12 days after the last application, the residue proved to always be much higher than the admitted maximum residue level (MRL 0.05 mg kg -1 according to SO G.U.. 292, issued on Dec. 14 2004). The residue of rotenone found in the olives 12 days after a single treatment was 0.40 mg kg -1 (2005) and 0.37 mg kg -1 (2006), whereas, after three consecutive treatments, it reached 0.52 mg kg -1 (2005) and 0.46 mg kg -1 (2006). In the 2006 trial including three consecutive treatments, samples were collected up to 30 days after the last application; even at a such long time after application the residue concentration was still high (0.26 mg kg -1 ). The analysis of the decay rates indicates that, within 5 days after the application, rotenone degrades quite rapidly, and then the decay process goes on very slowly. In our experimental conditions, rotenone halflife on the olives (DT 50 = 4.5-5.5 days; R 2 = 0.9367-0.9951) proved to be slightly higher than that found by [START_REF] Cabras | Rotenone residues on olives and in olive oil[END_REF]. The rotenone concentration found in the oil samples was always higher than that estimated on the olives (Table VI). Generally, a proportionality was observed between the number of treatments and the residue amount in the oil; indeed, even 15 days after the last treatment, rotenone residues in the oil were 16 (0.82 mg kg -1 ) to 54 (2.69 mg kg -1 ) times higher than the MRL (0.05 mg kg -1 ). Residues in the oil were higher than those on the olives by a concentration factor between 2.0 and 5.5.

As regards pyrethrins, the results were expressed as the sum of pyrethrin I and II concentrations. Pyrethrins decay and half -life on olive samples are illustrated in Tables VII-IX. In general, a rapid decay (DT 50 = 1.0-1.5 days, R 2 = 0.94-0.9971) was observed and, from the second day after each application, it resulted into a residue below the admitted MRL on the olives (1 mg kg -1 ).

No accumulation effect was reported on the olives and no residue was found in the oil. The extraction technique applied quantified copper on the olive surface, regardless to the oxidation state of the metal. Results concerning copper on the olives are reported in Tables X-XII. In any trial and number of treatments tested, no decay of the copper ion was observed, even 50 days after applications. The accumulation effect was proportional to the number of applications. At the second application, the copper residue already exceeds its MRL on olives (30 mg/kg). In contrast, in both 2006 and 2005 trials, copper was detected in the oil at much lower concentrations than those detected on the olives: concentration ranges in the oil were 0.09-0.13 mg kg -1 in 2005 trial and 0.03-0.17 mg kg -1 in 2006 trial (Table XIII).

Discussion

The study reported here has allowed monitoring of residue rates for pyrethrins, rotenone and copper in olives and olive oil samples after different treatment procedures. Although azadirachtin was one of the chemicals used in the experiments, it was not possible to detect it on the treated olives, even when sampling was performed within 24 hours after application, probably because of the inadequate sensitivity of the method and the rapid decay of this chemical. However, the actual concentrations of azadirachtin on the treated olives must have been below the detection limit of the method (0.08 mg kg -1 ) and the MRL for this chemical is in fact 0.5 mg kg -1 (Commission Regulation (EC) No 149/2008). Thus, the quantities of azadirachtin used with the described treatments applied to olive trees would result in residue levels of no concern at any time after application. Also pyrethrins proved to be readily degradable and the residue, regardless of the matrix, was always lower than the accepted maximum limit (1 mg kg -1 ). Therefore, the use of pyrethrins and azadirachtin, within the tested conditions, may be feasible with respect to the current needs of consumers and demands for environmental safety: given the rapid decay observed, these substances can be applied also close to harvest, if necessary. Conversely, copper was the chemical which showed the slowest decay on the olives. Twenty days after the last application, as indicated in the label the residue level found was similar to that detected immediately after the application; moreover, the active ingredient increased on the olives according to the number of applications. Considering that copper-based products are generally applied to control plant pathogenic bacteria and fungi, and taking into account the strong accumulation effect on the olives, copper should only be used as an insecticide in the framework of an integrated management programme, especially when olives are to be marketed for fresh consumption.

Indeed, in the oil, the metal was always found at concentrations lower than the accepted maximum limits. Since copper is concentrated in the olive aqueous fraction, it might be removed from the paste in the extracted water. It is necessary to validate this hypothesis above all for the appropriate management of vegetation water, which might contribute to copper ion accumulation when it is disposed in the soil.

Rotenone was found to be a very stable compound on olives. Its decay, was quite rapid in the early days after its application, decreased when the concentration on the olives reached 0.4-0.5 mg kg -1 (values 10-fold over the limit) and continued slowly, without ever going below the maximum residue limit, not even 12 days after the treatment (two days more than the postharvest interval). For rotenone, as well, a slight accumulation effect was found in the olives and olive oil, when two or three applications were consecutively performed. The highly lipophilic molecule [START_REF] Cabras | Rotenone residues on olives and in olive oil[END_REF] markedly concentrated in the oil, where it reached an amount even five times higher than that detected on the olives (1.2-2.7 mg kg -1 ). The effective biological activity of rotenone makes it a key active ingredient in the control of Bactrocera oleae, mainly in the case of heavy infection. However, a possible involvement of rotenone in Parkinson's disease was recently reported [START_REF] Sherer | Mechanism of toxicity in rotenone models of Parkinson's disease[END_REF]). Thus, because of the possible hazard to human health, the slow degradation of rotenone on olives has caused concern about its use in the organic cropping of olive trees, as the residue concentration never falls below the legal limits, even finding alternatives to the use of rotenone is urgent for bio-farms producing olives for the fresh market and/or olive oil. The much more rapid decay observed with pyrethrins and that it was not possible to even detect azadirachtin residues provided to olive trees in this study, taking into account the positive data on the efficacy against the olive fly, suggest that these active ingredients may represent more ecologically-sound control strategies, which may be beneficial to the consumer, the environment, the operators and the host-pest system.

  All the procedures applied to field studies were carried out under Good Laboratory Practice (GLP). The trials were carried out, in 2005 and in 2006, on two different organically cropped olive tree fields: in 2005, the field, cropped with cv. Nocellara del Belice was at the bio-farm "La Pizzica" in the area of Crispiano (Taranto, Italy); in 2006, the field cropped with cv. Coratina, was located inside the Mediterranean Agronomic Institute, in the area of Valenzano (Bari, Italy). Trees from the two cultivars were about 10 years old, and cropped, vase-trained, with a 6 x 6 (cv. Nocellara) or 8 x 8 (cv. Coratina) m planting distance. Twelve plots were identified per cultivar, each including 6 plants, which were submitted to one to three treatments with azadirachtin (Trial A), rotenone (Trial B), pyrethrum (Trial C) and copper (Trial D). Treatments were carried out with a knapsack sprayer, applying 6 or 10 L per tree, respectively in 2005 and in 2006. The product characteristics, -harvest interval are reported in Table

  to 50 mL with distilled water, was eluted through a Supelclean NVI-CARB SP 500 mg column.At least three different extractions were carried out on each olive and oil sample.

(

  250 x 4.6 mm, 5µm) column. The gradients for the pesticide separation were 80 H 2 O:20 acetonitrile up to 18 min, 50 H 2 O:50 acetonitrile up to 22 min, 40 H 2 O: 60 acetonitrile, up to 27 min, 20 H 2 O: 80 acetonitrile until the end of the run. Samples of 20 µL were injected, elution set at 1 mL min -1 flow rate and the column kept at 25°C. LC analysis for azadirachtin, rotenone and pyrethrins was carried out at 215, 295 and 230 nm, respectively, according to the absorbance peak of the relative UV spectra. Calibration curves were plotted by the External Standard method,

  treatment is carried out. Thus, from April 10, 2008, the EU Commission recently removed rotenone from Annex I of the Directive 91-414-CEE, although, its use is still permitted until October 10 2009. Therefore,

Table I .

