Jaroslav Dobiáš

Lenka Votavová

Kamila Klaudisová

Keywords: Packaging -food simulants, Packaging -inks, Packaging -migration modelling food packaging, printing inks, migration, food safety

The occurrence of extractible ink residuals in packaging

materials used in the Czech Republic.

Introduction

Printing inks used in food packaging production contain many substances that have not been toxicologically evaluated, since they are not coming into direct contact with food. However, these substances (e.g. pigments, plasticisers, solvents, photoinitiators, stabilizers, their breakdown products, etc.) may be transferred from the printing to the food contact surface either by penetration through the packaging material or via the set-off transfer phenomenon (transport of printing ink constituents from printed surface to the unprinted side of another sheet in a stack or roll).

When the printing ink components reach the food contact layer of the package, they could migrate into the packaged food due to extraction by food, evaporation and/or steam distillation during storage and/or heating in original package (Sun Chemical 2007). As printing is not in a direct contact with food no specific legislation concerning printing inks for food packaging has been agreed so far in EU. The Framework Regulation (EC 2004) applicable to all food packages only states that printing for food packages in EU will be in future controlled by particular system of directives and regulations. At present materials and articles intended to come in contact with foods should comply with the general criteria laid down in Art. 3 of above mentioned Framework Regulation, i.e. should not transfer their constituents in food in quantities, which could endanger the human health or bring about unacceptable changes in composition or characteristics of foodstuffs. These criteria are also reiterated in the Council of Europe Resolution (Council of Europe 2005). To prove the conformity with above mentioned demand the risk assessment is necessary. This is the responsibility of packaging material and/or food contact article producer. The Framework Regulation (EC 2004) also requires the traceability of food contact materials and articles at all stages as well as production by Good Manufacturing Practice (EC 2006, EuPIA 2008, Sun Chemical 2007).

The influence of the printing system on food packaging safety, mainly migration of solvents, plasticizers and photoinitiators, has been already studied. Skjevrak et al (2005) tested migration from 75 samples of plastic laminates for food packaging into oil and/or distilled water and they found eight major migrants with level of migration above 0.3 mg/kg, six of them originated (or could have originated) from printing and/or coating, i.e. dioctyl adipate, diisooctyl adipate, 2-(2-butoxyethoxy)ethanol, bisphenol A diglycidyl ether (BADGE), methyl ester of dehydroabietic acid and N-ethyl-toluenesulfonamide. They also identified four primary aromatic amines originated from printing (2-methyl-aniline, 2,4,-dimethylaniline, 2methoxyaniline and 4,4´-methylene-dianiline) at migration level <0.02 mg/kg. Nevertheless their results showed that migration of non-authorized substances in found level did not present a risk to consumer health. Migration of plasticizers (mainly esters of phthalic acid, adipic acid, sebacic acid, citric acid and mellitic acid, but also N-methyl-and N-ethyltoluensulfonamide) from printing inks into packaged foodstuffs was also reported [START_REF] Nerin | Plasticizers from printing inks in a selection of food packaging and their migration to food[END_REF][START_REF] Balafas | Phthalate and adipate esters in Australian packaging materials[END_REF], Piergiovanni et al. 1999).

The danger of residues of photoinitiators (PI) migrating from UV-cured printing of food packages has been reported [START_REF] Castle | Migration studies from paper and board food packaging materials. II. Szrvey for residues of dialkylamino benzophenone UV-cure ink photoiniciators[END_REF]. Their transfer by set-off caused significant damage for producers of beverages like milk, cocoa or juice packaged in paperboard packages in 2005, when the substances 2-isopropyl thioxanthone (ITX) and 2-ethylhexyl-4- dimethylaminobenzoate (EHDAB) were detected in packaged products at levels up to 300 µg/kg food. (EFSA 2005, EuPIA 2005b) These findings often exceeded a maximum migration level of 50 µg/kg food, which was announced by the European Food Safety Authority (EFSA) as an acceptable threshold for substances with no genotoxic potential (EFSA 2005[START_REF] Gil-Vergara | Determination of 2-isopropyl thioxantone and 2ethylhexyl-4-dimethylaminobenzoate in milk: comparison of gas and liquid chromatography with mass spectrometry[END_REF][START_REF] Benetti | A packaging contaminant: isopropyl thioxantone (ITX) in dairy products[END_REF]. [START_REF] Kleinschnitz | Identification and semi-quantitative determination of a migration contaminant from beverage carton packages into mineral water by on-line solid phase extraction gas chromatography-mass spectrometry (SPE-GC-MS)[END_REF] reported the determination of 2,4,7,9-tetramethyl-5-decyne-4,7-diol in mineral water packaged in paperboard beverage boxes up to levels of 50 µg/l. The origin of this substance was a surfactant in water-based printing inks. [START_REF] Aurela | Migration of alkylbenzenes from packaging into food and Tenax ®[END_REF] described the identification of alkylbenzenes (alkyl chain C 10 -C 13 ) in 10 out of 15 samples of offset-printed food packaging made of paperboard. Alkylbenzenes are used as solvent components in some offset printing inks and the authors found migration into packaged product up to 2 mg/kg.

The main aim of this study was to screen polymer printed packaging materials commercially used in Czech Republic to obtain objective data for an evaluation of possible contamination of packaged food with printing ink constituents.

Materials and methods

Packaging materials:

Printing ink residuals testing. 94 food packaging materials were tested. All of them are currently used for food packaging in Czech Republic. The set of samples included: i. 3 films made of low density polyethylene (LDPE) and 2 samples from polypropylene (PP) with direct printing on outside surface. Concerning printing techniques used for tested packaging materials production 53 samples and 26 packaging materials were printed with flexography using UV-cured and not UV cured inks, respectively. 15 samples were produced using gravure print.

Analytical methods:

Determination of free printing ink constituents by diethyl ether extraction. 1 dm 2 of tested packaging material was extracted with 50 ml of diethyl ether (p.a., Sigma-Aldrich Chemie,GmbH) in Soxtec TM 2043 extractor (Foss Analytical, DK) for two hours. Diethyl ether extract was evaporated to dryness at 40 °C using a vacuum evaporator and redissolved in 2 ml of diethyl ether. 1 µl of this solution was injected into a gas chromatograph coupled with a mass spectrometry detector and analysed at following conditions: gas chromatograph -Agilent 6890N (Agilent Technologies); detector -Agilent 5973N mass detector, electron impact ionisation 70 eV, temperature 280 °C; capillary column -DB-5 (30 m x 0.25 mm x 0.25 µm); mobile phase -He, flow rate 0.6 ml/min, linear speed 28.9 cm/s; injection -1 µl using split 1 : 100, temperature 300°C; column temperature -70 °C for 5 minutes, increase 15 °C/min to 300 °C, 300 °C to the analysis end. Identification of separated substances consisted in comparison of obtained mass spectra with the spectrum library of used chromatography software (NIST MS Search 2.0) and its confirmation using the mass spectra of analysed standards. The quantification was done using diethyl phthalate (p.a., standard for GC, Sigma-Aldrich Chemie, GmbH) as the internal standard. At least three parallel samples for each tested packaging film were analysed. The results in the next text are expressed as mean values± standard deviations.

The analytical deviation for reference compounds at 10 mg/l (according with migration level of 10 µg/dm 2 ) concentration level in diethylether solution typically corresponded to 5-10% RSD and at 100mg/l (100 µg/dm 2 ) to 3-5% RSD.

Results and discussion

Having planned this study, we had to overpass some problems. Firstly we did not get either interest for cooperation from printing ink producers so that we had to obtain the packaging material samples from food and packaging material producers, i.e. we could not exploit any

Identification of ink components released from tested films

From the tested films 50 substances (see Table I) were isolated and identified in amount exceeding 0.001 mg/dm 2 . According to the published data, these substances could originate from printing inks even it is obvious that extraction with diethyl ether did not enable to distinguish between additives released from polymers and those really extracted from printing as only the printed packaging materials were tested. Extractible levels of identified substances ranged from 0.0015 ± 0.0002 mg/dm 2 for lauramide to 0.898 ± 0.017 mg/dm 2 for acetyl tributyl citrate.

The presence of substances obviously originated from printing, which are not allowed for polymer food contact materials, is interesting, e.g. dichlor benzenamine (probably the breakdown product of aryl pigments), six photoinitiators and/or their breakdown derivatives (isopropyl thioxantone, 4-phenyl benzophenone, 4-hydroxy benzophenone, ethyl-4-dimethyl aminobenzoate, 2-ethylhexyl-4-dimethylaminobenzoate and trimethyl benzaldehyde). On the other hand there were substances present in amount exceeding 0.500 mg/dm 2 , e.g. diethylhexyl adipate (up to 0.698 ± 0.014 mg/dm 2 ), 2-ethylhexyl diphenyl phosphate (0.873 ± 0.026 mg/dm 2 ) and acetyl tributyl citrate (0.898 ± 0.017 mg/dm 2 ). The most frequent substance found in tested samples of packaging materials was diethylhexyl adipate, which was identified in 24 samples included beverage board boxes, laminated and co-extruded packaging films and polyethylene terephthalate films for sealing of cups with yoghourt. The maximum content 0.698 ± 0.014 mg/dm 2 was found in beverage board box printed using flexography. In 23 samples (paper board boxes, laminated and/or co-extruded films) oleamide was identified in maximal level of 0.220 ± 0.004 mg/dm 2 . The highest concentration of printing ink constituent (i.e. 0.898 ± 0.017 mg/dm 2 ) was found for acetyl tributyl citrate in co-extruded film for biscuit packaging. This substance was identified in twelve more packaging films. 2-ethylhexyl diphenyl phosphate was identified in 8 samples of co-extruded and/or laminated films for biscuit packaging at maximum level of 0.873 ± 0.026 mg/dm 2 . Erucamide was found in one sample of beverage board box at level 0.232 ± 0.007 mg/dm 2 .

