Diane Fuegel
email: diane.fuegel@cvuas.bwl.de

Markus Baumann

Thorsten Rothenbacher

D Fügel

2-Isopropylthioxanthone (2-ITX) in food and food packaging materials on the German market

Keywords: Chromatography -HPLC Additives/Contaminants: Food contact materials, Migration, Packaging Food Types: Baby food, Beverages 2-isopropylthioxanthone, 2-ITX, 2, 4-diethylthioxanthone, 2, 4-DTX, HPLC, UV curing inks, photo-initiator, juice, dairy products, baby food

2-Isopropylthioxanthone (2-ITX) in food and food packaging materials on the German market

Introduction

In September 2005 the Italian authorities informed the European Commission by a notification transmitted through the Rapid Alert System for Food and Feed (RASFF) that they found baby milk contaminated with a substance called 2-isopropylthioxanthone (2-ITX, Compared to solvent-based inks, UV curing seemed to be a good alternative because the packaging material was no longer able to contaminate food with residues of organic solvents from the printing process. But there are new possible contaminants in the packaging material, particularly with regard to acrylates and photo-initiators [START_REF] Papilloud | Analysis of food packaging UV inks for chemicals with potential to migrate into food simulants[END_REF].

Generally 2-ITX can transfer from the packaging into the foods by migration. As intermediate layers of aluminium do not allow ink components to pass through packaging material, it was assumed that in this particular case, 2-ITX contaminated the food by the so-called set-off effect. When the printed material is rolled into spools (e.g. carton base packaging materials) or stacked (e.g. plastic cups), the external layer comes into contact with the internal layer.

During this storage 2-ITX is transferred to the surface intended to come into contact with food and finally can migrate into the foodstuff after packaging. At the beginning of this study 2-ITX was detected only in food packed in cartons. It is even unknown if food, that is packed in materials other than cartons can be affected. To assess the contamination of food by this photo-initiator, there is need to investigate food packed in cartons as well as in other packaging materials like plastic cups or foils.

[Insert Figure 1 Inks applied to the outer surface of food packaging materials are not covered by specific European legislation. One exception is the Commission Directive 93/10/EEC (EEC 1993a).

According to this the printed surfaces of regenerated cellulose film must not come into contact with the foodstuffs. However, the only European legislation concerning 2-ITX in food and packaging materials other than regenerated cellulose film are the Framework Regulation (EC) No 1935/2004(EC 2004), the Regulation (EEC) No 315/93 (EEC 1993b) Manufacturers of food packaging materials are responsible for ensuring that their products comply with the above mentioned regulations and that, from a technological viewpoint, they are suitable for the use for which they are intended. Therefore, the European Printing Ink Association (EuPIA) defined a guideline on printing inks (EUPIA 2006). According to this guideline, if only insufficient toxicological data are available, a substance is acceptable whether its specific migration does not exceed 10 µg kg -1 . If three negative mutagenicity tests as requested by the EFSA-Guidelines are available, as in the case of 2-ITX, the specific migration limit is raised up to 50 µg kg -1 . To assess the migration of 2-ITX into food, several methods have been developed. With respect to UV curing inks and the determination of major acrylates and widely used photo-initiators migrating into simulating solvents, gas chromatography coupled to a mass selective detector (GC-MS) was applied successfully with a recovery rate of 70-100% depending on the simulant used [START_REF] Papilloud | Analysis of food packaging UV inks for chemicals with potential to migrate into food simulants[END_REF]. In milk, yoghurt and fat 2-ITX was determined using accelerated solvent extraction and high performance thin layer chromatography (HPTLC)-fluorescence detection; confirmation of the results was done by HPTLC-mass spectrometry [START_REF] Morlock | Determination of isopropylthioxanthone (ITX) in milk, yoghurt and fat by HPTLC-FLD, HPTLC-ESI/MS and HPTLC-DART/MS[END_REF]. The recoveries of this method ranged from 6-70%, corrected by internal standard up to 70-130% with a limit of detection of 1 µg kg -1 in butter. A method to determine 2-ITX in fruit juices using pressurized liquid extraction and high performance liquid chromatography (HPLC) coupled to a single quadrupole, ion trap, or triple quadrupole MS detection systems led to recoveries of about 70% and detection limits up to 0.05 µg l -1 [START_REF] Sagratini | Determination of isopropyl thioxanthone (ITX) in fruit juices by pressurized liquid extraction and liquid chromatography-mass[END_REF].

In this paper, a fast and reliable method to determine 2-ITX in food and food contact materials to enable an effective routine surveillance of commodities on the German market is described. Due to the UV-activity of photo-initiators it is conceivable to determine this compound in a fast and reliable way using its characteristic UV-spectra and fluorescence activity. Therefore, a method based on HPLC coupled to a diode array (DAD) and a fluorescence detector (FLD) for the quantification of 2-ITX and other photo-initiators (Figure 1) was developed. Following a step-wise procedure, identification was carried out in food contact materials (multi-layer cartons, plastic cups, and foil) at first and in case of positive findings analysis was carried out in the wrapped foodstuffs also.

Experimental

Chemicals

HPLC grade acetonitrile was purchased from Mallinckrodt Chemicals (Griesheim, Germany) and hexafluoro-2-propanol from Sigma-Aldrich (Taufkirchen, Germany). All further solvents were of gradient grade or distilled prior to use. Dist. water was produced by a Milli-Q water purification system (Millipore, Schwalbach, Germany). The sorbent used for the cleanup Bondesil-PSA (40 µm) came from Varian (Darmstadt, Germany). Analytical standard of 2-isopropylthioxanthone (2-ITX, purity 98%) was provided by IGM Resins (Krefeld, Germany)and 2,4-diethylthioxanthone (2,4-DTX, Figure 1) as internal standard (purity 98%) by Sigma-Aldrich (Taufkirchen, Germany). All further chemicals were minimum of analytical quality.

For preparation of 0.1 M citrate phosphate buffer (pH 6.0) 21 g citric acid monohydrate and 14.2 g disodium hydrogen phosphate are dissolved in approximately 900 ml water. After adjusting to pH 6.0 with concentrated sodium hydroxide, the solution is diluted up to 1000 ml with water. Solutions of 2-ITX and 2,4-DTX were prepared in acetonitrile and stored at 4 °C in the dark. Samples 137 samples of fatty and non-fatty food were collected since October 2005 on the German market at retail randomly and partly direct from the food manufacturer. Following a stepwise procedure the food contact materials were tested for 2-ITX at first while storing the homogenized fillings at -18 °C for further analysis in case of positive findings.

Sample preparation

Food contact material. After separation of the food contact material from the filling approximately 4 square centimeters of the printed packaging material are cut into small pieces and extracted with 1 ml hexafluoro-2-propanol (EU DG XII Research Programme AIR 941025 (1994-1997) 1997) in an ultrasonic bath for 45 min. Then 4 ml ethanol are added and the mixture is shaken intensively for 1 min. The precipitate is removed by filtration prior to HPLC analysis. If 2,4-DTX was found originally in food packaging materials, no internal standard was added during the sample preparation (as described below) and both photoinitiators were determined by external standard calculation.

Non-fatty foods (e.g. juices, tomato puree). Sample preparation for non-fatty foods was based on the QuEChERS-method [START_REF] Anastassiades | Fast and easy multiresidue method employing acetonitrile extraction/partitioning and "dispersive solid-phase extraction" for the determination of pesticide residues in produce[END_REF]. To 10 g of the homogenized sample material 10 ml acetonitrile was added. Extraction was carried out by shaking intensively for 1 min. Then 4 g magnesium sulfate and 1 g sodium chloride was added and the mixture was shaken again intensively for 1 min. After addition of the internal standard 2,4-diethylthioxanthone and gently shaking for 30 s the mixture was centrifuged for 5 min at 3000 rpm. An aliquot (8 ml) of the supernatant was mixed with 1.2 g anhydrous magnesium sulfate and 200 mg PSA. After shaking intensively for 30 s, the mixture was centrifuged for 1 min at 5300 rpm. The supernatant was directly subjected to HPLC analysis.

Fatty foods (e.g. yogurt, milk, sausage). To approximately 5 g of homogenized fatty food 5 ml 0.1 M buffer solution (pH 6.0) was added and the sample was extracted by shaking intensively for 1 min. After addition of the internal standard and 30 ml acetonitrile the mixture was shaken for 10 min and quantitatively filtrated through a filter paper. Flask and filter paper were rinsed with 10 ml acetonitrile/water (3/1 v/v) respectively. After addition of 1.5 g sodium chloride and intensively shaking for 30 s the filtrate was mixed with 20 ml tert.-butyl methyl ether/isohexane (80/20 v/v) before gently shaking again. The lower aqueous phase was discarded. The organic layer was washed twice with 20 ml water. After addition of 10 ml tert.-butyl methyl ether/isohexane (50/50 v/v) and, if necessary, separation of the aqueous phase, the organic layer was dried over anhydrous sodium sulfate. The solution was evaporated to dryness under vacuum. Finally, the residue was dissolved in 1 ml acetonitrile and directly subjected to HPLC analysis.

High performance liquid chromatography with diode array and fluorescence detection (HPLC-DAD/FLD)

Analysis was performed on a Agilent 1100 high performance liquid chromatograph (Agilent, Waldbronn, Germany) equipped with a LC-PAH Supelcosil (250 mm; 4.6 mm ID; 5 µm) column (Sigma-Aldrich, Taufkirchen, Germany) coupled to a diode array (260 nm; spectra recorded from 200 up to 500 nm) and a fluorescence detector (excitation 272 nm/emission 440 nm) connected in series. The system was run at 40 °C (stop time 10 min) in isocratic mode (dist. water/acetonitrile 15/85 v/v) with a flow rate of 1 ml min -1 and an injection volume set to 10 µl. The limit of detection and quantitation (2-ITX) as well as the linearity range (2-ITX and 2,4-DTX) were determined by HPLC-FLD analysis of solutions of the photo-initiators in acetonitrile in absence of matrix interferences according to DIN standard 32645 (DIN 1994).