 I Characteristics of the chemicals applied to the olive trees

					Food Additives and Contaminants	Page 16 of 26
	Table II. Treatments, sampling and harvesting times in 2005 and 2006 trials
	Active ingredient	Trial	Active Ingredient Trial	Commercial product Treatment schedule a Company Name 2005	Product Sampling after each treatment Pre-harvest Harvesting time a (days) a.i. (%) dosage interval (days)
	Azadirachtin Rotenone Pyrethrins Copper Azadirachtin Rotenone Pyrethrins Copper a days/month	A B C D	Azadirachtin Rotenone Pyrethrins Copper F o r BioIntrachem Italia BioPirenPlus® Serbios Diractin® Serbios Rotena® Caffaro Cuprocaffaro® P e e r R e v i e 3 6 2 50 A-I --03/12 A-II -17/11 03/12 A-III 01/11 17/11 03/12 B-I --28/11 0, 4, 8, 12, 16 150 ml/hl 300 ml/hl 150 ml/hl 400 g/hl 0, 1, 2, 5, 9 0, 1, 2, 5 0, 1, 2 B-II -12/11 28/11 0, 4, 8, 12, 16 B-III 27/10 12/11 28/11 0, 4, 8, 12, 16 C-I --03/12 0, 1, 2, 3, 9 C-II -17/11 03/12 0,1, 2, 3, 9 C-III 01/11 17/11 03/12 0,1, 2, 3 D-I --14/11 0, 5, 21 D-II -29/10 14/11 0, 5,10,15 D-III 13/10 29/10 14/11 0, 10 2006 A-I --03/11 0,1,2,3 A-II -18/10 03/11 0,1, 2,3 A-III 02/10 18/10 03/11 0,1, 2, 3 B-I --31/10 0, 4 , 8, 12, 16, 30 B-II -15/10 31/10 0, 4, 8, 12, 16, 30 B-III 29/09 15/10 31/10 0, 4, 8, 12, 16, 30 C-I --03/11 0, 1, 2, 3 C-II -18/10 03/11 0, 1, 2, 3 C-III 02/10 18/10 03/11 0, 1, 2, 3 D-I --15/10 0, 5, 10, 15, 21 D-II -29/09 15/10 0, 5, 10, 15 D-III 13/09 29/09 15/10 0, 5, 10, 15, 20, 29 e i v F o r P e e r R e Table III. Rotenone decay on olives after one single application in 2006 and 2005 trials a concentration expressed as mg Kg -1 ± standard deviation nd = not determined Experimental Time (days) 2006 Trial 2005 Trial 0 2,2± 0,15 a 2,09± 0,37 4 1.07±0,12 1.04±0,25 8 0,76± 0,14 0,81± 0,2 12 0,37± 0,05 0,40± 0,26 16 0,29± 0,1 nd DT 50 5,4 5,3 R 2 0,9767 0,9713 Table IV. Rotenone decay on olives after two consecutive applications in 2006 and 2005 trials 3 10 2 20 07/12 12/12 06/12 06/12 08/11 15/11-05/12 08/11 07/11 O n l y Experimental Time (days) First application 2006 First application 2005 Experimental Time (days) Second application 2006 Second application 2005 0 2,42± 0,33 a 2,20± 0,4 16 2,73±0,45 2,5±0,4 4 1,09±0,22 0,98±0,13 20 1,02 ±0,23 1,01 ±0,22 8 0,734± 0,12 0,71± 0,14 24 nd 0,733±0,05 12 0,39± 0,09 0,42± 0,13 28 0,42±0,07 0,52±0,07 16 0,26± 0,08 0,24± 0,11 32 0,30±0,04 nd DT 50 5,0 5,2 DT 50 5,2 4,8 R 2 0,9865 0,9852 R 2 0,9595 0,9636 a concentration expressed as mg Kg -1 ± standard deviation F o r P e e r R e v i e w nd = not determined
						w w
						O n l O n l
						y y

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Page 17 of 26 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants F o r P e e r R e v i e w O n l yTable V .

 V Rotenone decay on olives after three consecutive applications in 2006 and 2005 trials Table VI. Rotenone residues on olives at harvest and in the extracted oil in 2006 and 2005 trials

	Food Additives and Contaminants	Page 18 of 26
	a concentration expressed as mg Kg -1 ± standard deviation nd = not determined Experimental Time (days) First application 2006 First application 2005 Experimental Time (days) 0 2,56± 0,32 a 2,05± 0,3 16 4 1,0± 0,25 nd 20 8 0,72± 0,17 0,71± 0,2 24 12 0,35± 0,13 0,44± 0,12 28 16 0,23± 0,15 0,21± 0,14 32 DT 50 4,7 5.0 DT 50 R 2 0,9773 0,9934 R 2 Table VII. Pyrethrin decay on olives after one single application in 2006 and 2005 trials Experimental Time (days) 2006 Trial 2005 Trial F o r P e e r R e v i e w Second application 2006 Second application 2005 2,74±0,21 2,40±0,3 1,54±0,29 1,19±0,3 0,67±0,11 0,67±0,12 0,42±0,18 nd 0,29±0,12 0,32±0,14 4,7 5,3 0,9815 0,9809 e r R e a concentration expressed as mg Kg -1 ± standard deviation e dala = days after last application; CF = concentration factor 2005 Trial Number of applications Olives (12 dala) Oil (15 dala) CF Olives (12 dala) Oil (15 dala) 1 0,37±0,05 a 1,18±0,06 3.2 0,41±0,01 0,82±0,05 2 0,41±0,06 2,25±0,05 5,5 0,42±0,06 2,14±0,06 0 0,75± 0,09 a 0,77± 0,15 1 0,33±0,04 0,38±0,13 F 2 0,27± 0,1 0,22± 0,1 3 0,18± 0,1 0,17± 0,08 o r 1,7 1,5 DT 50 R 2 0,9479 0,9684 3 0,56±0,08 2,69±0,08 4,8 0,46±0,02 2,50±0,05 P a concentration expressed as mg Kg -1 ± standard deviation	Experimental Time (days) 32 36 40 44 48 DT 50 R 2 O n l y Third application 2006 3,03±0,23 1,83±0,32 0,87±0,14 0,46±0,08 0,28±0,13 4,5 0,9952 2006 Trial CF Olives (30 dala) Oil (30 dala) Third application 2005 2,60±0,3 1,33±0,32 nd 0,52±0,18 0,31±0,09 5,3 0,9956 CF 2,0 0,13±0,03 0,27±0,02 2,1 5,1 0,13±0,01 0,68±0,01 5,2 5,4 0,26±0,05 1,04±0,01 4,0
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Table VIII .

 VIII Pyrethrin decay on olives after two consecutive applications in 2006 and 2005 trials

	Experimental	First application	First application	Experimental	Second	Second
	Time	2006	2005	Time	application	application
	(days)			(days)	2006	2005
	0	1,2± 0,1 a	1,03± 0,11	16	0.88±0,11	0.8±0,11
	1	0,62±0,1	0,57±0,09	17	0,54 ±0,05	0,65 ±0,05
	2	0,46± 0,08	0,34± 0,08	18	0,36±0,03	0,29±0,03
	3	0,22± 0,2	0,14± 0,02	19	0,16±0,1	0,16±0,08
	DT 50 R 2	1,25 0,9767	1,06 0,9854	DT 50 R 2	1,26 0,9756	1,07 0,943
	a concentration expressed as mg Kg -1 ± standard deviation

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 IX Pyrethrin decay on olives after three consecutive applications in 2006 and 2005 trials

	Experimental	First	First	Experimental	Second	Second	Experimental	Third	Third
	Time	application	application	Time	application	application	Time	application	application
	(days)	2006	2005	(days)	2006	2005	(days)	2006	2005
	0	1,2± 0,12 aa	1,3± 0,11	16	1,3±0,2	1,03±0,11	32	0,74±0,13	0,83±0,1
	1	0,65± 0,13	0,63± 0,09	17	0,69±0,1	0,52±0,07	33	0,33±0,12	0,42±0,1
	2	0,49± 0,11	0,45± 0,08	18	0,45±0,13	0,30±0,1	34	0,21±0,1	0,25±0,1
	3	0,21± 0,11	0,26± 0,07	19	0,27±0,08	0,24±0,1	35	0,13±0,04	0,11±0,08
	DT 50 R 2	1,26 0,9658	1,3 0,9871	DT 50 R 2	1,35 0,994	1,41 0,9586	DT 50 R 2	1,22 0,9791	1,05 0,9928
	a concentration expressed as mg Kg -1 ± standard deviation

Page 22 of 26 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants F o r P e e r R e v i e w O n l yTable X .

 X Copper concentration on olives after three consecutive applications in 2006 and 2005 trials

	Experimental	First	First	Experimental	Second	Second	Experimental	Third	Third
	Time	application	application	Time	application	application	Time	application	application
	(days)	2006	2005	(days)	2006	2005	(days)	2006	2005
	0	18,4± 3,4 a	16,4± 3,7	15		32,3±3,4	30		51,0±3,5
	5	18,5± 2,8	nd	20	31,9±3,1	30,9±3,1	35	54,9±2,3	50,7±5,7
	10	18,6± 3,0	15,5± 2,4	25	30,4±2,1	32,5±4,5	40	nd	51,2±4,1
	15	18,6± 2,2	nd	30	30,7±1,5	31,6±3,4	41	53,2±3,7	nd
	20	18,0±2,8	nd	35	29,1±3,3	33,0±4,4	45	nd	52,6±3,4
							54	nd	49,3±2,2
							56	52,3±4,4	nd
	a concentration expressed as mg Kg -1 ± standard deviation
	nd = not determined

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Table XI .