Seven PIs were identified in samples printed with UV curable inks. Ethyl 4dimethylaminobenzoate was the identified in ten samples of PET films for sealing of polystyrene cups at maximal level of 0.151 ± 0.005 mg/dm 2 , 4-phenyl benzophenone was found in 9 samples including laminated films for packaging of spices, soup condiment and/or dehydrated soups as well as PET films and aluminium foils for sealing polystyrene cups at maximal level 0.139 ± 0.006 mg/dm 2 . The more known PIs 2-isopropyl thioxanthone (ITX) and 2-ethylhexyl 4-dimethylaminobenzoate (EHDAB) were found only in 4 and 2 cases, respectively. This fact is probably the consequence of the attitude of food producers in Czech Republic, whose generally refuse packaging materials printed with inks containing ITX and EHDAB. The highest found contents of ITX and EHDAB were 0.182 ± 0.010 mg/dm 2 and 0.0759 ± 0.0023 mg/dm 2 , respectively.

Tributyl aconitate was identified in 14 samples of packaging materials (beverage boxes, laminated films) at level up to 0.0764 ± 0.0046 mg/dm 2 , toluene-2,4-diisocyanate in 12 samples (PET and aluminium sealing foils, co-extruded and laminated films) up to 0.0611 ± 0.0048 mg/dm 2 , dibutyl sebacate in two films for biscuit and spice packaging up to 0.0616 ± 0.0043 mg/dm 2 and 2-butoxy ethyl oleate in one laminated film for vanilla sugar packaging at level 0.0616 ± 0.0037 mg/dm 2 . The above mentioned results are generally comparable with findings of Skjevrak et al. (2005) for laminated food packaging films. The main difference is an absence of N-ethyl-toluene sulfonamide (NETSA). NETSA was used as plasticizer in resins, cellulose acetate, nitrocellulose, lacquers adhesives and inks and in Norwegian study this substance was isolated from printed laminated films in by far the highest amount. Its absence in materials tested in this study should certify the intent of packaging film producers not to use NETSA in printing inks (Skjevrak et al. 2005).

Besides the above mentioned residues, fifteen substances were detected but not identified, and so far were characterised only by mass spectra, but were present at quite significant levels (about 50-100 µg/dm 2 ), too. Their identification will pose one of the main tasks for the authors.

Evaluation of possible risk of potential migrants originated from printing inks

When migration of substances not included in the EU positive list of monomers and/or additives for food contact polymer is identified, assessments of health risks are usually carried out. In this study the content of substances which could be released and potentially transferred into packaged product was determined, but no data on real migration were obtained. It means that no proper health risk assessment could be done. Nevertheless the possible danger due to presence of identified substances could be estimated.

From substances not included in the EU positive list identified in Table 1 the occurrence of four PIs (ethyl 4-dimethylaminobenzoate, EHDAB, 4-phenyl-benzophenone and ITX) at levels up to 0.182 mg/dm 2 could pose problems. These substances were identified in the printing of barrier films for spice, condiment and or dehydrated soups packaging. These materials eliminate the possibility of PIs penetration through the package. Considering the extent of set-off transfer in agreement with this study results, i.e. 1 % of PI amount in lacquer per month at 40 °C, we can expect the safety of packaging material for approximately 2 year storage at 20 °C. Similar situation can be expected for aluminium foils for thermo sealing of polystyrene cups with yoghourt, moreover there is advantage in smaller area of foil in contact with food. In the PET films for thermo sealing of polystyrene cups the maximal found amount of PI reached the level 0.151 mg of ethyl 4-dimethylaminobenzoate per dm 2 . Due to good barrier parameters of PET film and the layer of thermo sealable lacquer on the base of polyvinyl dichloride (PVdC) as well as the fact that the area of such packaging material in the contact with food is quite limited the real risk for consumer could be considered as quite low.

Nevertheless the migration tests of these materials would be advisable.

Acetyl tributyl citrate was identified in different types of packaging materials including coextruded PP/LDPE films in levels up to 0.898 mg/dm 2 . Due to the absence of efficient barrier it would be suitable to verify migration of this additive through the tested packaging films even if acetyl tributyl citrate is accounted the material of low toxicity. Similar situation could be stated for tributyl aconitate, which was found in 14 tested samples in maximal level 0.0764 mg/dm 2 , and 2-butoxyethyl oleate that was identified only in one tested sample in amount 0.0616 ± 0.0037 mg/dm 2 .

Considering identified substances included in EU positive list the high content of 2-ethylhexyl diphenyl phosphate in five tested samples of co-extruded films (up to 0.873 mg/dm 2 ) is suspicious. The set specific migration limit for this additive is 2.4 mg/kg, i.e. 0.4 mg/dm 2 , and in agreement with the results obtained by this study it could be surpassed in contact with food simulants. In this case also the migration test of relevant packaging materials should be done for final assessment.

The migration tests of PIs, acetyl tributyl citrate, tributyl aconitate, 2-butoxyethyl oleate and 2-ethylhexyl diphenyl phosphate from tested packaging materials samples will be carried out in the next part of project solution.

Conclusion

The main results of carried out experiments can be summed as follows:

i)

The ,6,10,15,19,23hexamethyltetracosa-2,6,10,14,18,22hexaene) 7683

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

  on tested printing parameters. Unfortunately there was no possibility to obtain and test unprinted packaging materials as controls. As printing inks form quite complex chemical systems and due to little information on tested printings composition, we decided to analyze the packaging materials samples using the gas chromatography coupled with mass spectroscopy detector as this technique offers the most convenient identification of separated substances. Moreover the potential migrants capable of transfer from printing to packaged food, which could present the threat for consumer, should be first of all organic substances of lower molecular weight with sufficiently low boiling temperature.Three techniques of isolation of printing ink constituents were originally tested, i.e. adsorption on Tenax TA sorbent, application of solid phase micro extraction method (SPME) and direct extraction with diethyl ether. The last procedure was found the most efficient, as the number of isolated and identified substances was higher comparing with the other techniques. The examples of typical chromatograms are shown in Figure1

Figure 1 .

 1 Figure 1. Chromatograms of diethylether extracts from co-extruded polypropylene film with flexography print used for bisquit packaging (A) and laminated paper/Al/LDPE film with UV cured flexography printing used for spice packaging (B) (BHT -buty lhydroxy toluene, IS -internal standard = diethyl phthalate)

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  Council of Europe 2005. Council of Europe Resolution ResAP (2005)2 of 14 th September 2005 on packaging inks applied to the non-food contact of food packaging materials and articles intended to come into contact with foodstuffs. EuPIA 2008. Guideline on Printing Inks applied on the non-food contact surface of food Papilloud S, Baudraz D. 2002. Analysis of food packaging UV inks for chemicals with potential to migrate into food simulants. Food Additives and Contaminants, 16(8): 353-359. Safety data for squalane. Physical and Theoretical Chemistry Lab at Oxford University, web site. ( http://msds.chem.ox.ac.uk/SQ/squalane.html , 31.12.2008) PTCL Safety lab 2007. Safety data for cetyl alkohol. Physical and Theoretical Chemistry Lab at Oxford University, web site.( http://msds.chem.ox.ac.uk/CE/cetyl_alcohol.html, 5.1.2009)