Spiking procedure

Spiking of homogenized blank samples was performed by adding 2-ITX standard solution directly into the matrix. The spiked matrices were shaken briefly and left to stand quite a time (2 min) before extraction to enable the photo-initiator to distribute. For non-atty foods the recovery tests were conducted on blank homogenized orange and vegetable juice by spiking (5 times each matrix and level) with 0.05 and 0.5 mg kg -1 of 2-ITX. For fatty foods blank homogenized milk and oil was spiked (6 times each matrix and level) with 0.1 mg kg -1 of 2-ITX (Table 1).

Results and Discussion

Validation of the method Limits of detection and quantitation. The method for the determination of 2-ITX with HPLC-DAD/FLD (Figure 2) in acetonitrile was linear in the range of 6 up to 120 µg L -1 (Mandel test of linearity) for the FLD-signal with a correlation coefficient of 0.9995. Due to the similar molecular structure and chemical properties 2,4-DTX was used as internal standard (linearity 5 up to 100 µg L -1 ). According the DIN standard 32645 (DIN 1994), the limit of detection and quantification of 2-ITX by fluorescence detection were determined to 2 and 5 µg L -1 in acetonitrile, respectively. Confirmation of 2-ITX by its UV-spectrum could be achieved above 12 µg L -1 . The results of the analyzed samples were only accepted, if the presence of the photo-initiator was approved by the characteristic UV spectrum (Figure 2).

[Insert Figure 2 about here] Recoveries. For non-fatty foods the recovery rates were above 85% (data not shown). By application of the internal standard 2,4-DTX the recovery was increased to 94 -106% with a relative standard deviation (RSD) below 1.1% (Table I). For fatty foods, there was partial interference with the signal of the internal standard. Therefore, determination of 2-ITX in fatty foods was carried out by external calibration (Table I).

[Insert Table I

Analysis of real samples

Samples from the German market (137) were analyzed for 2-ITX by the method reported here (Figure 3). Most of the samples were packed in multi-layer cartons and plastic cups, but also in plastic foil like butter and sausages. The results of the foodstuffs analyzed are shown in Table II. Of all the packages analyzed, 2-ITX was detected in 36 samples (26%, Figure 4).

In 27 of 36 positive tested food packaging materials (75%), significant migration of 2-ITX into the food could be observed with the highest amounts of 2-ITX found in orange juice (357 µg kg -1 ) and baby food (208 µg kg -1 ). In 13 samples (10%) the recommended migration level of 50 µg kg -1 was exceeded.

[Insert Figure 4 about here]

[Insert Table II about here] Furthermore, the migration of 2-ITX into food was not only limited to printed multilayer cartons, but also occurs in food packed in printed plastic cups and foils (Figure 4). However, in these fillings analyzed the migration levels were always below 50 µg kg -1 food, at which level 2-ITX is not likely to pose a health risk (BfR 2005).

The latest results showed that the internal standard 2,4-DTX used in this study can be found in food packaging materials also, e.g. in plastic cups of yoghurt. The concentration of this contaminant in yoghurts was determined to be in the range of 15 to 48 µg kg -1 without addition of the internal standard as described in the experimental section. Like 2-ITX, complete toxicological data concerning 2,4-DTX is not available so far.

Conclusion

With the method presented above, it is possible to determine 2-ITX as well as 2,4-DTX in various food packaging materials and foods in a quick and reliable way. By the strategy chosen to analyze at first the wrappings -and only in case of positive findings -the corresponding fillings, a rapid throughput on a routine basis could be achieved. The recovery rates of 2-ITX in food ranged between 94 and 105% with a relative standard deviation between 0.4 and 8.5%. In practice, the limit of quantification for 2-ITX was below 50 µg kg -1 and thus allowed effective control of the maximum migration level of 50 µg kg -1 recommended by the German Federal Institute of Risk Assessment [START_REF] Bfr | Constituents of printing inks in beverages from cartons[END_REF]. Significant migration of 2-ITX from packaging materials into foodstuff could be detected in 20 % of the samples from the German market investigated, particularly up to 357 µg kg -1 in orange juice and 208 µg kg -1 in baby food. The occurrence of 2-ITX and 2,4-DTX in various food packaging materials, not being limited to multilayer cartons, should guide the industry to apply other, less-migrating photo-initiators. Apart from the efforts of the industry, the implementation of legislative standards for good manufacturing practice with a positive list for printing inks and maximum migration limits especially for substances with incomplete toxicological assessment is inevitable.

Figure 1 ,

 1 Figure 1, RASFF 2005).

  about here] According to the opinion No 044/2005 of the German Federal Institute of Risk Assessment (BfR) on the 25 th November 2005 (BfR 2005) and the press statement of the European Food Safety Authority (EFSA) on the 9 th December 2005 (EFSA 2005), 2-ITX toxicological data was still not sufficient. However, the existing in vivo genotoxicity studies do not indicate a genotoxic potential for 2-ITX (EFSA 2005) and at a maximum migration level of 50 µg kg -1 food 2-ITX is not likely to pose a health risk (BfR 2005).

Figure 1 .Figure 2 .Figure 3 .Figure 4 .

 1234 Figure 1. Chemical structures of the photo-initiators 2-ITX (1), and 2,4-DTX (2).113x35mm (600 x 600 DPI)

Table I .

 I Recoveries rates and relative standard deviations (RSDs) from food samples spiked with 2-ITX.

	food	fortification level	replicates	recovery rates	mean	RSD
		[mg kg -1 ]		[%]	[%]	[%]
	orange juice	0.05	5	95, 96, 96, 95, 95	95	0.4
		0.50	5	104, 104, 105, 103, 104	104	0.6
	vegetable juice	0.05	5	94, 97, 95, 97, 97	96	1.1
		0.50	5	104, 105, 106, 105, 106	105	0.8
	milk	0.10	6	80, 105, 93, 94, 100, 94	94	8.5
	oil	0.10	6	95, 95, 90, 94, 95, 94	94	2.0

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Acknowledgement

We wish to thank Ms Pechstein, Ms Drewnik and Ms Moser for the skilful technical assistance, Mr. Dr. Weißhaar and Mr. Altkofer for valuable discussions and IGM resins for providing 2-ITX.


Pauline ; Mounjouenpou

Noël Durand

Bernard Guyot

Joseph-Pierre P Guiraud
email: guiraud@univ-montp2.fr

Effect of operating conditions on Ochratoxin A extraction in roasted coffee

Keywords: Chromatography -HPLC, Extraction Roasted coffee, OTA extraction, ochratoxin

Confirmation was

extraction time (5, 20, 30, 40, 50, 60 and 80 min). The alkaline solvent that is used in the 20 method recommended by the European Union, extracted OTA better, but a maximum content 21 was obtained at 60°C after 50 min. At least a 100% improvement in extraction was obtained 22 when compared to the EU usual quantification method that is carried out at ambient 23 temperature. It turned out that the OTA extraction parameters for roasted coffee, as defined by 24 that method, were not optimum and needed to be modified. These results were verified in 25 double extraction experiments showing that OTA is not completely extracted by this method. 26 effects [START_REF] Höhler | Ochratoxin A in food and feed: occurrence, legislation and mode of action[END_REF][START_REF] Pfohl-Leszkowicz | L'Ochratoxine A. In: Les mycotoxines dans 192 l'alimentation: Evolution et gestion des risques[END_REF]. Its existence in coffee 36 was reported for the first time by [START_REF] Levi | Study of the occurrence of Ochratoxin A in green coffee 186 beans[END_REF]. Since then, a great deal of work has been 37

undertaken to study what happens to the toxin during technological processing of coffee, such 38 as roasting and beverage preparation [START_REF] Viani | Fate of Ochratoxin A (OTA) during processing of coffee[END_REF][START_REF] Blanc | Behaviour of ochratoxin A during green 172 coffee roasting and soluble coffee manufacture[END_REF]Van der Stegen et al. 39 2001). Some work has indicated a higher OTA content in the beverage (prepared hot) 40 compared to that found in the roasted coffee used for beverage preparation (Tsubouchi et al. 41 1987;[START_REF] Studer-Rohr | The occurrence of Ochratoxin A in 198 coffee[END_REF][START_REF] Suàrez-Quiroz | The impact of roasting on the Ochratoxin A content of coffee[END_REF]. As the usual method used to 42 quantify OTA in roasted coffee (European Union method prEN 14132: 2002 E) The basic OTA quantification method used was that published by [START_REF] Pittet | Liquid chromatographic determination of 195 Ochratoxin A in pure adulterated soluble coffee using an immuno-affinity column cleanup 196 procedure[END_REF] for 62 soluble coffee, an adaptation of the method developed by [START_REF] Nakajima | Determination of Ochratoxin A in coffee beans and coffee products by 189 monoclonal antibody affinity chromatography[END_REF]. The sample 63 of roasted coffee (10 g) was extracted at ambient temperature for 30 min with 100 ml of 64 alkaline solvent. For that study and all the extraction variants, the filtered extract (5 ml) was 65 diluted in 40 ml of alkaline phosphate buffer (PBS) and the mixture was purified on an 66 immuno-affinity column (Ochraprep, Rhône Diagnostics, Glasgow, UK). The toxin was 67 eluted from the column with 3 ml of methanol and the eluant was evaporated till dry in a 68 stream of nitrogen at 70°C. The dry extract was re-suspended in 1 ml of mobile phase 69 consisting of distilled water, acetonitrile and acetic acid (51/48/1; v/v/v). Quantification was 70 carried out by HPLC (Shimadzu LC-10ADVP, Japan) with fluorimetric detection. The 71 operating conditions were as follows: 100 µl injection loop, C18 reverse phase HPLC 72 column, ODS particle size 5 µm (Lichrospher 50DS2, Interchim, Montluçon, France) with 73 identical precolumn, thermostatically controlled at 35°C, isocratic flow of 1ml/min, excitation 74 wavelength of 333 nm and emission wavelength of 460 nm. The contents were calculated 75 from a calibration curve established from a standard (1 000 ng ml irrespective of the extraction time, and which were almost identical (10.3 ± 0.5 µg kg -1 ), the 103 difference in extraction falling within the sensitivity limit of the method (0.03 µg kg -1 ). 104