 XI Copper concentration on olives after two consecutive applications in 2006 and 2005 trials a concentration expressed as mg Kg -1 ± standard deviation nd = not determined

	Experimental Time (days) 0 5 10 15	First application 2006 Table XII. Copper concentration on olives after one First application 2005 Experimental Time (days) single application in 2006 and 2005 trials 17,9± 3,6 a 16,3± 3,2 15 Experimental 2006 Trial 2005 Trial 17,4±4,3 14,9±2,4 20 Time 16,6± 3,2 14,7± 3,2 25 (days) 16,5± 2,8 14,3± 3,1 30 34 39 0 14,6± 1,6 a 15,0±1,8 5 13,9±3,3 14,0±2,3 F 10 nd 14,4±1,1 15 nd 14,9±2,2 o 21 14,0± 2,5 14,4± 2,3 r a concentration expressed as mg Kg -1 ± standard deviation application Second 2006 34,1±4,1 nd nd 33,8±3,7 nd	Second application 2005 33,2±2,6 33,1±4,0 30,4±2,7 30,9±2,1 nd 29,7±3,0
		P nd = not determined
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	Table XIII. Copper concentration in the oil in 2005 and
	2006 trials
	Number of	2005 Trial	2006 Trial
	applications
	3 2 1 a concentration expressed as mg Kg -1 ± standard deviation 0,075± 0,002 a 0,029±0,0025 0,029± 0,003 0,17±0,0045 F r 0,011± 0,0013 0,077±0,0031 o
		P
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		R
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			v i e
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			O n l
			y

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Acknowledgements

The present work was carried out within the framework of the Interreg III A Project Italy-Albania 2000-2006 "Progetto finalizzato integrato per la diffusione e assistenza tecnica all'implementazione di metodologie per la produzione di prodotti biologici"; Acronym: (PAB). The authors wish to thank Prof. P. Cabras from the Toxicology Dept. of Cagliari University, and Dr S.

Pollastro from the Plant Protection Dept. of Bari University, for the scientific review of this paper.


Laura Anfossi

Claudio Baggiani

Cristina Giovannoli

Gianfranco Giraudi

DEVELOPMENT OF ENZYME-LINKED IMMUNOSORBENT ASSAYS FOR SUDAN DYES IN CHILLI POWDER, KETCHUP AND EGG YOLK

Keywords: Sudan dyes, ELISA, chilli powder, egg yolk

d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Sudan I, II, II and IV (Figure 1) are non-ionic fat-soluble dyes used as additives in gasoline, grease, oils, plastics, printing inks, and floor polishes [START_REF] Mejia | Determination of banned Sudan dyes in chili powder by capillary electrophoresis[END_REF] These dyes are classified by the International Agency for Research on Cancer as category 3 carcinogens to humans (IARC, 1975), and universally the use of Sudan I in foodstuffs is not permitted by food regulations (EC Regulation No 178/2002[START_REF] Donna | Assay of Sudan I contamination of foodstuff by atmospheric pressure chemical ionization tandem mass spectrometry and isotope dilution[END_REF]. Unfortunately, these dyes are still illegally used to intensify and maintain the colour of food products such as chilli, curry, and palm oil-containing foodstuffs.

From May 2003, warnings about chilli powder from Asia and alerts concerning a variety of foodstuffs contaminated with Sudan dyes were issued throughout Europe [START_REF] Han | Development of a higly sensitive and specific enzyme-linked immunosorbent assay for detection of Sudan I in food samples[END_REF] and, more recently, Sudan I was found in China in various commercial foodstuffs, including chilli sauce, meat and eggs [START_REF] He | Determination of Sudan dye residues in eggs by liquid chromatography and gas chromatography-mass spectrometry[END_REF]. This illegal use of Sudan dyes is a severe danger to public health, therefore simple, rapid and reliable analytical methods for the determination of these banned dyes in foodstuffs are required. The need for a method is also testified by the large number of papers concerning the development of extraction and detection methods for Sudan dyes in food (particularly, in chilli powder) which were published in 2007. The vast majority of analytical methods which have been described are based on HPLC with optical or mass spectrometric detection (Di [START_REF] Donna | Assay of Sudan I contamination of foodstuff by atmospheric pressure chemical ionization tandem mass spectrometry and isotope dilution[END_REF][START_REF] Ma | Simultaneous determination of water-soluble and fatsoluble synthetic colorants in foodstuff by high-performance liquid chromatography-diode array detection-electrospray mass spectrometry[END_REF][START_REF] Cornet | Development of a fast analytical method for the determination of Sudan dyes in chili-and curry-containing foodstuffs by high-performance liquid chromatography-photodiode array detection[END_REF][START_REF] Mazzetti | Determination of 1phenylazo-2-naphtol (Sudan I) in chilli powder and in chilli-containing food products by GPC clean-up and HPLC with LC/MS confirmation[END_REF], however, some gas chromatographic and capillary electrophoresis methods have also been reported [START_REF] Mejia | Determination of banned Sudan dyes in chili powder by capillary electrophoresis[END_REF][START_REF] He | Determination of Sudan dye residues in eggs by liquid chromatography and gas chromatography-mass spectrometry[END_REF]. Generally, chromatographic methods are expensive and time-consuming, therefore other techniques for detecting Sudan dyes have been developed. Among these, [START_REF] Han | Development of a higly sensitive and specific enzyme-linked immunosorbent assay for detection of Sudan I in food samples[END_REF] described the first enzyme immunoassay and applied it to the detection of Sudan I in some food samples (tomato sauce and juice, chilli sauce and powder). They prepared two hapten derivatives with different lengths of carboxylic spacers and used them to synthesize the immunogen and the coating antigen. The optimized indirect competitive ELISA showed itself to be sensitive and specific for Sudan I. However, when applied to samples, limits of detection were in the mg/kg range, which is dramatically higher than the detection capability of the chromatographic methods, 3 mainly because of the need for diluting the sample extracts from 1:100 to 1:1000 before analysis, in order to offset the matrix effect. Very recently, the preparation of a monoclonal antibody towards Sudan I, III and Para red and its use in the development of a sensitive competitive indirect ELISA has been described [START_REF] Ju | Enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody as a new tool to detect Sudan dyes and Para red[END_REF]. The determination of Sudan I in eggs by an immunoassay exploiting the resonance scattering of gold nanoparticles has also been reported [START_REF] Jiang | A higly sensitive and selective immunonanogold resonance scattering spectral assay for Sudan I[END_REF].

Nevertheless, the extraction of the dye from eggs requires a two-day protocol.

In this study, a Sudan I hapten derivative was synthesized, with the objective of firstly raising a polyclonal antibody characterized by low selectivity towards the other three Sudan dyes (II, III, and IV) and secondly to develop a competitive immunoassay able to detect the entire group of banned Sudan dyes. Different assay formats were compared: a direct and an indirect competitive ELISA were developed, and the simultaneous or subsequent addition of reagents was evaluated. Moreover, the extraction step and the solvent used to dissolve Sudan dyes were optimized with the aim of minimizing matrix interference without the contemporary need for an excessive dilution of extracts.

The assay which was developed was validated through comparison with an HPLC-UV method and proved to allow the determination of Sudan dyes in very different foodstuffs (chilli powder, ketchup and egg yolk) . Polyclonal antibodies (ammonium sulphate precipitated IgG fraction) were obtained from Davids Biotechnologie (Germany). Antibodies were prepared using the standard immunization protocol (Davids Biotechnologie, Germany) and sera were collected after 70 days from the first injection.

MATERIALS AND METHODS

Materials

Sudan dye stock solutions were prepared by dissolving the powders in chloroform and immediately diluting them in acetonitrile to the final concentration of 20 mg L -1 . Standard solutions were prepared by daily diluting the stock solutions with DMSO:water (10:90, v/v).

Synthesis of hapten and hapten-protein conjugates

The hapten used for immunization, antigen coating and enzyme labelling is depicted in Figure 1.

Carboxy-Sudan I (CSudan I) was synthesized as previously reported, [START_REF] Ju | Enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody as a new tool to detect Sudan dyes and Para red[END_REF]. CSudan I was then conjugated with BSA and HRP by the DCC/NHS ester method, as reported in the literature [START_REF] Hosoda | The preparation of steroid Nhydroxysuccinimide esters and their reactivities with bovine serum albumin[END_REF]. Briefly, equimolar amounts of CSudan I, DCC and NHS were dissolved in anhydrous DMF and the mixture was incubated at room temperature for 1 hour. Proper amounts of the mixture were then added to protein solutions prepared in 0.13 M NaHCO 3 , to obtain a final molar ratio of 50:1; 100:1; 200:1 (CSudan I:BSA), 20:1; and 10:1 (CSudan I:HRP). BSA conjugates were incubated overnight at room temperature, while HRP conjugates were reacted for 1 hour at room temperature. Separation of conjugates from by-products and the excess of reagents was carried out by gel filtration low pressure chromatography on Sephadex G-25 resin (mobile phase: phosphate buffer). Finally, sodium azide was added to BSA conjugates, which were stored at -18°C. The 200:1 conjugate was used as the immunogen, while conjugates with lower substitution degree were used as coating antigens. HRP conjugates were stored at 4°C, with 33% (v/v) of glycerol added. goat-antirabbit immunoglobulin conjugated to horseradish peroxidase was used to detect the immobilized rabbit antibody. The incubation time was 30 min, followed by three well washes.