		oleate and 2-ethylhexyl diphenyl phosphate should be completed with information on
	real migration into food and or food simulants. package materials and articles,	April	2008.
	Piergiovanni L, Fava P, Schiraldi A. 1999. Study of diffusion through LDPE film of di-n-
	Science Lab 2008. Dioctyl adipade MSDS. Science Lab.com web site. butyl phthalate. Food Additives and Contaminants, 19(2): 168-175.
	(www.sciencelab.com/xMSDS-Dioctyl_Adipate-9923845 , 31.12.2008) PTCL Safety lab 2003a. Safety data for erucamide. Physical and Theoretical Chemistry Lab
	Dow Chemical Company. 2008. Glycol ethers for intermediates. Product Information, Form at Oxford University, web site. (http://msds.chem.ox.ac.uk/ER/erucamide.html , 31.12.2008)
	F o r P e e r R e v i e w F o r P e e r R e v i e w (www.dow.com/webapps/include/getdoc.aspx?objectid=&filepath=oxysolvents/pdfs/noreg/11 No. 110-00756-0304. 0-00756.pdf&pdf=true , 31.12.2008) EC 2002. Commission Directive 2002/72/EC relating to plastic materials and articles intended to come into contact with foodstuffs. The Commission of the European Communities, 6.8.2002. European Communities, 22.12.2006. EFSA 2005. Opinion of the scientific panel on food additives, flavourings, processing aids and materials in contact with food on a request from the Commission related to 2-isopropyl thioxanthone (ITX) and 2-ethylhexyl-4-dimethylaminobenoate (EHDAB) in food F o r P e e r R e v i e w F o r P e e r R e v i e PTCL Safety lab 2003b. Sun Chemical 2007. Printing for packaging -Low migration printing. Best Practice Guide, w revised version 2, December 2007. Sun Chemical Ltd., St Mary Cray, Orpington, United
	O n l O n l EFSA 2007. Scientific Opinion of the Panel on food additives, flavourings, processing aids contactmaterials. The EFSA Journal, 293: 1-15. and materials in contact with food (AFC) on a request related to a 14 th list os substances for O n l Kingdom. O TCI Europe 2009. Photopolymerization Initiators. TCI Europe web site. n l (http://www.tcieurope.eu/en/product/materials-chem/F013.shtml, 6.1.2009)
	y y food contact materials Question N° EFSA-Q-2006-1, EFSA-Q-2006-178, EFSA-Q-2006-116. y y TradeWinds 2007. TriForceTM Canine Squeeze On MSDS. TradeWinds web site.
	The EFSA Journal, 452 to 454: 1-10. (http://www.ginnyweaver.com/duff/triforce/website/download/CanineMSDS.pdf , 6.1.2009)
	EFSA 2008. Scientific Opinion of the Panel on food contact materials, enzymes, flavourings Wikipedia 2009. E numer. Wikipedia, the free encyklopedia. determination of residues of printing inks in 94 food packaging materials commercially used in Czech Republic did not indicate any acute health risk for food (http://en.wikipedia.org/wiki/E_number#E1000.E2.80.93E1999_.28additional_chemicals.29 , and processing aids (CEF) on 21 st list of substance for food contact materials. The EFSA 5.1.2009)
	consumer. Journal (2008), 888-890, 1-14.
	ii) EuPIA 2005. Opinion on isopropyl thioxantone (ITX) in food packaging inks. December Suspicious findings of higher levels of PIs in PET films for thermosealing of
	2005.	polystyrene cups as well as acetyl tributyl citrate, tributyl aconitate, 2-butoxyethyl

EC 2004. Regulation (EC) No 1935/2004 on materials and articles intended to come into contact with food and repealing Directives 80/590/EEC and 89/109/EEC. The European Parliament and the Council of the European Union, 2004. EC 2006. Commission Regulation (EC) No 2023/2006 on good manufacture practice for materials and articles intended to come into contact with food. The Commission of the Skjevrak I, Due A, Gjerstad KO, Herikstad H. 2003. Volatile organic components migraing from plastic pipes (HDPE, PEX and PVC) into drinking water. Water Research, 37:1912-1920. Skjevrak I, Brede C, Steffensen I-L, Mikalsen A, Alexander J, Fjeldal P, Herikstad H. 2005. Non-targeted multi-component analytical surveillance of plastic food contact materials: Identification of substances not included in EU positive list and their risk assessment. Food Additives and Contaminants, 22(10): 1012-1022.

Table I -

 I List of identified potential residuals from printing inks found in 94 examined samples of packaging materials extracted in level above 0.001 mg/dm 2 (PL = included in EU positive list of Directive 2002/72/EC, SCF-L = Scientific Committee of Food list as defined e.g.in EFSA 2008, TSCA = Toxic Substances Control Act )

	No.	Printing ink residue	CAS-no.	Toxicological data	highest content (mg/dm 2 )	number of occurance	origin/function	Included in EU positive list for polymers (4, 24)
				• LD50
	1	Acetyl tributyl citrate (1,2,3-Tributyl 2-acetyloxypropane-1,2,3-tricarboxylate)	77-90-7	intraperitoneal, mouse: >4g/kg; • low toxicity by ingestion, intraperitoneal routes; skin contact, and	0.898±0. 017	13	Solvent, film-former coatings (1) and plasticizer for inks, adhesives,	PL
				• TSCA listed (1)
	2	Butyl hydroxyl toluene (4-methyl-2,6-ditert-butyl-phenol)	128-37-0	• ADI 0.05 mg/kg bw/d (5)	0.020±0. 002	7	Antioxidant	SML = 0.5 mg/dm 2 (SCF-L 1)
				• Maximised Survey-
				derived Daily Intakes (MSDI-
				EU) : 0.79 µg/capita/day;
	3	Butyl palmitate (butyl hexadecanoate)	111-06-8	• modified Theoretical Added Maximum Daily Intake (mTAMDI) :	0.0045±0 .0005	1	Lubricant (1)	PL (SCF-L 3)
				3500 µg/person/day;
				• Threshold of concern :
				1800 µg/person/day (2)
	4	2-Butoxyethyl oleate (2-butoxyethyl cis-octadec-9-enoate)	109-39-7		0.0616±0 .0037	1	Plasticizer, lubricant

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Acknowledgement

The work was done in the framework of the project number 525/07/1153 supported by Czech Grant Agency.


Valentina Meucci
email: vmeucci@vet.unipi.it

Elisabetta Razzuoli

Giulio Soldani

Francesco Massart

MYCOTOXIN DETECTION IN INFANT FORMULA MILKS IN ITALY

Keywords: Mycotoxins, Mycotoxins -aflatoxins, Mycotoxins -ochratoxin Baby food, Infant formulae, Milk ochratoxin A, aflatoxin M1, milk, infant formula, Italy, child health ), Aflatoxin B1 (AFB1), High Performance Liquid Chromatography (HPLC), Immunoaffinity Columns (IAC), Ochratoxin

After birth, infant formulas constitute an important or often sole food source for infants during the first months of life. In this study, a survey on the presence of aflatoxin M 1 (AFM1) and ochratoxin A (OTA) in the 14 leading brands of infant formulas marketed in Italy was conducted. Mycotoxins were determined by immunoaffinity column clean-up and HPLC with fluorescence detection. AFM1 was found in 2 out of 185 samples but at levels below the European legislation limit of 25 ng/L. OTA was detected in 133 (72%) samples (range 35.1-689.5 ng/L). It has been observed that OTA contamination was 80% in the ready-to-use preparations and 63% in the powdered samples. The Scientific Committee for Food (SCF) reviewed the toxicology on OTA and concluded that it would be prudent to reduce exposure to OTA ensuring that exposure is towards the lower end of the range of tolerable daily intakes of 1.2-14 ng/kg b.w. per day. OTA was also evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and a provisional tolerable weekly intake (PTWI) of 100 ng/kg b.w. was established. The OTA levels in pre-term ready-to-use infant formulas were sufficient to cause a higher OTA intake than the suggested TDI. Our results point out the need to perform controls for prevention programs especially when attempting to identify risk markers of the infant feed quality.

as class 2B, possible human carcinogens (IARC, 1993a;1993b). AFM1 level in milk and in milk products is regulated in several countries; the European Union (EU) limit of 0.05 µg/kg being one of the lowest in the world (EC, 2003a). In contrast, no regulation for OTA in milk exists, even though it has been suggested that OTA level in cow's milk may exceed the tolerable daily intake (TDI) of 5 ng/kg b.w. per day for small children in some areas in Norway [START_REF] Skaug | Analysis of Norwegian milk and infant formulas for ochratoxin A[END_REF].

The Scientific Committee for Food (SCF) reviewed the toxicology on OTA and concluded in its opinion on OTA in food on the 17 th September 1998 (SCF, 1998) that there is an increasing concern about potential genotoxicity of OTA and its mechanism of action as a carcinogen. Therefore the Committee considered it would be prudent to reduce exposure to OTA as much as possible, ensuring that exposures are towards the lower end of the range of tolerable daily intakes of 1.2-14 ng/kg b.w. per day. OTA was also evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and a provisional tolerable weekly intake (PTWI) of 100 ng/kg b.w. was established (FAO/ [START_REF] Fao | Ochratoxin A. In: Safety evaluation of certain mycotoxins in food, Prepared by the 56th Meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA)[END_REF]. The European Food Safety Authority (EFSA) Scientific Panel on Contaminants in the Food Chain in considering all the toxicological and mechanistic data, established a Tolerable Weekly Intake (TWI) of 120 ng/kg body weight [START_REF] Efsa | Opinion of the scientific panel on contaminants in the food chain on a request from the commission related to ochratoxin a in food[END_REF].

Aflatoxins are predominantly produced in hot climates. In temperate countries, the strict control of imported feeds has limited the cases of milk contamination above the maximum tolerable limits. However, the presence of aflatoxins was recently reported in feeds (EC, 2003b;[START_REF] Efsa | Opinion of the scientific panel on contaminants in the food chain on a request from the commission related to aflatoxin B1 as undesirable substance in animal feed[END_REF]. The emergence of aflatoxins in the European continent is concurrent with the increase in the annual average temperature registered in the past decade [START_REF] Anderson | Emerging infectious diseases of plants: Pathogen pollution, climate change and agriculture drivers[END_REF].