Consequently, at ambient temperature, there was no significant difference depending on 105 whether extraction was carried out in an alkaline or neutral medium. At 60°C, we found a 106 gradual and continual rise in OTA content in line with the extraction time. After 80 min, the 107 content extracted was 24.4 ± 0.2 µg kg -1 . The extraction improvement was therefore 137% 108 compared to ambient temperature, but the amount extracted was smaller than that obtained in 109 an alkaline medium. At 85°C, the quantities extracted increased in line with the extraction 110 time, up to 50 min. At that extraction maximum, the OTA content was 23.1 ± 0.1 µg kg -1 , 111 which was greater than for extraction at ambient temperature, but was still below the 112 maximum obtained in an alkaline medium. 113 The residue resulting from OTA extraction by the usual method (ambient temperature, i.e. 129 23°C for 30 min) was recovered and re-quantified under optimum conditions, i.e. at 60°C for 130 50 min. The OTA content found for the first extraction was 16.5 ± 0.2 µg kg -1 (higher content 131 than for the first batch of coffee), whereas that found in the residue after the second extraction 132 was 9.1 ± 0.1 µg kg -1 , i.e. a total of 25.6 µg kg -1 . Direct extraction at 60°C gave 26.1 ± 0.6 µg 133 kg -1 for the same batch. These results show that OTA extraction conditions at 23°C were not 134 optimum and largely underestimated the OTA content. Both the temperature and the 135 extraction time were inadequate. 136 137 Verification of the method on several samples of roasted coffee 138 Twenty-four different brands of roasted, ground coffee were bought commercially and 139 quantified using the method recommended by the EU (alkaline solvent at 23°C for 30 min) 140 and the alternative method proposed (alkaline solvent at 60°C for 50 min). method for all the coffee samples contaminated with OTA. The degree of improvement in the 145 extraction rate obtained with the new method varied from sample to sample. That variation 146 could be explained by the type of coffee (arabica, robusta, or blend) and the degree of roasting 147 (light, medium, dark). During roasting, reactions leading to reversible chelation of OTA with 148 other molecules such as proteins can occur [START_REF] Il'ichev | Interaction of Ochratoxin A with 180 human serum albumin. Binding sites localized by competitive interactions with the native 181 protein and its recombinant fragments[END_REF]. Figure 4 shows a 149 chromatograms of a commercial sample of roasted coffee, a same spiked sample (with 2, 5 µg 150 kg -1 added), and a sample of coffee beverage. 151

[Insert Figure 4 about here] 152 153

The purpose of this study was to explain the increase in OTA content found in a coffee 154 beverage compared to that in the roasted coffee used to prepare it. The increase was due to the 155 operating conditions used. The solvent (pH), temperature and extraction time affected the 156 OTA content obtained. The EU method (alkaline solvent at ambient temperature for 30 min) 157

gave unsatisfactory OTA extraction rates. Alkaline solvent was best for extracting OTA from 158 roasted coffee, provided it was extracted at 60°C for 50 min. An improvement of at least 159 100% was observed. The results of OTA analysis in roasted coffee or the beverage by the 160 conventional technique therefore need to be treated with caution. As the legislation fixes a 161 tolerable limit of 5 µg kg -1 , it would be advisable to revise the analysis conditions. 162

  , neutral, acid), temperature (ambient temperature/23°C, 60°C and 85°C) and 19

[

  Figure1shows the curves for OTA extraction in an alkaline medium, depending on the 84

Figure 3

 3 Figure 3 gives the curves for OTA extraction in an acid medium for the same batch of coffee. 117

Figure 1 .Figure 2 .Figure 3 .

 123 Figure captions 214

Table 1

 1 An improvement in OTA extraction, ranging from 100 to 200%, was obtained with the new 144

	gives the

prepare it. Three extraction parameters were studied for roasted coffee : type of extraction

Ochratoxin A (OTA) is a mycotoxin produced by moulds of the genera Aspergillus and

Penicillium. In coffee, the OTA-producing strains most frequently found are Aspergillus

niger, Aspergillus carbonarius and Aspergillus ochraceus[START_REF] Frank | On the activity of fungi in coffee in relation to Ochratoxin A production[END_REF] Joosten et al. 2001;

[START_REF] Suàrez-Quiroz | Study of Ochratoxin A producing strains in coffee processing[END_REF]). OTA has nephrotoxic, immunotoxic, teratogenic and carcinogenic

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Acknowledgements 164


Malgorzata A Majcher

Henryk H Jeleñ
email: henrykj@au.poznan.pl

Małgorzata A Majcher

Henryk H Jeleń

Acrylamide formation in low-fat potato snacks and its correlation with color development

Keywords: low-fat potato snacks, acrylamide, colour development, gas 38 chromatography-mass spectrometry, (GC-MS/MS)

Acrylamide formation in low-fat potato snacks and its correlation with color development

The high temperature used for the preparation of potato snacks to bring a pleasant 42 flavor and colour can also at the same time cause the development of toxic substances 43 such as acrylamide (2-propenamide). Since the year 2002 when a group of Swedish 44 researchers announced the presence of acrylamide in a wide range of fried or oven-45 treated foods, many product types have been investigated. The highest amounts of 46 acrylamide were found to be in fried and baked potato products followed by cereal 47 products, whereas only low amounts of acrylamide were found in meat products (Ahn 48 et al. 2002[START_REF] Becalski | Acrylamide in foods: 300 Occurrence, sources and modeling[END_REF]. Since acrylamide is known to be a neurotoxin, a 49 carcinogen in animals and a probable carcinogen to humans as defined by the 50 International Agency for Research on Cancer (IARC, 1994) it is a concern of food 51 technologists to decrease its amount to the lowest possible levels.

53

There are several hypotheses concerning formation of acrylamide in cooked foods, 54 among which the Maillard browning reaction involving free asparagine and reducing 55 sugars is the most likely and frequently described mechanism (Mottram et al., 2004).

56

The second pathway considered for acrylamide formation is degradation of glycerol to 57 acroleine and later to acrylic acid, which in reaction with ammonia can develop into 58 acrylamide [START_REF] Becalski | Acrylamide in foods: 300 Occurrence, sources and modeling[END_REF]. The optimal temperature for acrylamide formation 59 has been shown to range from 140°C to 180°C (Freidman 2003[START_REF] Elmore | The 309 effect of cooking on acrylamide and its precursors in potato, wheat and rye[END_REF].

60

The Maillard reaction is being used intentionally to develop a nice, desirable texture, 61 flavor and colour of the food product. Several factors are known to influence the colour 62 formation, such as pH, water content and activity, oxygen level but most important is 63 temperature and length of heating time. Increase in temperature by 10°C speeds up a 64 non-enzymatic browning reaction by factors of 3-4 times [START_REF] Belitz | Food Chemistry[END_REF]. The analysis was based on a previously described [START_REF] Castle | Determination of acrylamide monomer in mushrooms grown on 303 polyacrylamide gel[END_REF] method based on 110 bromination of acrylamide into 2-bromopropenamide, followed by GC/MS/MS analysis.

111

Extruded and roasted potato snacks (20 g) were ground and mixed with water (150 ml), 112 spiked with 100 µl of 100 mg l -1 d 3 -acrylamide internal standard, homogenized during 5 113 min and centrifuged after at 14j000 rpm for 15 min. 20 ml of the organic extract was 114 transferred to 100 ml screw-top vial and bromination reagents were added: 5 g of 115 potassium bromide; 0.2 ml of hydrobromic acid; 4 ml of saturated bromine water. The

  ) and d3-acrylamide (isotopic purity 98%) were commercially available 95 from Aldrich (Poznań, Poland) and Cambridge Isotope Laboratories (Andover, MA, 96 USA), respectively; acetonitrile, potassium bromide, hydrobromic acid were obtained 97 from Sigma-Aldrich (Poznań, Poland); n-hexane, sodium thiosulfite, ethyl acetate, 98 saturated bromine water were from Chempur (Tarnowskie Góry, Poland); anhydrous 99 sodium sulfide was obtained from POCh (Gliwice, Poland). All chemicals were of 100 analytical grade. SEP-PAK Silica cartridges for extract purification were purchased 101 from Waters (Warsaw, Poland). 102 Preparation of extruded potato snacks 103 Experiments were conducted with extruded potato snack produced according a recipe 104 developed by us using a procedure described in the aforementioned paper (Majcher 105 and Jeleń 2005). After extrusion, products were cut into small pieces and heated in an 106 oven at 85°C for 15 min followed by 45 min of roasting at 130°C to develop