Competitive indirect

Colour development was obtained by a 30 min incubation with TMB (200 µl per well). One hundred microlitres of sulphuric acid (1M) was used as a stop solution and absorbance was recorded at 450 nm. All standards and samples were measured in duplicate.

Competitive direct ELISA (simultaneous addition of reagents)

The immunoreactive solid phase was obtained by coating wells with 300 µl of a goat-antirabbit IgG solution (10 mg L -1 ) [START_REF] Giraudi | Effect of homologous and heterologous spacer arms of progesterone-horse radish peroxidase conjugates on the equilibrium constants for an immobilized anti-progesterone antiserum[END_REF]. Antiserum and CSudan I -HRP were diluted in PBSA.

A volume of 100 µl of diluted antiserum (1:15000, v/v) was added to 50 µl of 0.75 mg L -1 of CSudan I-HRP (20:1) and to 50 µl of Sudan I at concentrations ranging from 0 to 100 µg L -1 . The mixture was incubated 15 min in immunoreactive wells. Then the wells were washed with washing solution. Colour development and absorbance readings were obtained as described above.

Competitive direct ELISA (pre-immobilization of the antibody)

The same immunoreactive solid phase used for the format with the simultaneous addition of reagents was used in this case. In addition, 200 µl of diluted antiserum (1:50000, v/v) in PBSA was Colour development and absorbance readings were obtained as described above.

Calculations

Sudan I concentrations were determined by interpolation on the calibration curve, where the signal was plotted against the log of analyte concentration. For each experiment, a calibration curve was determined by a nonlinear regression analysis of the data of the standards using the four-parameter logistic equation [START_REF] Findlay | Validation of immunoassays for bioanalysis: a pharmaceutical industry perspective[END_REF].

Relative cross-reactivity was calculated as follows: CR% = (IC 50 Sudan I / IC 50 Sudan dye)*100 where IC50 is the Sudan dye concentration which cause 50% inhibition of the maximum observed signal.

Samples and sample preparation

The following commercial samples: 5 kinds of ketchup, 5 kinds of eggs, 10 kinds of chilli powder and paprika were purchased in large stores. One sample for each category that did not show any detectable residues of target dyes when analyzed by HPLC was taken as the blank for matrix interference experiment and fortification experiments. Sudan dye extraction was carried out as reported in the literature [START_REF] Ma | Simultaneous determination of water-soluble and fatsoluble synthetic colorants in foodstuff by high-performance liquid chromatography-diode array detection-electrospray mass spectrometry[END_REF]

HPLC analysis

To evaluate the accuracy and to validate the method, a comparative study using both the developed ELISA method and a HPLC reference procedure [START_REF] Thompson | Harmonized guidelines for single-laboratory validation of methods of analysis (IUPAC technical report)[END_REF] was performed. Samples were extracted as described above, except that DMSO extracts were diluted 1:5 (v:v) with the same solvent and filtered through a 0.45 µm nylon membrane filter, according to the literature [START_REF] Ma | Simultaneous determination of water-soluble and fatsoluble synthetic colorants in foodstuff by high-performance liquid chromatography-diode array detection-electrospray mass spectrometry[END_REF]. Each extract was injected in triplicate. An HPLC system (LaChrom Elite, VWR-Hitachi, Darmstadt, Germany) equipped with a C-18 Chromolith Performance 100-4.6 mm column (VWR, Darmstadt,Germany) was used. The mobile phase consisted of acetonitrile and water at a volume ratio of 80:20, delivered to the column at a rate of 1 ml min -1 . Detection was made by a UV detector (472 nm). Sudan I concentrations were calculated by interpolation on an external calibration curve.

RESULTS AND DISCUSSION

Production of polyclonal antibodies

Two rabbit antisera were raised against CSudan I-BSA (200:1). These were tested by a noncompetitive indirect ELISA, following the protocol described in the experimental section, with the difference that no Sudan I was added. Both antisera show very similar performance (Figure 1) and high titres on the coating antigen (IC 50 at 1:80 000 and 1:100 000, v/v, antibody dilution for rabbit 1 and 2 respectively). The non-specific binding of antisera towards the carrier protein was also evaluated, but resulted negligible in comparison with the binding towards the hapten (Figure 1).

Arbitrarily, rabbit 1 antiserum was used for this study. (v/v) and the coating antigen CSudan-BSA 50:1 at 0.2 mg L -1 was selected as the most suitable on the basis of the IC 50 value that was the lowest. Since Sudan dyes are very hydrophobic compounds, they should be dissolved and kept in organic solvent. A rapid literature review permitted us to identify a wide variety of solvents used to dissolve and extract Sudan dyes from food samples for determination by chromatographic techniques [START_REF] Mejia | Determination of banned Sudan dyes in chili powder by capillary electrophoresis[END_REF][START_REF] Ma | Simultaneous determination of water-soluble and fatsoluble synthetic colorants in foodstuff by high-performance liquid chromatography-diode array detection-electrospray mass spectrometry[END_REF][START_REF] Cornet | Development of a fast analytical method for the determination of Sudan dyes in chili-and curry-containing foodstuffs by high-performance liquid chromatography-photodiode array detection[END_REF][START_REF] Mazzetti | Determination of 1phenylazo-2-naphtol (Sudan I) in chilli powder and in chilli-containing food products by GPC clean-up and HPLC with LC/MS confirmation[END_REF]. The application as the analyte solvent in immunochemical methods of analysis narrowed the field, but acetonitrile and dimethylsulfoxide (DMSA) remained as possible candidates. Even if DMSO is rarely associated with ELISA measurement, both organic solvents were considered as the Sudan diluent to set up the standard curve. The solvent effect on the developed assay performance was studied by diluting the Sudan I standard solution in PBSA/DMSO and PBSA/acetonitrile (5, 10 and 20%, v/v) and carrying out the standard curve in the optimized condition. Low amounts of acetonitrile (below 10%) allowed an increase in the absolute signal to be observed, but was associated with limited detectability (IC 50 above 20 µg L -1 ).

On the contrary, the presence of DMSO (below 10%) lowered the signal of the blank, but allowed us to reach decidedly higher sensitivity (IC 50 below 10 µg L -1 ). The increase of the organic solvent amount from 5 to 10% produced a parallel doubling of the IC 50 value, whereas when the solvent percentage was increased up to 20%, the IC 50 increased more dramatically. Finally, 10% (v/v) of DMSO in PBSA was chosen as the solvent to prepare the dilute Sudan I standards. The time of incubation for the competitive reaction was then optimized and 15 min was selected as a satisfying compromise between the lowest IC 50 and a blank signal around 1-1.5 UA. Figure 3 shows a typical inhibition curve obtained under optimized conditions. The IC 50 value of the assay was 4 µg L -1 with a detection limit of 0.2 µg L -1 and a dynamic range of 0.4-100 µg L -1 .

Competitive direct ELISA

Competition experiments were carried out under various combinations of antibody and enzyme tracer concentrations. Two formats were optimized: the first involving the simultaneous reaction between antibody, tracer and analyte (dissolved in 10% DMSO) and a second one, which was based on a long pre-immobilization reaction of the antibody, followed by a short incubation of the mixture of the analyte and the tracer into functionalized wells. Figure 3 shows typical inhibition curves obtained under optimized conditions. Antibody dilutions, enzyme tracer concentrations and incubation times are summarized in Table 1, together with the analytical performance of the optimized assay in both formats. As expected, the pre-immobilization of the antibody allowed the use of lower amounts of antibody and competitor (the enzyme tracer) and consequently the improvement of assay sensitivity (lower IC 50 value). Nevertheless, the preparation of functionalized wells by the pre-immobilization of the antibody involves overnight incubation before carrying out the assay or the stabilization of functionalized wells.

Selectivity of the assays

Since, from a legal point of view, the occurrence of all four Sudan dyes (I-IV) should be assessed in food, the assay would be virtually able to cross-react with the four target analytes. To assess the influence of the assay format on the selectivity, cross-reactivity towards Sudan II has been measured by the three developed assay formats. The manipulation of the format of the immunoassay slightly influences the selectivity: the competitive direct ELISA with the preimmobilized antibody is the most selective assay (CR%=26%), whereas the other two formats show about the same selectivity (CR%=34% for the direct assay and 35% for the indirect ELISA). Since the format finally chosen to validate the method was the direct ELISA with the simultaneous addition of reagents, a more extensive study of selectivity was made using this assay format.