Investigations on milk AFM1 contamination are regularly conducted in European countries [START_REF] Efsa | Opinion of the scientific panel on contaminants in the food chain on a request from the commission related to aflatoxin B1 as undesirable substance in animal feed[END_REF], but there is little information on the milk contamination by other mycotoxins. In particular, there is scarce information on the presence of OTA in cow's milk [START_REF] Skaug | Analysis of Norwegian milk and infant formulas for ochratoxin A[END_REF][START_REF] Breitholtz-Emanuelsson | Ochratoxin A in cow's milk and in human milk with corresponding human blood samples[END_REF][START_REF] Valenta | Determination of ochratoxin A in regional samples of cow's milk from Germany[END_REF], and no data are available in Italy concerning infant milk-based formula contamination by this mycotoxin.

Consumption of cow's milk has not previously been considered as possible source of OTA intake, because of the microflora in the rumen hydrolyses OTA to the less toxic metabolite ochratoxin α [START_REF] Hult | Degradation of ochratoxin A by a ruminant[END_REF][START_REF] Sreemannarayana | Studies of the tolerance and disposition of ochratoxin A in young calves[END_REF]. However, OTA has been found in bovine milk, in low concentrations, indicating that small amounts may escape rumen metabolism and are absorbed [START_REF] Skaug | Analysis of Norwegian milk and infant formulas for ochratoxin A[END_REF][START_REF] Breitholtz-Emanuelsson | Ochratoxin A in cow's milk and in human milk with corresponding human blood samples[END_REF]. The OTA concentration in cow's milk is expected to be very low, but even low concentrations might markedly increase OTA daily intake by consumers of large quantities of milk such as children, and contribute to significant portion of the total dietary intake [START_REF] Skaug | Analysis of Norwegian milk and infant formulas for ochratoxin A[END_REF].

After birth, infant formulas constitute an important or often sole source of food for newborns and infants during the first months of life. Thus, these formulas have a special role in the infant diet because they should serve as substitutes for human milk. It is desirable that infant formulas should be proportionally similar or inferior in AFM1 and OTA levels to those found by human milk. In general, AFM1 and OTA concentrations in breast milk show marked variability depending on mother's life style and dietary habits. OTA was detected in milk from healthy mothers in Germany, Norway, Sweden, Hungary, Italy, Australia and Sierra Leone [START_REF] Scott | Biomarkers of human exposure to ochratoxin A[END_REF]. Previous data from Italy had indicated a high prevalence of OTA in human milk samples with concentrations ranging between 1700-6600 ng/L and 100-12000 [START_REF] Micco | Contamination of human milk with ochratoxin A[END_REF][START_REF] Micco | Evaluation of ochratoxin A in human milk in Italy[END_REF]. A second study has been carried out in Italy [START_REF] Turconi | Evaluation of xenobiotics in human milk and ingestion by the newborn--an epidemiological survey in Lombardy (Northern Italy)[END_REF]. OTA was detected in 198 samples (85.7%) at an average value of 6.01 ng/L. More recently, data from milk samples collected from 82 Italian women indicated that 74% of human milk samples contained OTA in the range < 5 ng/L-405 ng/L (Galvano et al., 2008). In the same study AFM1 was detected in four (5%) of milk samples (ranging from < 7 ng/L to 140 ng/L) (Galvano et al., 2008). Relatively little work on the topic of mycotoxins in infant food has been reported in the literature [START_REF] Galvano | Survey of the occurrence of aflatoxin M1 in dairy products marketed in Italy[END_REF]2001;[START_REF] Lombaert | Mycotoxins in infant cereal foods from the Canadian retail market[END_REF][START_REF] Tam | Survey of breakfast and infant cereals for aflatoxins B1, B2, G1 and G2[END_REF][START_REF] Beretta | Ochratoxin A in cereal-based baby foods: occurrence and safety evaluation[END_REF]. Most data have only been part of larger surveys of cereal foods [START_REF] Lombaert | Mycotoxins in infant cereal foods from the Canadian retail market[END_REF][START_REF] Tam | Survey of breakfast and infant cereals for aflatoxins B1, B2, G1 and G2[END_REF][START_REF] Beretta | Ochratoxin A in cereal-based baby foods: occurrence and safety evaluation[END_REF].

For these reasons, the aim of the following work was to determine AFM1 and OTA levels in the leading brands of infant formulas marketed in Italy, and then, to assess their repercussion in the provisional tolerable weekly intake of OTA and of AFM1. Table 1 and 2 report their main characteristics.

Standard preparation

OTA (from Aspergillus ochraceus) (M 403.8) and AFM1 (from Aspergillus flavus) (M 328.2) reference standards, acetic acid (analytical reagent grade) and Celite were purchased from Sigma (Milan, Italy). The OTA standard was dissolved in a toluene-acetic acid mixture (99:1 %, v/v) to give a stock solution of 200 µg/ml which was stored at -20 C until use. Working solutions were prepared by diluting the stock solution with the mobile phase consisting of methanol-sodium phosphate buffer (pH 7.5) 45:55 v/v. The AFM1 standard was dissolved in a acetonitrile to give a stock solution of 10 µg/ml which was stored at -20 C until use.

Working solutions were prepared by diluting the stock solution with the mobile phase consisting of acetonitrile-water 23:77 v/v. HPLC-grade water, methanol and acetonitrile were supplied by LABSCAN (Hasselt, Belgium). The immunOTAffinity columns (IAC) AflaStar® and OchraStar® were purchased from Tecna Srl (Trieste, Italy).

Mycotoxins are highly toxic substances, precautions must be observed when handling these substances, safety instructions for working with mycotoxins are given in [START_REF] Valenta | Chromathographic methods for the determination of ochratoxin A in animal and human tissues and fluids[END_REF]. All mycotoxins waste and contaminated laboratory materials were kept for 24 h in sodium hypochlorite solution (household bleach) for decontamination.

Samples preparation

OTA. Five ml of infant formula samples were mixed with 15 ml of extraction solution (water/methanol 50/50 v/v). Extraction was done by shaking for 5 min on horizontal shaker and centrifuging at 3000 rpm for 10 min. The extract (4 ml) was diluted with 46 ml of phosphate-buffered saline (PBS, pH 7.4). The diluted sample was filtered through Whatman filter paper (Millipore Corporation, Maid Stone, UK). This solution (50 ml) was passed through IAC at flow-rate of 1-2 drops s-1. The column was washed with 20 ml of PBS (1-2 drops s-1). Elution was performed with 3 ml of methanol and acetic acid 98/2 v/v. The elute was evaporated to dryness under nitrogen stream. The residue was re-dissolved in 250 µl of HPLC mobile phase and injected into HPLC system. For samples of powdered milk 5 g were suspended in 30 ml of distilled water, heated to 50°C, homogenized and then processed as for liquid milk.

AFM1. Milk samples were previously skimmed by means of addition of Celite and centrifugation at 3000 rpm for 10 min. After elimination of the fat layer, 50 ml of milk was filtered through Whatman filter paper (Millipore Corporation, Maid Stone, UK) and then passed through IAC at flow-rate of 1-2 drops s -1 . The column was washed with 20 ml of PBS (1-2 drops s -1 ). Elution was performed with 3 ml of methanol. The elute was evaporated to dryness under stream of nitrogen. The residue was re-dissolved in 250 µl of HPLC mobile phase and injected into the HPLC system. For samples of powdered milk 5 g were suspended in 30 ml of distilled water, heated to 50°C, homogenized and then processed as for liquid milk. 8 milk, after thoroughly mixing for 30 min, the mycotoxins fortified milks were left for at least 2 hours at room temperature to enable equilibration and used to assay the cleaning procedures prior to HPLC analysis. For samples of powdered milk, spiked samples were prepared by adding appropriate volume of the spiking solution of mycotoxins to the milk suspension (described above) and then processed as for liquid milk.

High-performance liquid chromatography

The chromatographic system consisted of Jasco880 pump and Jasco821 fluorescence detector (Jasco, Tokyo, Japan). JascoBorwin software was used for data processing. The excitation wavelength (λex) and emission wavelength (λem) were set at 380 and 420 nm for OTA and at 365 and 435 nm for AFM1, respectively. The reversed-phase column was a Luna C18 ODS2, 3 µm, (4.6 x 150mm) (Phenomenex® Torrance, CA, USA). The column was kept at room temperature. The HPLC was operated with mobile phase system consisting of methanolphosphate buffer solution pH 7.5 (0.03 M Na2HPO4, 0.007M NaH2PO4) 50/50 % v/v at flow rate of 1 ml/min and acetonitrile-water 23/77 % v/v at flow rate of 1 ml/min, for OTA and AFM1, respectively. Calibration curves were based on the analysis of triplicate standards solution at 5 concentration levels in matrix and resulted in good linear relationship between peak area and mycotoxins concentration (R 2 >0.999). The limits of detection (LOD) and quantitation (LOQ), calculated as threefold and tenfold the standard deviation of the intercepts of calibration curves are reported in table 1. The recovery (percentage of standard added to sample that is recovered after extraction and clean-up) of the extraction method was determined by sample fortification (Table 2). Intra-day reproducibility was evaluated by analyzing three milk samples with mycotoxins to obtain three different concentrations (10, 50, and 100 ng/L). Day-to-day reproducibility was determined by analysis of three replicate samples of the three concentrations (10, 50, and 100 ng/L) on seven consecutive days. From the data obtained, intra-and interday coefficient of deviations (CV) were calculated. Selectivity studies have been expressed as the ability to asses unequivocally mycotoxins in the presence of components which may be expected to be present: it has been evaluated by the comparison of free-mycotoxin milk samples vs spiked milk for both mycotoxins. Typical chromathograms obtained from mycotoxins free milk samples are reported in figure 1 and2 and compared with that obtained from spiked samples with mycotoxins. Absence of any interfering peak at the retention times of OTA and AFM1 are clearly evident.