  116 entire mixture was stirred and set aside in a refrigerator at 4°C overnight. Excess 117 bromine was then neutralized by the addition of 0.7 M sodium thiosulfite solution until 118 yellow colour disappeared. The mixture was then extracted with ethyl acetate (2 x 5 ml) for 10 min. After separation the organic layer was collected 120 and filtered through anhydrous sodium sulfate and concentrated to ~30 µl under a soft 121 stream of nitrogen. To remove the last traces of ethyl acetate, 200 µl of hexane was 122 added and than evaporated to dryness. The residue was re-dissolved in 20% v/v ethyl 123 acetate/hexane (250 µl) prior to cleanup. A SPE-silica cartridge was preconditioned 124 with 7 ml 20% ethyl acetate/hexane and the sample was applied. The cartridge was 125 eluted with 6 ml 30% ethyl acetate/hexane and the first 2 ml was discarded. The rest of 126 the eluate was collected in an amber vial, and finally evaporated to a volume of ~50 µl 127 ready for GC/MS/MS analysis. 128 Gas Chromatography/Mass Spectrometry and quantification 129 The acrylamide content was determined by gas chromatography -tandem mass 130 spectrometry (GC/MS/MS). Brominated sample extracts were analyzed on a 131 Thermoquest Trace 2000 gas chromatograph coupled to Finnigan PolarisQ ion trap 132 mass spectrometer. The chromatograph was equipped with fused silica column type with a Spectro-Pen (Dr Lange, Poland) in the L*, a*, b* modes, 147 which provide uniform colour differentiation in relation to visual differences. L*, a* and 148 b* are chromaticity coordinates, where L* = the lightness of the colour, positive a* = red 149 colour, negative a* = green, positive b* = yellow and negative b* = blue. Because the 150 surface of the potato snacks are not uniform in a colour and shape, colour 151 measurements were performed after milling of 10 g of the sample, where the optical 152 probe was placed over in a central position. Results are reported as an average of and temperature on acrylamide formation 156 Production of low-fat potato snacks consisted of two major technological processes:-157 extrusion cooking and roasting. As has been demonstrated in the aforementioned 158 paper (Majcher and Jeleń 2005) the roasting process is a crucial phase for developing 159 Maillard reaction flavor products. Therefore, the authors chose to leave the extrusion 160 parameters stable and consider only the influence of varying temperatures and roasting 161 times. Ten potato snacks samples were obtained. Five were obtained using a constant 162 time of 45 minutes, but varying the roasting temperatures: 130; 140; 150; 160; 170 o C. 163 Five were obtained using a constant temperature of 130 o C, but varying the roasting 164 times: 45; 55; 65; 75 and 85 min. For each product the acrylamide content was 165 calculated in triplicates and presented as a mean value with standard deviation. The 166 results are presented in Figure 1.

167[

  Insert Figure1about here] 168The average amount of acrylamide formed in low-fat potato snacks using our original 169 recipe when the roasting process is performed at 130°C for 45 min was estimated at 170 489 µg kg -1 ± 26 µg kg -1 . This amount is comparable to acrylamide content in products 171 biscuits, crackers (~ 423 µg kg -1 ) or French fries (~ 537 µg kg -1 ) but almost 172 three times less than in potato chips (~ 1312 µg kg -1 )(WHO 2002).173 174Changes in roasting conditions showed that the amount of acrylamide is highly 175 influenced by both temperature or heating time, however an increase in temperature 176 had a stronger effect than an increase in time. Ten minutes of additional roasting time 177 beyond 45 min doubled the amount of acrylamide, whereas 10 o C increase in 178 temperature from 130°C to 140°C increased the acrylamide content more than five 179 times. The longest time used for the roasting (85 min) caused a lower amount of 180 acrylamide formation in low-fat potato snacks (2010 µg kg -1 ± 64.5 µg kg -1 ) than the 181 smallest raise of temperature from 130°C to 140°C (2767 µg kg -1 ± 93.9 µg kg -1 ). This 182 work indicates that to have a lower acrylamide content in potato based products using 183 a longer time at a lower heating temperature is more satisfactory than using a higher 184 temperature. Ehling and Shibamoto in 2005 obtained similar results by performing 185 experiments on model systems using a different ratio of asparagine and D-glucose and 186 found the acrylamide content to be greater using higher temperatures rather than 187 longer heating times. 188 189 Further analysis of the results showed that the amount of acrylamide continued to 190 increase during entire experiment with variable time as well as increasing temperature 191 but only up to 160°C, at which point any further increase did not change significantly 192 the acrylamide formation. Compared to fried regular potato chips containing a high 193 amount of fat, described by Pedereshi at al (2005), the average acrylamide content 194 increased ~58 times as the frying temperature increased from 120°C to 180°C. Their 195 results gave no indication that acrylamide formation might slow significantly at some 196 point although the temperature continued to rise. This is opposite to the findings of the 197 authors, which indicate acrylamide formation recessed at 160°C At that moment either 198 substratum (i.e. asparagine or reducing sugars) had been all 199 exploited or acrylamide might take part in further Maillard reactions. Ehiling and 200 Shibamoto (2005) who experimented on model systems of asparagine and glucose 201 also noted an increase of acrylamide formation with a rise in temperature but only up to 202 a certain point, after which the level of acrylamide started to decrease. Their results 203 showed that heating above 160°C for a period of 45min reduced acrylamide level as it 204 started to decompose. Although it could be considered as a way to decrease 205 acrylamide content in foods, products obtained with such a high temperature or with 206 such a long time of heating are generally unacceptable to the consumer. 207 Correlation between colour and acrylamide content in low-fat potato snacks in a 208 function of roasting time and temperature 209 In a second experiment, the effect of colour development and acrylamide formation 210 were studied. Samples roasted at 5 different temperatures (130, 140, 150, 160 and 211 170°C) and 5 different times ( 45, 55, 65, 75 and 85 min) were measured for the 212 intensity of lightness (L*), redness (a*) and blueness (b*) and correlated with 213 acrylamide content at p<0.005. Each colour measurement was performed in triplicates 214 and presented as a mean value with calculated standard deviation. 215 216 Measurements of L* value showed that with increasing time of roasting, lightness of the 217 sample was decreasing as well as with temperature increase but only up to 160°C. 218 Higher temperatures did not change significantly the level of lightness. On the contrary 219 acrylamide content was increasing during time extension as well as with temperature 220 increase. However, by increasing the temperature from 160°C to 180°C the acrylamide 221 content did not rise. Calculated correlation coefficient equal to r 2 = -0.995 for time 222 variable and r 2 = -0.947 for temperature change proved that there is a high reverse 223 linear correlation between lightness of the product (L*) and acrylamide content (A) as a 224 function of time (t): ↑T → ↓L* ↑A and temperature (T): ↑t → ↓L* ↑A (Figure 2). value of low-fat potato snacks, when measured at different 228 temperatures and roasting times, demonstrated an increase of redness (a*) either with 229 temperature or time change. However, an increase in the red colour intensity was 230 much more constant with time prolongation whereas change in temperature resulted in 231 substantial increases at the beginning of the heating but only till it reached 150°C, after 232 which it remained without further difference. The analogous situation with acrylamide 233 concentration effected again with high correlation coefficients between a* value and 234 acrylamide content: r 2 = 0.996 for changes in a roasting time(↑t → ↑a* ↑A) and r 2 = 235 0.968 for roasting temperature (↑T → ↑a* ↑A) (Figure 3).

PageFigure 1 .Figure 2 .Figure 3 .Figure 4 .

 1234 Figure 1. Influence of roasting time (temperature even = 130°C) (A) and temperature (time even = 45 min) (B) on acrylamide formation during preparation of low-fat potato snacks.(A)

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

Pedreschi F., Moyano P., Kaack K., Granby K. (2005): Colour changes and acrylamide


M Navarro
email: nalarcon@ugr.es

C Velasco

A Jodral

C Terrés

M Olalla

H Lopez

& M C Lopez

Dr Miguel Navarro-Alarcón

Copper, zinc, calcium and magnesium content of alcoholic beverages and by-products from Spain: nutritional supply

Keywords: AAS, FAAS, Metals analysis, Reference materials Additives/Contaminants: Copper, Heavy metals, Metals -nutritional, Trace elements (nutritional) Food Types: Beer, Beverages, Processed foods, Wine Copper, zinc, calcium, magnesium, alcoholic beverages and by-products, dietary intake

Copper, zinc, calcium and magnesium contents in alcoholic beverages and by-products from Spain: nutritional supply

Miguel Navarro

Introduction

In the manufacturing process employed for most alcoholic beverages we distinguish between fermentation and distillation. In the second process, the distillate is sometimes obtained directly from a previously fermented product, which carries over the volatile compounds (water, alcohols, aromatic substances like acids, aldehides, ketones, esters, etc.) and modifies the composition previously existing in the raw product [START_REF] Olivieri | Preparación y acondicionamiento de los vinos[END_REF]. Due to the low volatility of the four elements considered in this paper (Cu, Zn, Ca, Mg), their levels in the resulting distilled alcoholic beverages have to be lower. In the distillation process a copper still 3 is usually employed because copper is very malleable, a good heat conductor, and resistant to corrosion, as well as playing a role as catalyser in certain chemical reactions and of complexing molecules unpleasant from the organoleptical point of view [START_REF] Olivieri | Preparación y acondicionamiento de los vinos[END_REF].

Consequently, an increase in the Cu levels of the resulting distilled brandies would be expected.

Additionally, it is known that in the ageing of brandies obtained via distillation, the activation of chemical reactions that require oxygen are probably enhanced by the existence of ions of heavy metals like Cu [START_REF] Puech | Barrica y envejecimiento de los aguardientes[END_REF].

Vinegar is one of the oldest known wine by-products that is common in our diet. To manufacture vinegar, oak barrels fitted with aeration holes are half-filled with wine for several months so that the wine's alcohol will be transformed into acetic acid by the Acetobacter bacteria [START_REF] Bourzeix | Productos de diversificación[END_REF]. This fermentation process could influence the mineral content of the resulting vinegar.