Results reported in Table 2 show that Sudan II is well recognized. Sudan III and IV and other Sudan dyes demonstrate a slight recognition, which means that the assay would be able to measure these compounds only at levels ten-times higher than Sudan I. This finding is not very far from the performance obtained by some instrumental methods of analysis based on chromatographic or electrophoretic determination of Sudan dyes [START_REF] Mejia | Determination of banned Sudan dyes in chili powder by capillary electrophoresis[END_REF][START_REF] Ma | Simultaneous determination of water-soluble and fatsoluble synthetic colorants in foodstuff by high-performance liquid chromatography-diode array detection-electrospray mass spectrometry[END_REF].

Several dyes, whose use is permitted in food (Department of Food Biosciences, University of Reading, UK), were also tested for cross-reactivity. The interference with the assay was found to be negligible for all tested compounds.

Better choice of format and its validation

To evaluate matrix interference on the three developed assays, blank samples of chilli powder, ketchup and egg yolk were analyzed in six replicates. Negligible interference was observed when extracts were analyzed by means of the direct ELISA format with the simultaneous addition of reagents. The other two formats needed a simple 1/2 (v/v) dilution of samples with the PBSA containing 10% of DMSO before the analysis to offset the matrix effect.

The LOD was calculated from interpolation of the Sudan I standard curve as the analyte concentration corresponding to the mean signal of the zero standard (obtained by averaging the signal of eight replicate sets) minus three times its standard deviation (according to IUPAC, ISO, and AOAC harmonized guidelines) [START_REF] Thompson | Harmonized guidelines for single-laboratory validation of methods of analysis (IUPAC technical report)[END_REF]. The LOD was found to be 0.2 µg L -1 for both the indirect and the direct ELISA with the pre-immobilization of the antibody. The direct ELISA with the simultaneous addition of reagents had lower sensitivity (calculated LOD 0.4 µg L - 1 ). This result can be simply explained by remembering that in the direct ELISA (with the 11 simultaneous addition of reagents) standards and samples are diluted 1/4 (v/v) in wells, while in the other two formats they are diluted 1/2. For the same reason, no further dilution was required to offset matrix interference in the direct ELISA with the simultaneous addition of reagents. On the contrary, the other two formats required an additional 1/2 (v/v) dilution, before analysis.

Consequently, in the end, the limits of detection of the three developed formats, compared to the solid sample, were substantially the same (10 µg kg -1 ).

Therefore, the direct competitive ELISA with the simultaneous addition of reagents, being rapid, sensitive and the easiest to carry out was chosen as the format to be fully validated. The LOD, recalculated for each different matrix by measuring and averaging data from the five blank samples, confirmed the previous estimation (10 µg kg -1 ) as regards ketchup and chilli powder.

The precision of the method was determined by extracting and analyzing replicates of Sudan I artificially contaminated samples (ketchup, chilli powder,), which were fortified with a Sudan I concentration of 25 (low level), 125 (medium level), and 625 (high level) µg kg -1 .

The assay was carried out in six replicates on the day for the evaluation of within-assay precision and on six different days for the evaluation of the between-assay precision. The values of RSD% were calculated at each nominal concentration level and ranged from 2 to 16%, which fulfilled FDA requirements for the validation of bioanalytical methods according to FDA guidance [START_REF] Viswanathan | Workshop/Conference Report -Quantitative bioanalytical methods validation and implementation: best practice for chromatographic and ligand binding assays[END_REF].

To evaluate the accuracy of the method, chilli powder and ketchup samples fortified with Sudan I (concentrations of 25, 125, and 625 µg kg -1 ), Sudan II, Sudan III, and Sudan IV (concentrations of 125 and 625 µg kg -1 ) were extracted and analyzed. Results are summarized in Table 3. Recovery values ranged from 70 to 103% for Sudan I, thus indicating a good accuracy of the assay when Fortification experiments were also conducted on egg yolk samples. This matrix has not been included in the work of [START_REF] Ma | Simultaneous determination of water-soluble and fatsoluble synthetic colorants in foodstuff by high-performance liquid chromatography-diode array detection-electrospray mass spectrometry[END_REF], thus no information regarding the possibility of extracting Sudan dyes from this kind of sample by means of the same protocol applied to other matrices was available. To our knowledge, this is also the first time that Sudan I has been measured via an immunoenzymatic method in egg yolk. Therefore, a blank sample of egg yolk was fortified with 25, 250 and 2500 µg kg -1 of Sudan I and extracted in triplicate according to the procedure described in the experimental section. Samples were analyzed both by the developed direct ELISA method and by HPLC and the obtained recovery values for both methods are reported in Table 4. The fortified sample at the lowest concentration could only be measured by means of the ELISA. The medium and high level fortified samples gave satisfactory recoveries and good agreement between the results obtained via the two methods. Generally speaking, the developed ELISA showed an increased imprecision of data, in comparison with fortification experiments made on samples belonging to different classes (chilli powder, paprika and ketchup), even if the relative standard deviation remained in the range considered acceptable for screening methods [START_REF] Viswanathan | Workshop/Conference Report -Quantitative bioanalytical methods validation and implementation: best practice for chromatographic and ligand binding assays[END_REF]. The LOD was, in this case, higher than for other considered matrices (15 µg kg -1 ), due to the greater data imprecision.

Comparison of ELISA and HPLC determination

A total of 20 samples (representative for all categories previously indicated) were analyzed with the newly developed direct competitive ELISA. Since no positive samples were found in the market, they were randomly fortified with Sudan I at different levels of contamination (from 50 to 2500 µg kg -1 ) and blindly analyzed by the two methods. Comparable results were obtained by the two methods: the linear regression analysis (Figure 4) yielded a good correlation between the methods (y=1.19x-10.0, r 2 = 0.991, n=20). A limited underestimation of the Sudan I concentration was observed at high contamination levels (above 500 µg kg -1 ), probably due to the low solubility of the analyte when diluted 1/10 in the PBSA for the analysis. In fact, if the two highest fortified samples are not included in the comparison between ELISA and HPLC, the agreement between methods further improves (y=1.09x-1.24, r 2 = 0.995, n=18). Thus, very highly contaminated samples, which can occur in the case of the illegal use of Sudan dyes [START_REF] Mazzetti | Determination of 1phenylazo-2-naphtol (Sudan I) in chilli powder and in chilli-containing food products by GPC clean-up and HPLC with LC/MS confirmation[END_REF], should be diluted in DMSO before diluting them in PBSA, in order to prevent Sudan I precipitation and low recoveries.

These results proved that the ELISA can be applied as screening methods for the detection of Sudan I in foodstuffs of very different kinds, with good accuracy and precision. The assay has good sensitive, sample extracts need a very limited dilution before being analyzed, and therefore the LOD in samples remains in the ten µg kg -1 range. In addition, the method is simple, rapid (the extraction requires 30 min and the assay is completed in 45 min), and applicable to very different matrices with very slight modifications, thus proving itself to be suitable as a first level screening method or for routine quality controls of various foodstuffs.

ACKOWLEDGMENT

All

  Sudan dyes, 4-aminobenzoic acid, 2-naphthol, bovine serum albumin (BSA), goat antiliquid substrate (TMB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sephadex G-25 resin and low chromatography apparatus were from GE Healthcare (Milan, Dimethylformamide (DMF), dimethylsulfoxide (DMSO), acetonitrile (HPLC grade) and all other chemicals and microtiter plates were obtained from VWR International (Milan, Italy).

  4°C. After the plates were washed three times with washing solution, they were used immediately. A volume of 100 µl of CSudan I-HRP (20:1) at 0.2 mg L -1 was added to 100 µl of Sudan I at concentrations ranging from 0 to 100 µg L -1 . The mixture was incubated for 15 min in wells coated with the anti-Sudan I antibody, then wells were washed with washing solution.

  Fortified samples were prepared by adding 25, 125, and 625 µg kg -1 of Sudan I and 125, 625 µg kg -1 of Sudan II, III, and IV to a blank sample before performing the extraction.

  Checkerboard assays, in which antiserum was titrated against varying amounts of two coating antigens (CSudan I-BSA 100:1 and CSudan I-BSA 50:1), were used to select appropriate antigen coating and antibody dilutions for the competitive indirect assay. An antibody diluted at 1:30000

  samples of very different compositions. Recovery values for Sudan II, III, and IV reflect their cross-reactivity and confirm that these dyes could be only detected in highly contaminated samples.