Statistical analysis

Values are expressed as mean ± standard deviation (SD). Statistical analysis was performed using two-tailed Student's t-test, Mann-Whitney test, Tukey test and one-way analysis of variance (ANOVA). Correlations between two parameters were determined by Pearson's correlation (r) coefficient analysis. Findings of p< 0.05 were considered significant.

RESULTS AND DISCUSSION

A total of 185 infant cow's milk-based formula samples were analyzed. AFM1 was detected only in 2 samples (range 11.8-15.3 ng/L) from the same brand (n. = 10). The AFM1 contamination levels and incidence in infant formula marketed in Italy were lower than those observed in previous studies regarding milk analysis [START_REF] Galvano | Survey of the occurrence of aflatoxin M1 in dairy products marketed in Italy[END_REF]2001). These results are probably due to the increased attention paid in European countries to the presence of aflatoxins in feeds and milk products and suggest that regulatory limits appear to be helpful.

OTA was detected in 133 (72%) samples (range 35.1-689.5 ng/L). The most contaminated one was a pre-term ready-to-use preparation containing 689.5 ng/L of OTA. In contrast to AFM1, no regulation for OTA in milk exists, even though it has been suggested that the level of this toxin in cow's milk may exceed the TDI of 5 ng/kg b.w. per day for small children in some areas in Norway. The European legislation limit of 0.50 ppb (µg/L) for [START_REF] Ec | Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs[END_REF]. Regarding OTA, [START_REF] Valenta | Determination of ochratoxin A in regional samples of cow's milk from Germany[END_REF] did not find OTA in any of the 121 cow's milk samples analyzed from a northern region of Germany, while OTA was present in a small percentage (10-15 %) of milk samples in Sweden and Norway in the range of 10-50 ng/L [START_REF] Skaug | Analysis of Norwegian milk and infant formulas for ochratoxin A[END_REF][START_REF] Breitholtz-Emanuelsson | Ochratoxin A in cow's milk and in human milk with corresponding human blood samples[END_REF]. The reason of our data could be a higher contamination of feedstuffs in Italy or the existence of other possible routes of OTA exposure such as inhalation of airborne particles containing mycotoxins, as suggested by [START_REF] Skaug | Analysis of Norwegian milk and infant formulas for ochratoxin A[END_REF]. Furthermore, there can be other possible sources of OTA in samples other than the original milk used for manufacturing infant formulas. Different ingredients have been added to infant formulae not only to simulate better the composition of breast milk, but also to impart health benefits. Raw material (cow's milk or isolated protein), processing methods, additives or mineral supplements, and storage will in part determine the levels of minor and trace constituents present in the final product. In the present study, correlations analysis between the OTA contamination levels of different types of infant formula and their main characteristics (lipid, protein and carbohydrates contents) showed no significant (p>0.05) correlation between parameters.

It has been observed that OTA contamination was 80% in the ready-to-use preparations and 63% in the powdered samples. Pre-term and starter ready-to-use formula preparations showed the same incidence of positive samples (80%), while pre-term powdered formula showed 100% incidence and starter formula the 60%, although the numbers of pre-term powdered formula tested were insufficient to obtain an accurate picture of the incidence contamination (Table 4).

Non-parametric statistical analyses (Mann Whitney test) were performed with OTA content for comparison of the different types of formulas, not establishing significant differences (p>0.05) between different groups. The OTA content in both powder and readyto-use formulas has been statistically evaluated to understand the influence of the aggregation state on final OTA level in formulas studied. OTA concentration found in 9 different formulas marked in both forms did not differ significantly, indicating that the most important factor of OTA levels was the formula ingredients. Furthermore, the present study shows a tendency for pre-term both powder and ready-to-use formula to have only slightly higher OTA levels than starter formulas.

Bar diagrams of OTA concentration distribution for all formulas commercialized by each brand analyzed (n. = 14) are shown in figures 3-4. A comparison of significant differences (ANOVA, p=0.0182) among ready-to-use preparations provided from the commercial brands was performed (Figure 3). Among the ready-to-use preparations, OTA level provided by brand 1 (n. = 15, 216.8±217.4 ng/L, range 54.9-689.5 ng/L) was significantly higher (Tukey test, p< 0.05) than those from most of the other brands. Regarding the powdered formulas, a comparison of significant differences (ANOVA, p=0.0438) among commercial brands was performed (Figure 4). OTA level provided by brand 1 (n. = 5, 195.2±138.2 ng/L, range 65.4-340.5 ng/L) was significantly higher (Tukey test, p< 0.05) than those from most of the other brands.

Non-parametric statistical analyses (Mann Whitney test) were performed with OTA content for comparison of the different types of formulas provided from the same commercial brand (pre-term vs. starter formula), not establishing significant differences (p>0.05) between different groups. The same statistical analyses were performed with OTA content for comparison of the different types of formulas provided from the same commercial brand (powdered vs. ready-to-use formula), not establishing significant differences (p>0.05) between different groups.

The OTA concentrations determined in the different types of formulas were used to calculate mean daily and weekly dietary OTA intakes by infants on the basis of feeding tables provided by brands, under the assumption that they receive only formulas. The mean daily 12 intakes for ready-to-use pre-term and starter formula were 16.08±3.93 and 12.29±2.12 ng/kg b.w., respectively; while corresponding weekly intakes were 112.56±27.51 and 86.03±14.84 ng/kg b.w., respectively. The mean daily and weekly intakes for powdered starter formula were 10.67±1.95 and 74.69±13.65 ng/kg b.w., respectively; while for powdered pre-term formula it has been impossible to calculate these values because of the few samples.

The Scientific Committee for Food (SCF) reviewed the toxicology on OTA and concluded that it would be prudent to reduce exposure to OTA ensuring that exposures are towards the lower end of the range of tolerable daily intakes of 1.2-14 ng/kg b.w. per day.

OTA was also evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and a provisional tolerable weekly intake (PTWI) of 100 ng/kg b.w. was established.

In the present investigation, the average OTA levels in pre-term ready-to-use infant formula were sufficient to cause a higher intake of OTA than the suggested TDI and PTWI, while other formulations were below these limits.

Interestingly, OTA was also detected in milk from healthy mothers in Germany, Norway, Sweden, Hungary, Italy, Australia and Sierra Leone [START_REF] Scott | Biomarkers of human exposure to ochratoxin A[END_REF]. The present results are comparable with those obtained by [START_REF] Turconi | Evaluation of xenobiotics in human milk and ingestion by the newborn--an epidemiological survey in Lombardy (Northern Italy)[END_REF] and Galvano et al. (2008) in two studies regarding human milk performed in Italy (198 of 231 samples contaminated with 1-57 ng/L and 74 of 82 samples contaminated with < 5 ng/L-405 ng/L, respectively), while are lower than those reported by [START_REF] Micco | Contamination of human milk with ochratoxin A[END_REF][START_REF] Micco | Evaluation of ochratoxin A in human milk in Italy[END_REF] (ranges 1700-6600 ng/L and 100-12000 ng/L, respectively).

Due to high toxicity and carcinogenic properties of OTA, its presence in milk is a concern. OTA is resistant to thermal inactivation and other varieties of food processing procedures, so to produce high quality milk, it is essential to keep feeds free from contamination by OTA. Mycotoxin concentrations in animal feed can be reduced by good manufacturing practice and good storage practices. On the other hand, infant formula

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

  are highly toxic secondary metabolites produced by several Aspergillus and Penicillium spp that can be found in different commodities including cow's milk. Aflatoxin M1 (AFM1), the major metabolite of aflatoxin B1 (AFB1), and ochratoxin A (OTA) are classified by the International Agency of Research on Cancer (IARC)

  brands commonly marketed in Italy, a total of 185 samples (5 different batch numbers for each product) of infant formula powders (n = 17) and ready-touse preparations (n = 20) were collected from 2006 to 2007 and analyzed. Cow's milk-based formulas were classified as pre-term formula (n = 11; 1 powder and 10 liquid) and starter formula for infants from the first day to 6 months of age (n = 26, 16 powder and 10 liquid).