Several techniques have been employed for the determination of minerals in wines and beverages: inductively coupled plasma mass spectrometry (ICP-MS) using a double-focusing sector fields; AAS with flame or electro thermal atomization; or electro analytical methods such as differential pulse anodic stripping voltammetry (DPASV) [START_REF] Gonzalez | Multivariate date analysis in classification of must and wine from chemical measurements[END_REF][START_REF] Mikkelsen | Voltammetry using a dental amalgam electrode for heavy metal monitoring of wines and spirits[END_REF][START_REF] Olalla | Optimized determination of calcium in grape juice, wines and other alcoholic beverages by atomic absorption spectrometry[END_REF][START_REF] Kokkinofta | Authenticity of the traditional Cypriot spirit "zivania" on the basis of metal content using a combination of coupled plasma spectroscopy and statistical analysis[END_REF][START_REF] Freire | Application of self-assembled monolayer-based electrode for voltammetric detemination of copper[END_REF]. In the available food composition and nutrition tables from Spain and other countries, the levels of the essential minerals studied, especially those for the trace elements Cu and Zn, are not usually collected. Therefore, the aim of this study was first to determine the Cu, Zn, Ca and Mg content of 124 alcoholic beverages most commonly produced and consumed in Spain (whiskies, gins, rums, liquors, brandies, wines and beers) and by-products (non-alcoholic liquors and vinegars) using the FAAS technique. Consequently, a common mineralization procedure for the analytical determination of the four elements in the referred samples was developed. We also studied the possible influence of the manufacturing process of alcoholic beverages and by-products on their element levels, as well as the evaluation of the existing relationships among these elements in the different samples analysed.

Additionally, we also hypothesised that the manufacturing process followed to obtain white or red wines could influence mineral content. Finally, taking into consideration the mean consumption of alcoholic beverages tested per person per day in Spain (Ministry of Agriculture, Fisheries and Food 2001), we have also calculated the supply from this source to the DDI of these elements per healthy adult in Spain. The results obtained will also prove very useful for compiling food composition tables, having noted the lack of data for these elements in existing tables.

Materials and methods

Apparatus

A Perkin-Elmer 1110B double beam atomic absorption spectrophotometer (Perkin-Elemer corp., CT) was used at a slit width of 0.7 nm, with a 5-mA (for Cu), 3-mA for Zn and 10-mA (for Ca and Mg) hollow cathode lamps for mineral measurements by FAAS. Samples were atomized for Cu, Zn, Ca and Mg determination at 324.7, 213.9, 285.2 and 422.7 nm, respectively. All analyses were performed in peak height mode to calculate absorbance values.

Wet digestion was carried out in a thermostated multi-place digestion block (Selecta S.A, Barcelona, Spain) for analytical samples (whisky, gin, rum, liquors, wines, beers and vinegars).

Reagents

All solutions were prepared from analytical reagent grade reagents: HNO 3 (65%), HClO 4 (60%), HCl (25%), V 2 O 5 (Merck Suprapur). Commercially available 1,000 µg/mL Cu [prepared from Cu(NO 3 ) 2 .3H 2 O in 0.5 M HNO 3 ], Zn [prepared from Zn(NO 3 ) 2 in 0.5 M HNO 3 ], and Ca and Mg dissolutions [prepared from Mg(NO 3 ) 2 .6H 2 O and Ca(NO 3 ) 2 .4H 2 O in 0.5 M HNO 3 , respectively] (Tritisol Merck) were used. The water employed for preparing the standards for calibration and dilutions was ultra pure water with a specific resistivity of 18 mΩ cm -1 obtained by filtering double-distilled water through a Milli-Q purifier (Millipore, Waters, Mildford, MA) immediately before use. The LaCl 3 solution used was prepared by dissolving 1.17 g La 2 O 3 in 5 ml HCl and adding water to 100 ml (1% w/v).

Sampling

The samples of alcoholic beverages and by-products analysed in the present study (n = 124) were purchased in local supermarkets in Granada (a city in south-eastern Spain). Distilled alcoholic beverages (n = 52) as well as by-products (n = 62) and beers (n = 10) were selected from the most popluar brands consumed by the local inhabitants taking into consideration their food habits. With this aim, a previous questionnaire of food consumption by individuals was prepared. The wines sampled correspond to the designations of origin of Sherry (n = 21), Rioja (n = 11) and la Mancha (n = 10). All vinegars considered (n = 10) were manufactured from

Sample treatment

The analytical procedures employed for the mineralization and determination of the Zn, Ca and Mg levels by the FAAS technique are similar to those previously optimised and published by our research group (Terres et al. 2001[START_REF] Jodral-Segado | Magnesium and calcium contents in foods from SE Spain: influencing factors and estimation of daily dietary intakes[END_REF]. For the beverages and byproducts studied, a 1-mL aliquot sample was placed in a glass tube and mineralized by addition of 5.0 ml of concentrated HNO 3 , 10 µg of V 2 O 5, and heating at 60 o C for 20 min in a multiplace mineralization block. Another 5.0 ml of 4:1 mixture of HNO 3 and HClO 4 was added and heated continuously at 90 o C for 45 min. Finally the temperature was raised to 120ºC and kept until sample was completely mineralized. Then, the digest was cooled and the resulting solution diluted with ultra pure water depending on the expected Zn, Ca and Mg concentrations. The adding of 0.2 ml of LaCl 3 solution as matrix modifier in order to avoid the phosphate interferences [START_REF] De La Fuente | Partition of main and trace minerals in milk: effect of ultracentrifugation, rennet coagulation, and dialysis on soluble phase separation[END_REF][START_REF] Moreno-Torres | Comparison of wet and dry mineralisation procedures for determining calcium and phosphorus in cow's milk[END_REF] was done for the Ca and Mg measurements [START_REF] Jodral-Segado | Magnesium and calcium contents in foods from SE Spain: influencing factors and estimation of daily dietary intakes[END_REF]. Zn, Ca and Mg determinations were carried out by direct aspiration into the flame atomic absorption spectrophotometer.

For the chemical analysis of Cu in alcoholic beverages and by-products another 1-ml aliquot was taken. The only difference with the previously described analytical procedure was that the final temperature selected in the digestion method was 125ºC, which was maintained until the digest became transparent.

The presence of matrix interferences was checked by comparing the slopes of the calibration graph and the standard addition methods applied to different samples from all groups of alcoholic beverages and by-products considered (data not shown). The existence of interference was observed only for Ca. Therefore, for this element, the samples were analysed by the standard addition method adding amounts of Ca ranging from 0.00 to 0.250 µg to 6 aliquots of the same sample. All Mg determinations were carried out by the linear calibration method. All samples and blanks were mineralized and diluted using the same procedure. The data were corrected for blank values which were usually very low for this method. The accuracy and precision were tested in 10 replicate assays with 2 CRM-certified reference materials (CRM 063R powered skimmed milk and CRM 278 mussel tissue) and 1 NISTstandard reference material (SRM 1572 citrus leaves) using the procedures previously described (Diaz-Alarcon et al. 1996[START_REF] Delgado-Andrade | Determination of total arsenic in levels by hydride generation atomic absorption spectrometry in foods from south-east Spain: estimation of daily dietary intake[END_REF]. Mineral concentrations found in the reference materials assayed were not significantly different from certified levels (p > 0.05, table I).

Analytical characteristics

Statistical analysis

For interpreting the obtained data the statistical package SPSS 12.0 for Windows program was employed. Results are expressed as arithmetic mean and standard deviation. They were analysed in order to evaluate the existence of statistical differences according to the category of alcoholic beverages and corresponding by-products. The normal distribution of variables and the homogeneity of variances were checked by the Kolmogorov-Smirnov and Bartlett's tests, respectively. The comparisons were done using the Student's t-test when the variable fulfilled parametric conditions, and the Kruskall-Wallis test when conditions were non-parametric.

Linear regression analyses among all mineral concentrations determined were also made.

Results and discussion

Levels of measured minerals (Cu, Zn, Ca and Mg) in alcoholic beverages and by-products from Spain are shown in table II. A high variability in the concentrations of the four elements analysed can be noted. This finding can be related to the drink type and manufacturing process, and also to the strong influence of the soil type and agricultural practices on the content of these elements in the wine grape [START_REF] Olalla | Nutritional study of copper and zinc in grapes and commercial grape juices from Spain[END_REF]). The mineral levels measured in alcoholic beverages and by-products were significantly different for the four elements studied (p < 0.001, table II). Additionally, from our regression analysis of the mineral levels in the alcoholic beverages, significant linear interrelationships (p ≤ 0.001) between Mg and Zn (r = -0.419) and between Cu and Ca (r = -0.365), were observed.

Drinks of high alcoholic content

Cu, Zn, Ca and Mg levels in the whisky, gin, rum, liquor, and brandy samples analysed are summarized in table II, where a broad variation can be observed among the different samples.

The variability is even more pronounced in the case of Cu. Mean Zn levels for alcoholic III). In drinks of high alcoholic content, mean Ca and Mg concentrations determined in the present study are considerably higher in whisky samples (table II). In liquors, although Ca contents measured by us are higher than those shown in table IV, mean Mg concentrations were lower than that indicated by [START_REF] Favier | Répertorie general des aliments[END_REF]. It is interesting to note that many of the food composition and nutrition tables included in table IV contain measurements of mineral content for spirits, although they only colect trace or 0.00 µg g -1 levels for Ca and Mg.