Figure 1 :

 1 Figure 1: Structure of the analytes; the hapten derivative used for immunization and preparation of

Figure 2 :Figure 3 :

 23 Figure 2: Non-competitive binding of antisera raised against CSudan towards the coating antigen,

Figure 4 :

 4 Figure 4: Correlation of results obtained by both ELISA and reference HPLC method for the Sudan

  were weighed and sonicated for 15 min with 50 ml of DMSO. Egg yolk and ketchup samples were previously mixed with 20 g of sodium sulphate to eliminate water. After being centrifuged for 15 min at 3000 rpm, extracts were diluted at 1:10 with PBSA and filtered through a 0.45 µm nylon membrane filter. Based on our experience, the use of a nylon membrane is mandatory to eliminate matrix interference from chilli powder and paprika samples.

	of homogenized sample
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with slight modifications. Egg yolk was manually separated and gently mixed before extractions. Briefly, 20 g

  This work was funded by the Italian Ministry for Universities(PRIN, prot. n. 2005030782_001)

	FIGURE CAPTIONS
	F o r F o r F o r F o r
	P P P P
	e e e e
	e r e r e r e r
	R R R R
	e e e e
	v i e v i e v i e v i e
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	y y y y

  Assay characteristics of the three competitive ELISA formats

	Page 23 of 26		Food Additives and Contaminants
		antibody	competitor	IC 50 (µg L -1 )	Time
		dilution in	concentration		requested to
		wells	(ng well)	carry out the
	Competitive Indirect ELISA Competitive Direct ELISA F 1/60000 1/30000 o r	40 37.5	3.8 8.3	assay (min) 75 45
	(simultaneous addition of	P
	reagents)	e
	Competitive Direct ELISA (pre-immobilization of the	1/50000	20 e r	4.9	45
	antibody)		R
			e
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				O n l
					y

TABLES

Table

1

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Table 2

 2 Cross-reactivity of target Sudan dyes, compounds structurally related to Sudan I, and dyes legally used in food determined by competitive direct ELISA

	Food Additives and Contaminants	Page 24 of 26
	Compound	Cross-reactivity (%)
	Sudan I	100
	Sudan II	34
	Sudan III	7
	Sudan IV	9
	Sudan red B	6
	Sudan red 7B	3
	Tartrazine	< 0.1
	Amaranth	< 0.1
	Allura red	0.1
	New coccine	< 0.1
	Sunset yellow	< 0.1

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Table 3

 3 Recovery of Sudan dyes from artificially contaminated samples as determined by ELISA detection

	Page 25 of 26			Food Additives and Contaminants
	Sample	Fortified	Sudan I	Sudan II	Sudan III	Sudan IV
		concentration
		(ng/kg)
			Recovery	RSD%	Recovery	RSD% Recovery	RSD% Recovery	RSD%
			(%)		(%)		(%)		(%)
										Deleted: /
	Ketchup	25	70	16.2	-a		-a			Formatted: Superscript
										Deleted: /
		125	82	4.4	35	2.8	nd		nd
		625	89	3.3	25	2.4	5	18.4	6	21.7
										Deleted: /
	Chilli	25	90	11.8	-a		-a			Deleted: /
	powder
		125	88	3.4	38	7.9	nd		nd
		625	103	2.3	20	4.0	5	20.8	6	23.9
										Formatted: Superscript
	a not measured

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Table 4

 4 Recovery of Sudan I from artificially contaminated egg yolk samples as determined by the reference HPLC method and by ELISA detection

					Page 26 of 26
	Fortified concentration		HPLC	ELISA
	(µg/kg)
		Recovery	RSD%	Recovery (%)	RSD%
		(%)
	0	nd		nd
	25	nd		97	22.7
	250	74	7.7	78	13.7
	2500	96	0.6	82	19.9

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Food Additives and Contaminants

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


Said Kinani

Stéphane Bouchonnet
email: stephane.bouchonnet@dcmr.polytechnique.fr

Janine Abjean

Christian Campargue

residues in milk cream by gas chromatography-mass spectrometry

Keywords: Clean-up, Extraction -ASE Additives/Contaminants: Environmental contaminants, Trace elements Polybromodiphenylethers, milk cream, gas chromatography, mass spectrometry, accelerated solvent extraction, solid phase extraction

Determination of polybromodiphenylethers residues in milk cream by gas chromatography-mass spectrometry

Said Kinani, Stéphane Bouchonnet, Janine Abjean, Christian Campargue

Introduction

Polybromodiphenylethers (PBDEs) are widely used as flame retardants in many industrial products; they are added to polymers which are used in plastics, TV, computers, building materials and textiles to slow down or inhibit fires [START_REF] Alaee | An overview of commercially used brominated flame retardants, their applications, their use patterns in different countries/regions and possible modes of release[END_REF]De Wit 1999). They are of environmental concern because of their high lipophilicity and high resistance to degradation processes; they readily bioaccumulate and have potential endocrine disrupting properties; they can induce genetic recombination, which causes a number of diseases including cancer (Rahman et al. 2001;[START_REF] Helleday | Brominated flame retardants induce intragenic recombination in mammalian cells[END_REF]. The main findings concerning toxicology of PBDEs are changes in liver weight accompanied by histological alterations in animals [START_REF] Fernlof | Lack of effects of some individual polybrominated diphenyl ethers and polychlorinated biphenyl congeners on human lymphocite functions in vitro[END_REF]. In vitro studies have shown that PBDEs can induct an estrogenic activity (Meerts et al. 2001). Recent studies indicate that the OH and H 3 CO-PBDEs main metabolites of PBDEs can interfer with CYP17, an enzyme which catalyzes an important step in sex steroidogenesis and is responsible for the biosynthesis of dehydroepiandrosterone (Rocío et al. 2006).

During the last few years, PBDEs have been detected in environmental matrices such as soil, sludge [START_REF] Eljarrat | Sample handling and analysis of brominated flame retardants in soil and sludge samples[END_REF]) and sediment (De la Cal et al. 2004;Zegers et al. 2003) as well as in biological matrices: human serum [START_REF] Covaci | Optimization of the determination of polybrominated diphenyl ethers in human serum using solid-phase extraction and gas chromatography-electron capture negative ionization mass spectrometry[END_REF], adipose and organ tissue [START_REF] Saito | Develoment of a accelerated solvent extraction and gel permeation chromatography analytical method for measuring persistent organohalogen compounds in adipose and organ tissue analysis[END_REF][START_REF] Li | Determination and occurrence of polybrominated diphenyl ethers in maternal adipose tissue from inhabitants of Singapore[END_REF]. Much research interest has recently been focused on the occurrence of PBDEs in mother's milk of countries such as Japan [START_REF] Akutsu | Timetrend (1973-2000) of polybrominated diphenyl ethers in Japanese mother's milk[END_REF]Eslami et al. 2005), Sweden [START_REF] Lind | Polybrominated diphenyl ethers in breast milk from Uppsala County, Sweden[END_REF], Norway [START_REF] Thomsen | Comparing electron ionization high resolution and electron capture low-resolution mass spectrometric determination of polybrominated diphenyl ethers in plasma, serum and milk[END_REF], Turkey [START_REF] Erdoğrul | Levels of organohalogenated persitent pollutants in human milk from Kahramanmaraş region, Turkey[END_REF]) and Czech Republic [START_REF] Kazda | Determination of polybrominated diphenyl ethers in human milk samples in the Czech Republic. Comparative study of negative chemical ionization mass spectrometry and time-of-flight high-resolution mass spectrometry[END_REF] The aim of the present study was to develop a method for PBDEs detection in milk cream, a raw material potentially higher in contamination than the finished product due to the lipophilic property of PBDEs. It was decided to validate the method on cream so that it could be used for the analysis of a variety of dairy products. Special emphasis was given to sample preparation since the development of GC-MS methods [START_REF] Pirard | New strategy for comprehensive analysis of polybrominated diphenyl ethers, polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans and polychlorinated biphenyls by gas chromatography coupled with mass spectrometry[END_REF][START_REF] Thomsen | Comparing electron ionization high resolution and electron capture low-resolution mass spectrometric determination of polybrominated diphenyl ethers in plasma, serum and milk[END_REF] (Tollbäck et al. 2003;[START_REF] Björklund | Influence of the injection technique and the column system on gas chromatographic determination of polybrominated diphenyl ethers[END_REF]) have been the object of several recent studies.

PBDEs have 209 possible congeners, depending on the number and positions of the bromine atoms on the two phenyl rings. Although the fully brominated congener deca-BDE is, by far, the main PBDE incorporated into manufactured products, the majority of PBDEs detected in biological samples are lower brominated compounds; this is assumed to result of the very poor capacity of highly halogenated compounds to accumulate in biological tissues [START_REF] Darnerud | Polybrominated diphenyl ethers: occurrence, dietary exposure and toxicology[END_REF], Jianwen et al. 2007). Six congeners were retained for this study because they are the most widely encountered in environmental matrices [START_REF] Darnerud | Polybrominated diphenyl ethers: occurrence, dietary exposure and toxicology[END_REF] and they are also suspected to be among the most toxic PBDEs (Rahman et al. 2001): the tri-BDE-28, tetra-BDE-47, penta-BDEs-99 and -100, hexa-BDEs-153 and -154 congeners. 2',, 2',4,, 2',4,4',2',4,4',, 2',4,4',5,2',4,4',6,6'hexabromodiphenyl ether) and 4,4'-dibromodiphenyl (Purity > 99%) were purchased from Cambridge Isotope Laboratories (Andover -USA). Hexane, nonane, acetone, methanol, acetonitrile, diethyl ether and methylene chloride were purchased from Sigma-Aldrich (Saint Quentin Fallavier -France); all were at least 99.9 % pure. Anhydrous Na 2 SO 4 was purchased from Merck (Fontenay Sous Bois, France) and "hydromatrix" (cleaned and sieved diatomaceous earth) from Varian (Les Ulis -France). The samples of milk cream were extracted from milk batches delivered from different regions. The analyzed cream cheese is a commercial product. Toxitubes ® A and B and C18 cartridges for solid phase extraction (SPE) were purchased from Varian (Les Ulis, France).