  Spiked samples. Samples spiked before extraction were used to check the performance of the extraction and clean-up procedure and to obtain validation parameters. Spiking solutions of mycotoxins were prepared daily by dilution with HPLC mobile phases. For samples of liquid

  infant foods was established

  to obtain substantial reductions in levels of contaminants, presumably through more careful selection of raw materials and/or avoidance of contamination during industrial processing.In conclusion, the results of this study firstly indicate the OTA presence in infant cow's milk-based formulas and therefore, they point out the need to perform occurrence surveys in this area. The present study also suggests the usefulness of prevention programs especially when attempting to identify risk markers of the infant feed quality. Analysis of infant cow's milk-based formulas samples hence may serve as a marker of exposure to OTA. At the same time, OTA levels in milk indicate the exposure of new-born infants, which needs to be considered in the overall risk characterization.

  FIGURE LEGENDS

Figure 2 .

 2 Figure 2. Chromatograms obtained from extracts of (a) a blank milk sample and (b) a

Figure 3 .

 3 Figure 3. OTA levels (ng/L) in the ready-to-use milk-based infant formulas provided by

Figure 4 .

 4 Figure 4. OTA levels (ng/L) in the powdered milk-based infant formulas provided by

Figure 1 .

 1 Figure 1. Chromatograms obtained from extracts of (a) a blank milk sample and (b) a milk sample spiked with AFM1.

Figure 2 .

 2 Figure 2. Chromatograms obtained from extracts of (a) a blank milk sample and (b) a milk sample spiked with OTA.

Figure 3 .

 3 Figure 3. OTA levels (ng/L) in the ready-to-use milk-based infant formulas provided by different manufacturers.

Figure 4 .

 4 Figure 4. OTA levels (ng/L) in the powdered milk-based infant formulas provided by different manufacturers.

Table 1 . Composition of liquid (ready-to-use) infant formula analyzed, (values are expressed as g/100 ml of product).

 1

	Brand Type	Lipids Proteins Carbohydrates Minerals
	5	Pre-term 3.75	2.03	8.32	0.37
	1	Pre-term 4.80	3.20	10.90	0.50
	1	Pre-term 3.80	2.20	10.10	0.40
	2	Pre-term 4.40	2.40	7.70	0.34
	3	Pre-term 4.20	2.30	8.60	0.47
	4	Pre-term 4.20	2.20	8.30	0.36
	4	Pre-term 4.00	2.00	7.80	0.35
	6	Pre-term 4.00	2.00	7.80	0.35
	6	Pre-term 4.00	2.30	7.50	0.32
	7	Pre-term 4.40	2.30	8.30	0.400
	2 4 6 3 8 9	Starter Starter F 3.60 4.10 Starter 3.70 Starter 3.60 o Starter 3.60 r 1.40 1.40 1.40 1.20 1.40 Starter 3.60 1.30	7.40 7.30 7.50 7.50 7.70 7.70	0.22 0.30 0.25 0.25 0.30 0.30
	4 5 1	Starter Starter Starter	3.50 3.50 3.1 0	1.70 1.50 P 1.80	7.20 8.30 8.30	0.22 0.32 0.30
	7	Starter	3.70	e 1.30 7.70	0.30
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/100 ml 3.60 1.20 7.50 0.24 Table 2. Composition of powder infant formula analyzed.

				AFM1
	Matrix	LOD	LOQ	Recovery % ± SD CV% intra-day CV% inter-day
	Liquid			75.0± ± ± ±0.3%	3	8
	Powders	2.5 ng/L	5 ng/L	69.5±0.2%	7	9
				OA
	Matrix Liquid Powders	LOD F o LOQ r 1.25 ng/L 6.25 ng/L	Recovery % ± SD CV% intra-day CV% inter-day 87.1± ± ± ±0.3% 2 7 79.5±0.5% 3 8
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Table 3 . Validation parameters for HPLC methods of AFM1 and OA, (LOD, limit of detection; LOQ, limit of quantitation; SD, standard deviation; CV, coefficient of variation).

 3

	Infant formula Number of samples Numbers of positives (%)	Mean±SE	Range
	Pre-term	55	45 (80%)	106.2±23.88 ng/L	<LOD-689.5 ng/L
	Liquid	50	40 (80%)	107.2±26.26 ng/L	<LOD-689.5 ng/L
	Powder	5	5 (100%)	96.10±15.59 ng/L	80.49-127.3 ng/L
	Starter	130	88 (68%)	69.77±8.81 ng/L	<LOD-355.8 ng/L
	Liquid	50	40 (80%)	65.57±11.33 ng/L	<LOD-299.1 ng/L
	Powder	80	48 (60%)	72.40±12.52 ng/L	<LOD-355.8 ng/L
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Table 4 . OA concentrations in different types of infant formulas investigated, (SE, standard error of the mean; LOD, limit of detection). Page 21 of 26 http

 4

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Food Additives and Contaminants


Anne-Catherine Huet

Caroline Charlier

Stefan Weigel

Samuel Godefroy

Philippe Delahaut

Anne-Catherine Huet

A.-C Huet

S Benrejeb Godefroy

Validation of an optical surface plasmon resonance biosensor 1 assay for screening (fluoro)quinolones in egg, fish, and poultry 2 meat 3 4

Keywords: Veterinary drug residues -fluoroquinolones Food Types: Animal products -meat, Eggs, Fish quinolones, biosensor, surface plasmon resonance, validation 28

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Validation of an optical surface plasmon resonance biosensor assay for screening (fluoro)quinolones in egg, fish, and poultry meat

Introduction 29

Over the last few decades, consumers, regulators, and industry have been very 30 concerned about the quality and safety of food. Several crises, mainly of animal 31 origin, have broken out in the last 20 years, causing a decline of consumption of 32 certain foodstuffs. To prevent such crises in the future and to ensure that food is 33 safe for consumers, effective monitoring is necessary. This concern has been 34 integrated into a European project aiming to develop new technologies to screen 35 for multiple chemical contaminants in foods (http://www.biocop.org/). Among 36 these contaminants, fluoroquinolones (FQs) have been chosen as one of the 37 priority residue groups. 38 39 FQs constitute an entirely synthetic class of antibiotics that have found wide 40 application in both human and veterinary clinical practice. The widespread use of 41

FQs in agriculture has resulted in the potential presence of residues of these 42 compounds in foodstuffs of animal origin. In parallel to exposure to low levels of 43 these compounds, an upsetting increase in resistant human pathogens has been 44

observed. This constitutes a public health hazard, primarily through the increased 45 risk of treatment failure [START_REF] Sarközy | Quinolones: a class of antimicrobial agents[END_REF]. The European Commission has thus 46 established maximum residue levels (MRLs) for various FQs in different tissues 47 (EMEA, 1998(EMEA, -2002)). Rapid diagnostic methods for screening for the presence of 48 all FQs in animal products are therefore needed for effective control, i.e. to ensure 49 the correct use of these antibiotics. 50 51 Recently, our team developed a generic immunoassay based on surface plasmon 52 4 resonance (SPR) biosensor technology for a set of 13 (fluoro)Qs in three matrixes 53 [START_REF] Huet | Development of an optical surface plasmon 388 resonance biosensor assay for (fluoro)quinolones in egg, fish, and poultry 389 meat[END_REF]. The current paper describes the validation of this qualitative 54 screening assay according to the criteria specified in European Commission 55 Decision 2002/657/EC (EEC, 2002). The validation parameters 56 specificity/selectivity, decision limit (CCα), detection capability (CCβ), 57 repeatability, ruggedness, and stability are presented in this work. In the literature, 58 two main references have been reported which dealt with validation of a 59 multiresidual method by SPR biosensor [START_REF] Traynor | Detection of multi-Beta-407 Agonist residue in liver matrix by use of a surface plasma resonance biosensor[END_REF]Gaudin et al., 60 2007). 61 62

Materials and methods

63

Apparatus and reagents

64

The Optical SPR Biosensor System Biacore Q and HBS-EP buffer were obtained 65 from Biacore AB (Uppsala, Sweden). 66 Sarafloxacin, ofloxacin, flumequine, norfloxacin, ciprofloxacin hydrochloride, 67 oxolinic acid, lomefloxacin, enoxacin, and nalidixic acid were provided by Sigma 68 (St Louis, MO, USA). Enrofloxacin was obtained from Bayer (Leverkusen, 69 Germany), difloxacin hydrochloride from Chemos GmbH (Regenstauf, Germany), 70 pefloxacin from Rhone Poulenc (Vitry sur Seine, France), danofloxacin from 71 Pfizer Inc (New York, USA), and marbofloxacin was kindly provided by UBE 72 Europe (Lure, France). The related compounds nicotinic acid, nicotinamide, and 73 ethoxyquin were purchased from Sigma-Aldrich (St Louis, MO, USA). 74

Acetonitrile was provided by Biosolve (Valkenswaard, the Netherlands); hexane 75 from Merck (Haar, Germany); sodium chloride, sodium hydroxide, di-sodium 76 hydrogen phosphate, and potassium dihydrogen phosphate from VWR 77 International (Leuven, Belgium); HPLC-grade water from Acros Organics (Geel,78 Belgium). 79