Due to the different manufacturing processes used to produce the liquors and complex brandies [simple brandies redistilled with vegetable matter and then aged (Olalla-Herrera and Lopez-Martínez (2005)], we performed a statistical study to see their influence on Cu, Zn, Ca and Mg concentrations. We discovered that mean Cu levels measured in liquors were statistically lower than those found in the group of complex brandies (p < 0.001) (table II). This result could be related to the use of copper stills for complex brandies (whiskies, rums and brandies) which increases Cu concentrations. Contrarily, mean Mg concentrations found in liquors were significantly higher (p < 0.005) than mean levels measured in complex brandies.

Distilled and fermented drinks

When a comparison was made between the mineral content of distilled products (whisky, gin, rum and alcoholic liquors) and fermented products (wine, beer and vinegar), significant differences were observed for all four minerals (p < 0.005). Specifically, the Zn, Mg and Ca concentrations in distilled drinks were statistically lower. That is to say that in a still, the volatilization process favors the transfer of alcohol, other aromatic and low molecular weight compounds through the condensation coils, and tends to leave behind the heavier metals (Zn, Ca and Mg). Therefore the final Zn, Ca, and Mg concentrations present would be significantly lower in distilled products. Conversely, for fermentation products, Zn, Ca and Mg transfer from the grape [START_REF] Maria | Heavy metals in grapes and tarnave vineyard[END_REF], Cabanis 2003) and barley grain is largely conserved.

Wines and by-products

The mean mineral contents measured in our study for wines, brandies and vinegars (table II) differ from those provided by the food composition tables for several countries (tables III and IV). Mineral levels for the different types of samples analysed (wine, brandy and vinegar are included in table II. Their statistical study in three sample groups demonstrated the existence of significant differences among them (p < 0.001). Specifically, Cu levels found in brandy samples (8.01 ± 1.58 µg ml -1 ) were significantly higher (p < 0.001). This finding is related with the distillation process necessary for their manufacturing. Contrarily for Zn, Ca and Mg mean concentrations found in wines were higher than those found in brandy and vinegar (table II). For Zn, mean levels found in vinegar samples were statistically lower (p < 0.001).

Mean Ca concentrations determined in brandy samples were significantly lower. Finally, mean Mg concentrations were statistically different among the three groups of samples considered.

Specifically, Mg levels in wines were significantly higher. These findings underline that the main source of Ca and Mg in wines and by-products is the grape itself. The presence of these minerals in the grapes (as with all vegetables) is derived from the soil [START_REF] Kokkinofta | Authenticity of the traditional Cypriot spirit "zivania" on the basis of metal content using a combination of coupled plasma spectroscopy and statistical analysis[END_REF], De Lima et al. 2003[START_REF] Salvo | Influence of different mineral and organic pesticide treatments on Cd(II), Cu(II), Pb(II), and Zn(II) contents determined by derivative potentiometric stripping analysis in Italian white and red wines[END_REF]) from which the available fractions of both elements are obtained by the plants. These minerals as cations are present in the grape juice and persist in the wine normally in lower concentrations due to their insolubility during the fermentation and ageing process [START_REF] Olivieri | Preparación y acondicionamiento de los vinos[END_REF].

In white wines analysed, mean Zn and Ca levels (1.386 ± 0.630 and 150.0 ± 51.7 µg ml -1 , respectively) were significantly higher to those found in the red wines (p < 0.05, table V).

Contrarily, Cu concentrations were statistically lower (0.349 ± 0.149 vs. 0.464 ± 0.124 µg ml -1 ). [START_REF] Olalla | Nutritional study of copper and zinc in grapes and commercial grape juices from Spain[END_REF] measured Zn and Cu levels in white and red grapes (0.455 ± 0.147 and 0.525 ± 0.270 µg g -1 ; and 0.479 ± 0.611 and 0.491 ± 0.137 µg g -1 ; respectively) from the most popular commercial brands in Spain. They did not find a significant difference between the two types of grapes. [START_REF] Salvo | Influence of different mineral and organic pesticide treatments on Cd(II), Cu(II), Pb(II), and Zn(II) contents determined by derivative potentiometric stripping analysis in Italian white and red wines[END_REF] noted that when organic pesticides are used on the grapes, a significant enhancement in mineral content is produced in the wine. These findings established that the type of soil, climatic, agronomic and agrochemical treatments influence the total mineral content in wines and are related with the high variability existing in Cu and Zn levels in wines from different countries and regions [START_REF] Salvo | Influence of different mineral and organic pesticide treatments on Cd(II), Cu(II), Pb(II), and Zn(II) contents determined by derivative potentiometric stripping analysis in Italian white and red wines[END_REF][START_REF] Gremaud | Characterization of Swiss vineyards using isotopic data in combination with trace elements and classical parameters[END_REF]).

Zn concentrations found in white and red wines (1.386 ± 0.630 and 0.878 ± 0.238 µg ml -1 , respectively) were lower than those shown in the food composition and nutrition tables (table III). Mean Ca contents measured by us in white and red wine samples (table V) are higher than those shown in the most commonly used food composition and nutrition tables considered (table IV) with the exception of levels indicated in Danish white wines (Food Informatics 2005) which were similar. For Mg we found that the data shown by most of the tables are nearly in agreement with our study (table V). In wines, it is important to know the exact levels of Ca in order to avoid the precipitation as Ca titrate. On the order hand, the Mg is a main component of chlorophyll.

When mineral levels determined in wines are compared taking into account their place of origin (Sherry, Rioja and la Mancha; Commission of the European Communities, 2006) no significant differences were observed for Mg and Cu concentrations (p > 0.05) (table VI). For Ca, levels measured in sherry wines were significantly higher (p < 0.005) than those found in Rioja wines. This finding is similar to that established for Slovakian, Bohemian and European wines [START_REF] Korenovska | Identification of some Slovakian and European wines origin by the use of factor analysis of elemental data[END_REF]Suhaj 2005, Sperkova and[START_REF] Sperkova | Multivariate classification of wines from different Bohemian regions (Czech Republic)[END_REF] for which these researchers found that Ca and Mg contents are the best markers that can be used for the identification of the wine region. For Zn, levels in sherry wines were significantly higher than Rioja and la Mancha wines.

Similarly to the results of others [START_REF] Camean | Metallic profiles of Sherry brandies[END_REF] the content of these metals was higher in Sherry wines than in Sherry brandies (table II), with the exception of Cu .

For wine vinegars, Cu and Zn levels determined in the present study (0.321 ± 0.132 µg ml -1 and 0.723 ± 0.108 µg ml -1 ) were higher than those published by the Danish Institute for food composition and nutrition tables. However, not all these data indicated the source of the vinegar samples.

Beers

The mean Cu, Zn, Ca and Mg contents in analysed beer samples are shown in table II. Mg was the mineral for which a lower variation was observed. The British food composition and nutrition tables [START_REF] Holland | Dietary References Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium and Zinc[END_REF] showed more different types of beer and gave a more precise and complete mean Cu (0.467 µg ml -1 ), Zn (0.183 µg ml -1 ), Ca (82.9 µg ml -1 ) and Mg (97.1 µg ml -1 ) levels. II), their contribution to the mean daily mineral intake in the Spanish diet was calculated by multiplying the mean element content of each drink by its mean consumption in Andalusia (Southern Spain) per person per day as collected in the foodstuff consumption tables in Spain [START_REF] Olalla | Nutritional study of copper and zinc in grapes and commercial grape juices from Spain[END_REF]). On the basis of results obtained, the daily dietary intake of every element for each drink could be estimated.

Estimation of DDI of Cu, Zn, Ca and Mg

Thusly, a daily dietary intake of 123.5 µg Cu, 204.5 µg Zn, 41.7 mg Ca and 20.8 mg Mg per day per person in the Spanish diet was calculated (table VII). Specifically, beer represents the alcoholic beverage which supplies more than 45% of Cu, Ca and Mg intakes in the daily diet (table VII). For Zn, wine constitutes the main source, with a DDI of 55%, followed by beer (40%). These findings are due to the higher beer and wine consumption in the daily diet, and to their higher Ca and Mg content, when compared with distilled alcoholic beverages and liquors.

The dietary reference intake tables for the measured elements included the recommended dietary allowances (RDA) for Cu, Zn, Ca and Mg in different life stage groups (Institute of Medicine 1997Medicine , 2002)). For male and female healthy adults these RDAs are 900 µg Cu day -1 , 11 and 8 mg Zn day -1 y, 1000 mg Ca day -1 and 400-420 and 310-320 mg Mg day -1 . Taking into consideration the daily dietary intakes measured for studied minerals (table VII), the percentages that they would cover of the established RDAs are 13.84%, 1.76 and 2.42%, 4.03%, and 4.84 and 6.31%, respectively. It is interesting to note that the only one element for which alcoholic beverages would constitute an important source is the Cu. However, only a small portion of the minerals ingested through alcoholic beverages in the daily diet is absorbed and transformed into a biologically active form [START_REF] Azenha | Assessment of the Pb and Cu in vitro availability in wines by means of speciation procedures[END_REF]. Consequently, future research will be needed in order to understand how specific factors, such as the manufacturing process and/or components of alcoholic beverages can influence the bioavailability of those mineral in the human gastrointestinal tract. p < 0.001. Cu concentrations measured in brandies were significantly higher to the remaining groups of samples; in rums, found Cu levels were also significantly higher to the other sample groups with the exception of brandies; in samples of whisky, Cu concentrations were significantly higher than those measured in the other groups with the exception of brandies and rums; and finally Cu levels measured in gin samples were significantly lower to the other samples groups.