Materials and methods

Standards and samples

Development of the sample preparation process

The sample preparation process requires two steps. The first one, referred as "extraction", consists in extracting the lipid content from the cream; the second one, referred as "cleanup", consists in separating PBDEs from the fat. Liquid-liquid extraction was carried out in a 100 mL separatory funnel. To 1 g of cream were added 10 mL of methanol and 10 mL of the extraction solvent: pentane (method 1) and hexane (method 2) were tested. The separatory funnel was manually agitated and the organic phase was transferred into a tube. 3 mL of a saturated solution of potassium oxalate were added drop to drop to break the emulsion. The methanolic phase was extracted again with 10 mL of the extraction solvent and the second organic phase was added to the first one. ASE extractions were carried out using a fully automated ASE 200 system (Dionex, Sunnyvale, CA, USA) fitted with 11-mL ASE cells. Two protocols were tested. Each one used 5 g of cream thoroughly mixed with 3 g of Hygromatrix. The first protocol was proposed by Saito et al. to extract lipids from human adipose tissue [START_REF] Saito | Develoment of a accelerated solvent extraction and gel permeation chromatography analytical method for measuring persistent organohalogen compounds in adipose and organ tissue analysis[END_REF]). It uses a binary methylene chloride / acetone (1:1, v:v) mixture at a temperature of 100°C and a pressure of 1500 psi; the ASE cell is purged during 60 s with 5.5 mL of solvent. This first protocol involves two extractions per sample. The second protocol was derived from an application note by Dionex to extract PBDE from human breast milk (Dionex application note 2004). It uses a tertiary hexane, methylene chloride, methanol (5:2:1, v:v:v) mixture at a temperature of 80°C and a pressure of 1500 psi; the ASE cell is purged during 240 s with 6.0 mL of solvent.

Extraction

The second extraction protocol includes three extractions per sample.

Extraction with toxitubes was performed according to the following protocol. 4 mL of water were added to x g of cream; thre values of x were tested: 0.10, 0.25 and 0.60. The mixture was transferred into a toxitube B, automatically mixed by inversion before being centrifuged at 3500 g during 15 min at 25 °C.

In all extraction processes, the resulting extract was dried with anhydrous Na 2 SO 4 . The Na 2 SO 4 residue was rinsed with two aliquots of 2 mL of the extraction solvent. The organic extract was then taken using a 20 mL syringe and filtered on two 25 mm Acrodisc® syringe filters (Pall Life Sciences -Ann Arbor -USA). The mass of extracted fat was determined by gravimetry, according to the following procedure. Before extraction, a collecting vial was dried in an oven at 100°C for 30 min, cooled in a desiccator for 1 hour and weighted. After

Cleanup

The cleanup involves a SPE protocol which has been derived from the one described by Bordet et al. for the cleanup of polychlorinated biphenyl, organochlorine pesticides and pyrethrinoids from fatty food [START_REF] Bordet | Dosage des pesticides organochlorés, pyrethrinoïdes et PCB (Polychlorobiphényles) dans les aliments gras -Note de service DGAL/SDSSA/SDRRCC/ N2003-8141[END_REF]. A C18 cartridge (cartridges containing 2 g and 5 g of phase were tested) was conditioned with 5 mL of diethyl ether, then 5 mL of acetone, then 10 mL of methanol and allowed to dry. The extracted fat was solubilized in 2 mL of acetonitrile which were loaded onto the cartridge. The tube was rinsed either with 20 mL of acetonitrile or with four aliquots of 5 mL of acetonitrile (recovery yields are compared in the results and discussion part). The elution was carried out at about one drop per second.

The solvent was then evaporated under a N 2 stream at 45 °C. 100 µL of nonane were added for GC-MS analysis.

The retained sample preparation process

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GC-MS analysis

All analyses were carried out on a Varian "CP3800" gas chromatograph fitted with a "CP8400" autosampler and connected to a Varian "1200" triple stage quadrupole mass spectrometer. The chromatographic separation was performed on a 60 m "Factor four VF-XMS" capillary column (internal diameter: 0.25 mm, film thickness: 0.25 µm) from Varian.

Helium was used as the GC carrier gas and the flow-rate was maintained constant at 1.4 mL/min owing to an electronic flow controller. All experiments were performed by automatically injecting the sample in the splitless mode at a rate of 50 µL/sec. 1 µL of sample was injected in the experiments concerning the development of the method but a value of 1.5 6 µL has been finally retained for the validation procedure according to the detection thresholds required. The injector temperature was 280°C. The split valve opened after 0.8 min, with a split ratio of 35/100. The capillary column was ramped from an initial temperature of 100°C, held for 1 min, increased at 20°C/min up to 300°C where it was held for 14 min. The total duration of GC analysis was 25 min. The transfer line was maintained at 300°C. The ion source and quadrupole temperatures were 150°C and 40°C, respectively. Tuning of the mass spectrometer was automatically performed using the ions resulting from ionization of perfluorotributylamine. Electron capture ionization was performed using methane as thermalization gas at a pressure of 800 Pa. The filament emission current and the electron energy were set to 150 µA and 150 eV, respectively. The multiplier voltage was automatically set to 1200 V. All experiments were performed in the negative mode, with a scan time of 0.4 sec/spectrum.

Results and discussion

GC-MS

In the electron attachment mode, all PBDEs dissociate to provide only Br -and HBr 2 -product ions, in agreement with results of previous work. Under electron attachment, 4,4'dibromodiphenyl mainly provides molecular ions M .-and Br -ions. A SIM (Selected Ion Monitoring) method has thus been developed. Three ions are monitored for PBDEs (m/z 78.9, m/z 80.9 and m/z 160.8) and for the internal standard (m/z 78.9, m/z 80.9 and m/z 311.9). The main disadvantage of this method is its lack of specificity since some brominated compounds may interfere with PBDE peaks in the chromatogram but several studies showed that electron attachment is the ionization mode, which provides by far the lowest detection thresholds for PBDEs. The analytical performance of the GC/MS method for solutions of standards are summarized in Table 1. Limits of quantification (LOQ) were determined for a signal-to-noise ratio of 10; they range from 10 pg/mL for tri and tetra-BDE to 80 pg/mL for hexa-BDE.

Coefficients of variation were determined for ten injections at two concentrations: 2.5 and 30.0 ng/mL and ranged between 0.5 and 17.7 %. For all the PBDEs which were investigated, the coefficient of correlation (r 2 ) was greater than 0.995 for a concentration range from the LOQ to 50 ng/mL. from the same extract purified on a SPE cartridge containing 5g of C18 phase; it permits the detection of the six analytes. 5g-cartridges have thus been retained for further experiments.

Please insert

Please insert Figure 4 near here

The recovery from the SPE cleanup step was determined for each PBDE using the GC/MS method described above. A cream extract was divided into six aliquots of 500 mg; 30 ng of each PBDE were added to each aliquot. Cleanup was carried out from each spiked sample according to the procedure described in the experimental part. The tube containing the ASE extract was rinsed with 20 mL of acetonitrile. Mean recovery yields from the cream extract ranged from 43 % for BDE-154 to 79 % for BDE-28 and the associated coefficients of variation for n=6 ranged from 6 to 26 %. Considering the poor recovery yields and the large values of the corresponding CV, the influence of the rinsing of the tube containing the ASE extract was studied. The SPE process was applied to extract a sample spiked in PBDEs at 50 ng/g of fat. The tube was rinsed eight times with 5 mL of acetonitrile. After each rinsing, the rinsing solution was deposited onto the cartridge and eluted. Each corresponding collected fraction was analyzed by GC/MS. In Figure 5 the recovery yields are plotted as a function of the number of 5 mL-eluted fraction(s) collected. For all PBDEs, the recovery increased with the number of collected fractions to reach a maximum for 5 fractions collected. Under these conditions, recoveries were between 90 and 96 %, according to the PBDE considered. The CV on cleanup recovery yields for five extractions ranged from 1.0 to 5.4 % according to the individual PBDE. This experiment showed the importance of carrying out several rinses and allowed excellent recovery to be achieved with a good CV. The influence of the concentration in PBDE on the recovery was studied as follows. The cleanup was applied to ten samples of 500 mg of extracted cream; five were spiked with 5 ng and five were spiked with 50 ng of each PBDE. Considering each PBDE, the difference in mean recovery yields (comparable to those obtained in the experience described in the previous paragraph) between both concentrations did not exceed 9 % and showed no evidence of a correlation between the recovery yield of the cleanup step and the initial PBDE concentration in the sample.