Antibodies and core-FQ sensor chip preparation 80

The polyclonal antibodies raised against norfloxacin hapten-flumequine-BSA 81 were produced in our animal facilities. They were purified by affinity 82 chromatography on a Protein A-Sepharose column (Amersham Biosciences, 83

Belgique) and then concentrated with a centricon (with a molecular weight cut-off 84 of 30,000) purchased from Millipore (Brussels, Belgium). Antibody concentration 85 was measured by the Bio-Rad protein assay (Bio-Rad Laboratories, Hemel 86 Hempstead, UK). Antibody stock solution was diluted 1/4 (v/v) with HBS-EP 87 buffer just before use. 88

89

The core-FQ derivative was immobilised by a standard amine-coupling procedure, 90 it was synthesized from starting product 6-fluoro-4-oxo-1,4-dihydro-3-91 quinolinecarboxylic acid (Apollo Scientific Limited, Bredbury, UK) on which a 3-92 aminopropyl was added at position 1. Between analyses the chips were stored over 93 desiccant at 4°C in a sealed container. 94

95

The full antibody production and FQ derivative immobilisation protocols have 96 been described in a previous publication [START_REF] Huet | Development of an optical surface plasmon 388 resonance biosensor assay for (fluoro)quinolones in egg, fish, and poultry 389 meat[END_REF]. 97 98

Preparation of standard curves in matrixes

99

A stock solution of norfloxacin at 100 µg mL -1 was prepared in methanol. From 100 this solution, spiking solutions were prepared in HBS-EP buffer. A set of five 101 matrix-matched standards at 0.1, 0.5, 1, 2.5 and 10 ng g -1 (poultry meat and egg) 102

Extraction of samples 106

Egg and poultry meat: 2g homogenised muscle or whole egg were extracted with 107 8 mL acetonitrile. Each tube was immediately vortexed before vigorous head-108 over-head shaking for 10 min. After centrifugation, all of the supernatant was 109 evaporated to dryness. The residue was reconstituted in 1 mL PBS pH 7.4 and 110 mixed. Then 1 mL hexane was added and the tubes were vortexed. The sample 111 was centrifuged and the hexane layer and any traces of emulsion at the interface 112 were removed. The washing step was repeated once again and the reconstituted 113 sample was quickly vortexed and centrifuged just before application to the 96-well 114 plate. 115 Fish: as above except that only a 2-mL aliquot of the supernatant was transferred 116 into a new tube and evaporated to dryness. 117

Sample analysis

118 Antiserum freshly diluted in HBS-EP buffer was mixed 25% : 75% with sample 119 extract and injected for 210 seconds over the sensor chip surface at a flow rate of 120 25µl min -1 . The chip surface was regenerated between cycles with a 30-second 121 pulse of 10mM NaOH prepared from 1M NaOH with HPLC grade water at a flow 122 rate of 10 µl min -1 . 123 Specificity means the ability of a method to distinguish between the analyte being 127 measured and other substances. In keeping with the EU Decision requirements, 128 two approaches were used to test this aspect. Firstly, 20 blank samples from 129 different origins of each of the three studied matrixes (egg, poultry meat, and fish) 130 were analysed by the biosensor procedure to detect the presence of possible 131 interferences from endogenous substances and matrix constituents. Secondly, 132 cross-reactivity (CR) profiles were determined for a range of potentially 133 interfering substances by spiking blank samples at relevant concentration (1000 ng 134 g -1 ) with ethoxyquin, nicotinamide, and nicotinic acid considered as FQ analogues. 135

Method validation

The CR profile of the polyclonal antibody was also determined for a range of 136 common (fluoro)Qs. Calibration curves were prepared in each matrix using the 137 compounds of interest in the range 0-1000 ng g -1 (0, 0.25, 1, 2.5, 10, 25, 100, and 138 1000). The CR was calculated from the relevant calibration curves as IC 50 139 (norfloxacin)/CR 50 (competitor) x 100, where IC 50 (inhibitory concentration) is the 140 concentration at midpoint of the standard curve and CR 50 is the concentration of 141 competitor required to cause the same decrease in signal as obtained with 142 norfloxacin used at its IC 50 concentration. The calculations were done with the 143 BIAevaluation software provided by Biacore AB (Uppsala, Sweden). 144 145The detection capability CCβ was determined for the reference molecule 146 norfloxacin. According to the Commission Decision, in the case of substances for 147 which no permitted limit has been established, where no quantitative results are 148 available (i.e. qualitative screening), the CCβ can be determined by investigation 149 of fortified blank material at and above the decision limit (CCα). In this case the 150 concentration level at which only ≤ 5% false compliant results remain equals the 151 keeping with the values just mentioned since they ranged from 0.8 to 1.2 ng g -1 for 198 poultry, from 0.9 to 2 ng g -1 for egg and from 2.6 to 4.1 ng g -1 for fish. Union has established a maximum residue limit (MRL) at 100 ng g -1 for oxolinic 212 acid in fish and chicken muscle, at 600 for flumequine in fish, and at 400 ng g -1 for 213 flumequine in poultry muscle. The cross-reactivity profile was already presented 214 in the previous article [START_REF] Huet | Development of an optical surface plasmon 388 resonance biosensor assay for (fluoro)quinolones in egg, fish, and poultry 389 meat[END_REF], the results were compared and some 215 differences appeared. The assay's sensitivity was better with lower IC 50 values in 216 this work. The two CR profiles for oxolinic acid were quite different, much lower 217 here. These differences are explained by the use of an antibody purified on a 218 protein A-Sepharose column in this publication whereas a crude antibody was 219 working last year. On the other hand, there was no measurable cross-reactivity 220 with the common FQ analogues ethoxyquin, nicotinamide, and nicotinic acid at 221 high concentration. 222

223

The specificity of the SPR assay was also demonstrated after analysis of 20 known 224 negative poultry meat, egg, and fish samples. These showed no interference, when 225 analyzed against a corresponding matrix curve. Determination of the CCα is not 226 Norfloxacin is also commonly used for treatment of animals in many countries 236 [START_REF] Webber | Quinolone resistance in Escherichia coli[END_REF] and very often mentioned in the literature. The 237 negative samples of fish, egg, and chicken muscle were fortified with norfloxacin 238 at 1.5, 1 and 0.5 ng g -1 respectively and then assayed in the biosensor assay. All 239 fortified samples were declared positive (above their respective cut-off values). 240

The CCβ of the assay thus proved inferior to the tested level of fortification. To 241 ascertain if a sample was negative or not, the cut-off level for each matrix was 242 determined from the graphs presented in figure 1, which combine the calculated 243 mean norfloxacin concentrations for blank samples and for samples spiked at the 244 appropriate CCβ level. The means calculated for blank and fortified samples were 245 found to be different and the variability of known negative samples was much 246 lower than that of samples containing norfloxacin. To limit the number of pseudo-247 false positives, all cut-off levels were set near the mean of the spiked samples, as 248 in the case of MRL-regulated compound/matrix combinations, a considerable rate 249 of pseudo-false positive results can be generated; for instance samples containing 250

FQs, but below the MRL. In fact, this high sensitivity of the assay is essential to 251 12 detect flumequine and oxolinic acid at satisfactory levels even if it implies the 252 acceptance of a certain pseudo-false positives rate. This aspect is well-known in 253 the field of immunochemical screening assays using a generic antibody 254 [START_REF] Scortichini | Validation 397 of an enzyme-linked immunosorbent assay screening for quinolones in egg, 398 poultry muscle and feed samples[END_REF]. The cut-off values were set at 1.5, 1, and 0.5 ng g -1 for 255 fish, egg, and poultry meat respectively. More precisely, when the calculated 256 concentration of an unknown sample was below the corresponding cut-off level, 257 this sample was considered negative, but when it was above or equal to the cut-off 258 level, the sample was considered suspect and to require analysis by a confirmatory 259 method for identifying and quantifying (fluoro)Qs. 260 261The determination of cross-reactivities with other (fluoro)Qs (specificity/ 262 selectivity section) then enabled us to calculate the detection capabilities of all 263 investigated compounds. This was done for each matrix according to the following 264 formula: CCβ compound = (CCβ norfloxacin / corresponding CR) X 100. The 265 obtained values are presented in table 2. They range from 0.9 to 3.7 ng g -1 for the 266 fish matrix, from 0.5 to 2.2 ng g -1 for the egg matrix, and from 0.4 to 1.9 ng g -1 for 267 chicken muscle, except for flumequine and oxolinic acid which show higher 268 values due to their lower specificity. The assay could nevertheless detect both 269 residues at or below their half-MRL levels. These results are satisfactory, since the 270