b p < 0.001. Zn levels found in samples of wine were significantly higher than those measured in non-alcoholic liquors and gins; in brandies, found element concentrations were significantly higher than those determined in gin and beer samples. c p < 0.01. Ca concentrations determined in wines and beers were significantly higher than those found in rum, non-alcoholic liquors and brandies; and in samples of vinegar, mean Ca concentrations were statistically higher than levels measured in brandies. d p < 0.01. Mean Mg levels measured in rums, non-alcoholic liquors and brandies were significantly lower than those found in liquor, wine, vinegar and beer samples; in samples of rums and brandies the mean Mg concentrations found, were statistically lower than levels determined in gin samples; in brandies, mean Mg levels were statistically higher than those found in rums; and finally in wines the mean Mg levels measured, were significantly higher to those determined in beers and liquors.

  samples were brought to the laboratory of the Department of Nutrition and Food Science where they were stored at -18ºC until analysed.

  previous research by other countries show only a trace or very low Zn concentrations (table

  Food and Veterinary Research [0.100 µg g -1 , (Food Informatics 2005)], the USDA Agricultural Research Service (2005) [0.067 and 0.667 µg g-1, respectively,] and those included in the Spanish food composition tables [1 µg g -1 , (Farran et al. 2004)]. Mean Ca and Mg levels in vinegar are included in the Danish [120 and 220 µg g -1 , respectively; (Food Informatics 2005)], American [66.7 µg g -1 , (USDA Agricultural Research Service 2005)], Spanish [150 and 200 µg g -1 , respectively; Farran et al. 2004] and Peruvian [70 µg g -1 only for Ca, (Zavaleta Martinez 1996)]

  the element concentrations in alcoholic beverages and by-products (table

  Accuracy of the spectrometric determination of Cu, Zn, Ca and Mg in reference materials (n = 10) Cu µg g -1 (dry weight)Zn µg g -1 (dry weight) Ca mg g -1 (dry weight) Mg µg g -1 (dry weight)Reference material Certified (mean ± SD) Measured (mean ± SD) Certified (mean ± SD) Measured (mean ± SD) Certified (mean ± SD) Measured (mean ± SD) Certified (mean ± SD) Measured (mean ± SD)CBR CRM 063R

Favier

Table II .

 II Cu, Zn, Ca and Mg contents in alcoholic beverages and by-products from Spain

	Page 15 of 21			Food Additives and Contaminants
	Sample	n	Mean Cu ± SD a	Mean Zn ± SD b	Mean Ca ± SD c	Mean Mg ± SD d
	CBR CRM 278 NIST SRM 1572 a,b,c,d p > 0.05 Drinks of high alcoholic 0.60 ± 0.02 9.60 ± 0.16 16.5 ± 1.0 content Whisky Gin Rum Liquor Brandy Fermented alcoholic drinks Wine Beer By-products Non alcoholic liquors Wine vinegars a	52 10 10 10 12 10 52 42 10 20 10 10	0.57 ± 0.07 9.26 ± 0.26 16.38 ± 0.55 (µg ml -1 ) 2.77 ± 3.18 1.01 ± 0.15 0.10 ± 0.23 2.34 ± 0.94 0.59 ± 0.29 8.01 ± 1.58 0.39 ± 0.15 0.39 ± 0.15 0.40 ± 0.15 0.40 ± 0.18 0.48 ± 0.18 0.32 ± 0.13 F o r P e e r R e v i e w 49.0 ± 0.60 136.6 ± 2.9 29.0 ± 2.0 48.50 ± 2.32 138.6 ± 2.10 27 6 ± 2.80 13.49 ± 0.10 1.07 ± 0.04 31.5 ± 1.00 O n l y 13.21 ± 0.05 1.14 ± 0.05 31.1 ± 1.30 (µg ml -1 ) (µg/ ml -1 ) (µg ml -1 ) 0.80 ± 0.42 0.67 ± 0.47 0.55 ± 0.13 0.62 ± 0.30 0.93 ± 0.47 1.03 ± 0.40 100.1 ± 57.8 105.1 ± 51.8 135.0 ± 53.7 84.7 ± 44.4 125.0 ± 77.0 65.7 ± 20.4 36.2 ± 32.1 34.8 ± 43.4 64.8 ± 27.6 11.4 ± 5.28 60.4 ± 30.5 18.4 ± 8.8 1.08 ± 0.58 1.20 ± 0.58 0.55 ± 0.20 145.9 ± 54.0 136.5 ± 48.1 185.2 ± 62.4 93.2 ± 21.9 97.5 ± 26.6 74.9 ± 11.5 0.67 ± 0.11 0.68 ± 0.09 0.72 ± 0.11 84.3 ± 31.8 64.8 ± 21.7 103.8 ± 28.6 45.3 ± 29.3 27.3± 13.5 63.4 ± 30.1	1262.5 ± 24.00 1.38 ± 0.02 5.80 ± 0.30	1258.0 ± 42.10 1.33 ± 0.09 5.34 ± 0.25
							16

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Table III .

 III Mean Cu and Zn contents in alcoholic beverages from the most frequently used food composition and nutrition tables from various countries

		Cu (µg g -1 )				Zn (µg g -1 )
				Wine				wine
	Reference (country)	Drinks of high alcoholic content	Red	White	Beer	Drinks of high alcoholic content	red	white	beer
	Souci et al. (1989, Germany)
	Jimenez Cruz et al. [1994, 2000 Spain]

Table V .

 V Cu, Zn, Ca and Mg levels measured in white and red wines from Spain

	Page 19 of 21
	Wine type	n	Mean Cu ± SD a	Mean Zn ± SD b	Mean Ca ± SD c	Mean Mg ± SD
			(µg ml -1 )	(µg ml -1 )	(µg ml -1 )	(µg ml -1 )
	White wine	27	0.35 ± 0.15	1.40 ± 0.63	150.0 ± 51.7	97.9 ± 14.9
	Red wine	15	0.46 ± 0.12	0.88 ± 0.24	112.2 ± 28.5	96.9 ± 30.9
	a,b,c p< 0.05

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uk Food Additives and Contaminants F o r P e e r R e v i e w O n l y 20Table VI .

 VI Cu, Zn, Ca and Mg concentrations in different Spanish wines depending of their designations of origin

						Page 20 of 21
	Designation of origin	n	Mean Cu ± SD	Mean Zn ± SD a	Mean Ca ± SD b	Mean Mg ± SD
			(µg ml -1 )	(µg ml -1 )	(µg ml -1 )	(µg ml -1 )
	Sherry	21	0.38 ± 0.15	1.51 ± 0.65	155.4 ± 54.1	97.0 ± 16.0
	Rioja	11	0.46 ± 0.15	0.81 ± 0.22	98.8 ± 20.4	96.2 ± 35.9
	La Mancha	10	0.33.± 0.13	0.99 ± 0.21	138.3 ± 31.5	100.0 ± 10.7
	a,b p < 0.05

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uk Food Additives and Contaminants F o r P e e r R e v i e w O n l y 21Table VII .

 VII Contribution of alcoholic beverages to the DDI of Cu, Zn, Ca and Mg in the Spanish diet

	Page 21 of 21
				Cu		Zn		Ca		Mg
	Sample	Daily consumption	Edible fraction	Mean (µg ml -1 ) Daily intake	Mean (µg ml -1 )	Daily intake	Mean (µg ml -1 )	Daily intake	Mean (µg ml -1 )	Daily intake
		(ml/person)			(µg person -1 )		(µg person -1 )		(mg person -1 )		(mg person -1 )
	Wines	83.8	100	0.390	32.7	1.20	100.6	136.5	11.4	97.5	8.2
	Beer	149	100	0.403	60.0	0.552	82.2	185.2	27.6	74.9	11.2
	Distilled alcoholic
	Beverages and liquors	13.7	100	2.330	31.9	0.769	10.5	93.3	1.28	34.5	0.473
	Total intake				124.6		193.3		40.3		19.87

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Food Additives and Contaminants

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Acknowledgements

The authors would like to thank Mr. J. Robert Mason for his help in editing the English manuscript.


Thomas Wenzl

Elke Anklam

EU database of acrylamide levels in food -update and critical review of data collection

Keywords: Bakery products, Coffee, Potatoes, Processed food

The European Commission's Directorate General Joint Research Centre has been collecting data produced by EU Member States on the acrylamide content of food since 2003. More than 9000 individual data points have been received from official food control laboratories directly or via their Competent Authorities, and from the food industry. Before being entered into the database

Introduction

Whenever health concerns related to food products arise, particularly their composition, reliable data is needed for a thorough evaluation of the magnitude of potential health effects. Hence the exposure of humans to the threat can be estimated. This implies that both data on the content of the substance/group of substances in food, and on the pattern of food consumption are required.

Frequently data are not available ab initio. Studies have to be planned to collect the required data and databases have to be designed in order to receive the data. The preparative steps prior to the data collection are of the utmost importance, since the scope of the database defines the extent of information that needs to be collected.

The finding of acrylamide in food led to a number of research projects worldwide on e.g. the formation, toxicology and mitigation of acrylamide in food. Compilations of activities have been published on the internet by both the European Commission's Directorate General Health and Consumer Protection (DG SANCO, 2005) and the World Health Organization (WHO, 2002). A part of the projects that aim to evaluate the health risk to humans comprises the collection of data on acrylamide levels in different kinds of foods. National as well as supranational organisations such as WHO (WHO, 2005) or the European Commission's Directorate General Joint Research Centre (JRC, 2006) have subsequently set up databases on acrylamide levels in food, and the JRC collects, evaluates and publishes data on a regular basis. A first overview of these two international databases was published by [START_REF] Lineback | Overview on acrylamide monitoring databases[END_REF]. In 2005, the Joint FAO/WHO Committee on Food Additives (JECFA, 2005) assessed the health risk caused by acrylamide on the basis of about 6750 data points for acrylamide levels in food collected on a worldwide scale, including about 4500 data points coming from the EU database. JECFA recommended continuation of the efforts to mitigate the acrylamide levels in food, especially for food that is consumed in developing countries.