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Figure 6 shows the mean recovery yields for the method overall, i.e. taking into consideration ASE extraction and SPE cleanup. They were determined by spiking ten samples of cream, five at 10 ng/g and five at 100 ng/g in each PBDE. Mean recovery yields range from 65 to 101 % for samples at 10 ng/g and from 69 to 106 % for samples at 100 ng/g. There is no evident concentration effect since recovery yields do not significantly differ comparing both concentrations for a given PBDE. The associated coefficients of correlation range from 1.1 to 5.5 % for the samples spiked at 10 ng/g and from 3.4 to 9.3 % for the samples spiked at 100 ng/g, showing the good repeatability of the sample preparation procedure. Gel permeation chromatography (GPC) [START_REF] Kazda | Determination of polybrominated diphenyl ethers in human milk samples in the Czech Republic. Comparative study of negative chemical ionization mass spectrometry and time-of-flight high-resolution mass spectrometry[END_REF][START_REF] Dodder | Concentrations and spatial variations of polybrominated diphenylethers in fish and air from the North-Eastern United States[END_REF]Allae et al 2001) and adsorption chromatography [START_REF] Päpke | Determination of polybrominated diphenylethers (PBDEs) in biological tissues with special emphasis on QC/QA measures[END_REF][START_REF] Kazda | Determination of polybrominated diphenyl ethers in human milk samples in the Czech Republic. Comparative study of negative chemical ionization mass spectrometry and time-of-flight high-resolution mass spectrometry[END_REF][START_REF] Boer | Polybrominated biphenyls and diphenyls ethers. New types of persistent halogenated compounds[END_REF], 2001, 2002) have been widely used for the cleanup of PBDEs extracted from biological compounds.

The former technique is not available in our laboratories. Nowadays, "classical" adsorption chromatography with silica and/or alumina columns tends to be replaced by SPE which reduces solvent consumption and allows total automation.

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Method validation

In Cream samples were spiked at six concentrations ranging from 0.1 to 10 ng/g of fat for BDEs-28, -47, -99 and -100, and from 0.2 to 20.0 ng/g of fat for BDEs-153 and -154 for which preliminary tests showed higher detection limits. To estimate possible matrix effects, standard solutions of PBDEs were prepared at the same concentrations. In each case the internal standard (dibromobiphenyl) was added at 0.10 ng/g. The chromatographic response was proportional to the PBDE concentration in cream on all the studied concentration range with r 2 > 0,995 for each congener. The comparison of calibration lines with and without matrix interferents shows a matrix effect so slight that it can be neglected. Accuracy and repeatability were estimated on four consecutive dosages at two concentrations in PBDEs: 0.6 and 6.0 ng/g of fat for BDEs-28, -47, -99 and -100, 1.2 and 12.0 ng/g for BDEs-153 and -154. In terms of repeatability, the method provides satisfying CV ranging from 6.3 to 14.5 % at the lowest concentration and from 3.5 to 4.6 % at the highest one. In terms of accuracy, the determined amounts are systematically lower than expected: between 78 and 93 % of the expected amount at the lowest concentration and between 78 and 87 % of the expected amount at the highest one. The method systematically under evaluates the PBDE concentration in cream by about 10 to 20 %. Given the good recovery ratios (> 90 %) of the cleanup process, this underevaluation suggests that all PBDEs are not in the fat and that some of them remain in the aqueous phase during the extraction step.

Application to real samples

Milk cream

Figure 7 shows the chromatograms obtained from batches of cream from four different suppliers. In all cases, traces amounts of BDE-47 were detected below the limit of quantition of the method. The mean value of BDE-47 in these cream extracts could very empirically estimated at 0.02 ng/g of fat. Other PBDEs among the six congeners were not detected.

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Cream cheese

The applicability of the method was also tested on cream cheese. Four samples of 22 g of cream cheese underwent ASE extraction with poor recovery yields of fat ranging from 45 to 47 %. After SPE cleanup, the four extracts were analyzed by GC-MS. Quantification was not possible because of the difficulty to get good fat recovery from cream cheese, but there were samples. In all cases, PBDEs were not detected at concentrations above the limit of quantification of the method (0.1 ng/g).

F

  . A European Commission Directive has prohibited the use of PBDEs in new electric and electronic components (Commission Directive RoHS 2002/95/EC) since July 1 st 2006. The detection of PBDEs is tarditionally performed by GC/MS.

  well as the influence of chromatographic injection parameters

  were tested and compared in terms of lipid recovery yields: liquidliquid extraction (LLE), accelerated solvent extraction (ASE) and extraction with toxitubes A and B.

  of extract was added in the vial; the solvent was evaporated under a N 2 stream at 35°C. The residue was then dried at 100°C for 1 hour, cooled in a desiccator for 1 hour and weighed to determine the exact mass of extracted fat.

Figure 1

 1 Figure 1 summarizes the experimental protocol used to develop the sample preparation process. It shows that ASE has been prefered to the other extractions modes; the ASE protocol number 2 has been retained. The cleanup of the ASE extract was finaly performed on the 5g-C18 SPE cartridge. The detailed results of method development are given in the "results and discussion" part.

  Figure4shows two chromatograms of the same extract of cream spiked at 0.5 ng/g for each PBDE. The upper chromatogram shows the results from an extract purified on a SPE cartridge containing 2 g of C18 phase; it is very noisy and shows evidence of insufficient selectivity of the cleanup using the 2g cartridge. The lower chromatogram shows the results

Figure

  preliminary tests, traces of PBDEs (especially BDE-47) were systematically detected in creams from different regions and contamination by plastic tubes has been suspected. The whole sample preparation method has been carried out two times on two cream samples. In the first extraction, Eppendorf plastic tubes were used to store samples; in the second one, plastic tubes were replaced by glass tubes. The comparison of results obtained by GC-MS analysis clearly showed a PBDE contamination of the sample by the plastic tubes. Even of PBDEs detected were very low (below the quantitation threshold of the method), plastic tubes were discarded of the sample extraction procedure in further work.

  the presence of BDEs-47, -99 and -100 at low levels when samples were analysed.ConclusionA method has been developed for routine analysis of six polybromodiphenyl ethers (PBDE) in milk cream. Special attention has been focused on PBDEs extraction from cream. Sample preparation involves two steps. The first one consists in extracting fat from cream by ASE.The second step, referred as "cleanup", uses SPE to separate PBDEs from fat. Recovery yields of the whole sample preparation process were estimated between 65 and 101 % at 10ng/g, with CV ranging from 3.5 % to 14.5 % for n=6, according to the PBDE considered. PBDEs were analyzed by GC-NCI-MS. The GC-MS method suffers a lack of selectivity because the PBDEs which were analyzed provided only Br-and HBr 2 -ions under electron attachment conditions, in agreement with previous work; nevertheless, it is very sensitive and permited to reach detection thresholds of 0.1 ng/g of fat for tri-, tetra-and penta-BDEs and 0.2 ng/g of fat for hexa-BDEs. In terms of accuracy, the method systematically under estimates the PBDE concentration in cream by about 10 to 20 %, suggesting that some PBDEs remain in the aqueous phase during the extraction step. Evidence has been found of sample contamination by the PBDEs contained in plastic parts of the apparatus, which must be removed from the sample preparation equipment and replaced by glass parts. The analysis of real samples of cream and cream cheese showed the reliability of the method. Some traces of BDE-47 were detected in different milk cream batches. BDEs-47, -99 and -100 were found in cream cheese

Figure 1 .

 1 Figure 1. Experimental protocol used to compare the methods of extraction and to optimize the C18-SPE purification step

Figure 3 .

 3 Figure 3. Recovery yields of fat and coefficients of variation associated to the SPE purification step as a function of the amount on fat deposited onto the 2g-C18 cartridge

Figure 5 .Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 5 .Figure 6 .

 5123456 Figure 5. Cumulated recovery yields, for each PBDE, as a function of the number of 5 mLeluted fractions collected

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	Analyte	LOD a (pg/mL)	LOQ b (pg/mL)	Linearity range r 2	CV (%) at 2.5 ng/mL at 50.0 ng/mL
	BDE-28	3	10	10 pg/mL 50 ng/mL	0.999	4.5	0.5
	BDE-47 F 3 BDE-99 8 o r BDE-100 8 P 10 20 20 e BDE-153 25 80 e 10 pg/mL 50 ng/mL 20 pg/mL 50 ng/mL 20 pg/mL 50 ng/mL 80 pg/mL 50 ng/mL r R BDE-154 25 80 80 pg/mL 50 ng/mL	0.998 0.998 0.997 0.997 0.998	6.2 5.8 5.2 17.7 10.1	0.7 1.1 2.2 3.6 3.3
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Table 1 .

 1 Main characteristics of the GC-NCI-MS method on standard solutions

a Limit of detection b Limit of quantitation

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