CCβ values for all (fluoro)Qs were well below the established MRL levels set by 271 EMEA for specified analyte/matrix combinations (table 1). Moreover, the assay 272 proved sensitive enough to detect also the non-MRL substances at levels below 10 273 As explained in the experimental section, six replicate samples were spiked with 277 norfloxacin at the appropriate CCβ level and then extracted simultaneously with 278 one calibration curve. The experiment was performed three different times for 279 each matrix of interest. The intra-(n = 6) and interday variations (n =3) calculated 280 are listed in tables 3 and 4, respectively. CVs varied from 2.8 to 5.8 % under 281 intraday repeatability-testing conditions and from 1.9 to 5.7 % under interday 282 repeatability-testing conditions. Since a CV of 15% was set for the desired 283 repeatability, these repeatability results are satisfactory. 284 285 Ruggedness 286 A series of key parameters (the pH of reconstitution buffer (PBS), the operator, 287 and the duration of evaporation) were investigated. Usually, reconstitution of 288 dried residues after evaporation is carried out in 1 ml PBS adjusted to pH 7.4. In 289 this experiment 16 samples per matrix, corresponding to 6 calibration points and 290 10 known negative samples of which 5 were fortified at the corresponding CCβ 291 level were reconstituted in 1 ml PBS at pH 6.4. All blank samples were declared 292 negative because the results were lower than the respective cut-off levels (0.5, 1, 293 and 1.5 ng g -1 respectively in poultry meat, egg, and fish). The result for one 294 sample of chicken muscle, however, was very near to the cut-off value for this 295 matrix. This suggests that reconstitution in PBS with a small change in pH could 296 generate a false positive in this matrix. All spiked samples were declared positive, 297 giving values slightly higher than with the unchanged protocol. 298 299 A different operator carried out all the steps described under the instructions for 300 use of the prototype kit: preparation of calibrant solutions and spiking solutions, 301 No stability study was performed since this SPR assay is a qualitative screening 314 method and could mean to unreliable observations. As described in a recent 315 publication (2007), the team of Gaudin et al. showed that their biosensor method 316 was not suitable for conducting stability studies due to the variability of 317 quantitative results. For instance, they observed an increase of residue 318 concentration in spiked samples stored at -20°C for 20 weeks; no scientific 319 explanation was found for this kind of phenomenon. 320

321

The literature does provide, however, some stability data worth mentioning here. 322

The stability of some FQs in solution has been demonstrated by Okerman et al., 323 2007. In this study, frozen stock solutions of flumequine, enrofloxacin, and 324 marbofloxacin were evaluated in an agar diffusion test. Diameters of inhibition 325 zones were measured at monthly intervals for 6 months, and the decline in active 326 method. This method comparison study demonstrated the performance of our 354 developed SPR biosensor screening assay, since all spiked and incurred samples 355 containing one of the investigated FQ antibiotics, sometimes even at very low 356 concentration, were identified as suspicious by the biosensor method. Further 357 practical details and complete results are presented in a publication (Weigel et al., 358 2009)

  100 ng g -1 (fish) were prepared by fortifying blank extracts. 103 Duplicate injections at each concentration were performed and the mean response 104 was used to construct a calibration curve. 105

  practice, the CCβ was chosen as the lowest tested fortification level 152 giving no negative result in an analysis of 20 spiked blanks. This decision should 153 avoid the problem of false negatives. At least twenty negative samples of fish, 154 egg, and chicken muscle were fortified with norfloxacin at 1.5, 1, and 0.5 ng g -1 155 respectively, and then assayed in the biosensor assay. 156 157 For the repeatability step, a standard curve and six samples fortified with 158 norfloxacin at the corresponding CCβ value were prepared in each studied matrix, 159 and then samples were extracted and injected. This experiment was realized three 160 times in three days, giving intra-and interday variability. The detected 161 concentration of each spiked sample was calculated as were the mean 162 concentrations, the standard deviations (STDEVPA), and the coefficients of 163 variation (CVs). 164 165 To prove the ruggedness of the developed SPR-based FQ assay, the same 166 experiment was performed three times by changing one factor per experiment, 167 namely (i) the pH of the buffer (phosphate-buffered saline, PBS) in which dried 168 residues were reconstituted: pH 6.4 instead of PBS pH 7.4; (ii) the operator; (iii) 169 the time of evaporation (it was doubled). Each experiment was conducted as 170 follows: one calibration curve of norfloxacin was extracted simultaneously with 171 five blank samples of different origins and five blank samples fortified with 172 norfloxacin at its CCβ value in the matrix used. The detected concentration of 173 each spiked sample and each known negative sample was calculated for the egg, 174 fish, and chicken muscle matrix. Spinks in 2000, the current need is to develop assays for more 178 than one analyte per test and to increase sample throughput. Progress in 179 immunoassay development is constrained by the availability of antibodies with 180 appropriate affinity and specificity characteristics. It was a real challenge to 181 develop a single efficient biosensor immunoassay for the entire class of FQ 182 antibiotics. Four immunogens and one engineered antibody were synthesised, and 183 three different FQ derivatives were immobilised on the sensor chip surface. Huet 184 et al. (2008) described all the tested approaches in detail, revealing for the first 185 time the new concept of the bi-active antibody. The most difficult aspect of 186 development was taking into account the fact that some substances have different 187 MRLs, others have no MRLs, and the use of some is prohibited in animals from 188 which eggs are produced for human consumption. 189 190 All samples were determined free of FQ residues by LC-MS/MS before being 191 used in the biosensor procedure. One norfloxacin calibration curve was prepared 192 and extracted for each experiment of the validation study (see experimental 193 section). The midpoints of all these standard curves were evaluated with the 194 BIAevaluation software and compared with the expected values, midpoints at 195 about 1, 1.5, and 3 ng g -1 having been previously determined for poultry muscle, 196 egg, and fish, respectively. All IC 50 values obtained in the validation study were in 197

  the entire procedure was investigated against the following 202 quinolones: norfloxacin, flumequine, enrofloxacin, ciprofloxacin, sarafloxacin, 203 pefloxacin, enoxacin, difloxacin, lomefloxacin, danofloxacin, ofloxacin, 204 marbofloxacin, and oxolinic acid. The calibrants selected for the test were added 205 at six levels concentration. The CR of the binding protein used in the assay is 206shown in table1. The specificity of the antibody towards norfloxacin is the 100% 207 reference. It was confirmed that the selected antibody could detect 11 208 fluoroquinolones with high cross-reactivities ranging from 26 to 183 %. Lower 209 cross-reactions (between 1 and 14 %) were observed for flumequine and oxolinic 210 acid. Although low, these cross-reactivities remain acceptable, as the European 211

  2002/657/EC but can easily be calculated from the data 227 obtained so far. The decision limit CCα, determined as the concentration 228 corresponding to the mean response for negative samples minus three times the 229 standard deviation, was 0.13 (poultry meat), 0.29 (egg), and 0.31 ng g -1 detection capability (CCβ), 20 known negative samples were 233 spiked with the reference molecule norfloxacin, chosen because the polyclonal 234 antibody used as binding protein was raised against a norfloxacin hapten. 235

  , preparation of solutions for the run, running assay. The 302 change of operator did not affect the performance of the method. The third 303 parameter, the drying time, being quite variable in the routine execution of the 304 assay, we multiplied it by two. No variation was observed except that fortified 305 samples gave values slightly higher than with the unchanged protocol. 306 307The midpoints of the calibration curves obtained in this ruggedness experiment are 308 summarized in the table 5. The sensitivity proved similar to that of the usual 309 method except less sensitivity for the fish and egg matrixes reconstituted in PBS at 310 lower pH. 311 312 Stability 313

  methods chosen were an FQ-specific microbiological screening 352 assay and a liquid chromatography-mass spectrometric (LC-MS) confirmatory 353

Figure 436 Figure 1 .

 4361 Figure

Table 1 .

 1 IC 50 values and CR profile of FQ binding protein with a range of 419

	359	.
	360
	361	Conclusions
	362	Multi-residue identification of 13 fluoroquinolones in fish, egg, and poultry
	363	muscle by means of our SPR-based biosensor assay has been successfully
	364	validated. Satisfactory results were obtained with respect to selectivity/specificity,
	365	detection capability, repeatability, and ruggedness of analysis in each matrix. The
	366	pH of the reconstitution buffer might, however, influence the chicken matrix and
	367	lead to false positives. Stability data from a bibliographical study are included in
	368	the paper.
	369

Table 3 .

 3 Intra-assay parameters determined for the FQs SPR-assay (n=6) 428

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		spiking concentration	fish	egg	chicken

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Table 4 .

 4 Inter-assay parameters determined for the FQs SPR-assay (n=3) 430

	spiking concentration	fish	egg	chicken
	norfloxacin, ng g -1	1.5	1	0.5
	mean, ng g -1	1.9	1.2	0.7
	standard deviation, ng g -1	0.11	0.07	0.01
	coefficient variation, %	5.7	5.4	1.9
	431

Table 5 .

 5 Summary of midpoints of curve coming from the calibration curves of 432 norfloxacin realized during the ruggedness study and calculated thanks the 433 BIAevaluation software. These values were expressed in ng g -1 434

		fish	egg	chicken
	standard method	3.0	1.5	1.0
	small change in pH	4.0	2.5	1.2
	operator	3.3	1.4	1.0
	extended evaporation	3.3	1.5	1.0
	435

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Acknowledgements

This project was financially supported by the European Commission, project 371 "New technologies to screen multiple chemical contaminants in foods". BioCop, 372 contract FOOD-CT-2004-06988.