Regarding mitigation, a so-called toolbox was developed in a joint effort by different sectors of the food industry (CIAA 2006). This toolbox comprises measures that could be applied for the mitigation of acrylamide in different kinds of food. It also points out where, from an industry perspective, reduction of acrylamide levels is feasible and where limitations have to be taken into The implementation of this toolbox concept is a complex challenge since it should not only be applied by the major food suppliers, but also by small-and medium-sized companies.

Continuous monitoring of acrylamide levels in food is needed to study the effectiveness of mitigation measures, monitoring databases such as the EU database is a very good way to study potential changes of the acrylamide content of particular food commodities or particular food categories with time. If this information is then combined with consumption data, changes in exposure of the population in general, or particular vulnerable subpopulations such as infants, elderly people or people with specific sicknesses can be estimated.

The current paper gives an update of the EU acrylamide database and reports on experience gained during the data collection. It also aims to act as a stimulus for the design of future data collection programmes.

Overview on the EU acrylamide database

The EU database was designed by the JRC in close collaboration with representatives of the Confederation of the European Food and Drink Industry (CIAA) and opened for submission of data in April 2003. Official and private food control laboratories were requested to submit data on acrylamide levels in all kinds of food. Reminders to enter data into the database were given regularly to the EU Member States and the food industry. So far the publication of the acrylamide database on the JRC website has been updated five times. The most recent update dates from June 2006. Data were received mainly from Germany (77.6 %) and from the CIAA (12.1%), which supplied data that were generated by food industry shortly after the discovery of acrylamide in food, but also from Austria, Belgium, Finland, Greece, Ireland, Italy, The Netherlands, Spain, and the United Kingdom (in total 10.3 %). To date, a total number of 9228 individual data sets have been put into the database. The data were produced between 2002 and 2006 and comprise a variety of food items. Most abundant food items/food groups are presented in Table 1.

[Insert Table 1 The highest number of data sets were reported for potato chips (French fries, 1666 data), followed by data on the acrylamide content of biscuits and fine bakery ware (1459 data), and gingerbread (1131 data). A considerable number of data points were also received for food for diabetics (475 data) and food specially intended for babies and infants (in total 384 data sets).

Data assessment

The assessment of the reliability of analysis data is not a standard procedure in the collection of data on residues/contaminants in food. The WHO for example, which collects data on a global scale via the Global Environment Monitoring System -Food Contamination Monitoring and Assessment Programme (GEMS/Food), asks for detailed information on sampling and analytical methods only if the data are to be submitted for the evaluation of the Joint FAO/WHO Meeting on Pesticide Residues (JMPR), JECFA or other FAO/WHO bodies. Other data collection and collation initiatives e.g. the Scientific Co-operations on Questions related to Food (SCOOP) frequently report problems with the comparability of data (SCOOP 2004). Significant differences in the expression of results and meaning of results that were reported below limit of detection (LOD) or limit of quantification (LOQ) led to the suggestion of harmonising further collection of data and of creating a common approach.

With the knowledge that acrylamide was only in processed food (Rosen 2002), and that analytical methods were still in the development phase in many laboratories, it was decided from the beginning to scrutinise the reliability and also comparability of reported data. Criteria that lead to the removal of data from the database were set up together with representatives of the food industry. The criteria are listed and explained in Table 2.

[Table 2

about here please]

The number of data points that were excluded from the database according to the respective criterion is also given in Table 2. Very often incomplete information caused data exclusion.

Although this does not imply that the data were wrong, they were sacrificed for comparability reasons. The same applied to data that were reported as below LOQ with an LOQ above 100 Although the performance in a PT, indicated by the z-score, is only a snapshot of the degree of proficiency a laboratory shows in the determination of a particular analyte at a particular content level in a particular matrix, the accumulation of a number of snapshots allows conservative extrapolation to the general level of expertise with the respective type of analysis. In contrast, unsatisfactory performance in a number of PTs supports questioning the reliability of the reported data. Hence such data were discarded from the database. A descriptor that was proposed to describe the overall performance of a laboratory in a number of PTs is the Running Z-Score (RZ) [START_REF] Thompson | International harmonized protocol for proficiency testing of (chemical) analytical laboratories[END_REF]. It represents the sum of z-scores gained in a number of PTs divided by the number of PTs. RZ scores were omitted in the most recent update of the guidelines "The International Harmonized Protocol for the Proficiency Testing of Analytical Laboratories" [START_REF] Thompson | The international harmonized protocol for the proficiency testing of analytical chemistry laboratories[END_REF] and other scores were proposed. However, since RZ scores have been applied since before 2006 for the evaluation of the data, they are kept for the moment for the assessment of data fed into the database. Data of laboratories for which RZ>|2| was found were removed from the data base. Only 12 data sets, which were put into the database shortly after its installation, complied with that criterion. The laboratory that submitted this data participated in three PTs, for which the results of two tests were far above the accepted range. However the reliability of more than 5800 data is supported by RZ values in the range between -2 and 2.

Evaluation of acrylamide data

From more than 9000 data received almost 7500 data passed the data assessment. Details of the most abundant food classes are listed in Table 1. Besides the overview of accepted records, accompanying information is given on the data distribution. In order to consider acrylamide contents that were reported "below LOD" respectively "below LOQ", this text information has been replaced by numeric values corresponding to half of the reported LOD respectively LOQ.

The minimum value is for almost all food classes formed by half of the LOD of the applied method. Analysis results above the LOQ were constantly found only for coffee and coffee The representation of other food categories in the database is more balanced. A comprehensive overview of the acrylamide levels and their distribution within the first 24 months (May 2002 to April 2004) and the second 24 months (May 2004 to April 2006) of the reporting period is given for 6 food categories (bakery products and biscuits, potato crisps, gingerbread, crisp bread, coffee and diabetics cakes and biscuits) in Table 3.

[Table 3 about here please] The data were grouped according to the date of sampling respectively date of analysis. A shift of the distribution towards lower values was found for potato crisps, crispbread and cakes and biscuits for diabetics, whereas similar values were found for gingerbread. An increase in the acrylamide contents was found for the category "bakery products and biscuits as well as for roasted coffee beans. The amount of data generated in each of the four years of data collection was quite balanced especially for potato crisps. Therefore the variation of the distribution of acrylamide content levels was studied. This variation from year-to-year is depicted in Figure 1.

[Figure 1 about here please] The most significant change for acrylamide content of potato crisps happened from 2002 to 2003.

The apex of the acrylamide content distribution moved from about 470 µg/kg in 2002 to about 300 µg/kg in 2004 and 2005. The distribution also became narrower, which means that the number of samples with high acrylamide contents was lower. The lower number or unbalanced sampling or analysis date prevented such evaluation for the other food categories. However, information based on the analysis respectively sampling date has to be interpreted with caution, especially for bakery products and biscuits. This category comprises a variety of different products with different composition and different manufacturing conditions. Precise information, especially on processing conditions and food composition, was mostly not reported. Another fact that needs attention is the analysis respectively sampling date, which is not necessarily closely linked to the production date This means that the analysis data could be wrongly categorised. However the analysis respectively sampling date is the most complete date specification in the database.

Information on either the production date or expiry date of the product is more suitable for estimation of time dependent trends. The declaration of one or the other depends on the nature of the product.

An expiry date is reported most often for potato crisp samples. Only a very low number of data sets contained information on the production date. Information on the expiry date was given for about 750 of 843 potato crisps samples. The plot of the acrylamide contents against the expiry date of these 750 potato crisps samples is depicted in Figure 2.

[Figure 2

about here please]

As mentioned before a shift towards lower levels could be established. Figure 2 shows the regression curve as well as the corresponding 95% confidence interval. The dotted curve indicates the 95% prediction level. Samples with acrylamide contents above 2500 µg/kg were considered in the calculations, but are not depicted. Unfortunately, the number of data with specific information on production or expiry was much lower for other food categories. Figure 3 shows the development for gingerbread.

[Figure 3 about here please] About 450 of more than 1000 data sets for gingerbread contain information on the expiry date of the product. Data at a level above 1500 µg/kg are not shown for better visibility of lower content levels. A slight decrease of the acrylamide content was found by regression analysis. However the uncertainty of this evaluation needs to be considered, before conclusions can be drawn. Therefore, regression analysis was not performed for all food categories which are represented in the database. The biggest problem was the access to data produced in various laboratories. Much data has been generated, but was never submitted to the database. Evidence of these data can be found in different publications, on the internet or in printed media. It is only due to the strong request of the Competent Authorities that data were received for the evaluation. A major reason for not reporting data is that the filling of the spreadsheet for data submission is considered as too laborious by many laboratories, although it must be stressed that only a third of the entries were compulsory.

Critical review on the data collection

However, at the time of the design of the database, when experiences with the determination of acrylamide in food were still low in many laboratories, the requested information was considered necessary.

The conception of the database layout is a crucial step in the whole data collection activity.

Whatever the purpose of a database might be, the balance between desired information and reasonable workload for supplying this information needs to be respected. The accessibility and usefulness of information that is gathered shall be questioned, especially if the level of uncertainty of the information would nearly prohibit the drawing of conclusion. To illustrate, the influence of process conditions e.g. frying temperature of potato chips/crisps, on the acrylamide content of the final product can be easily studied in model experiments within one laboratory working under repeatability conditions. If such influence shall be studied from the collected data, two events are likely to happen. First, the analysing and data reporting food control laboratory has limited access to such level of detail of information, and second, even if such information is provided by different laboratories, it is questionable whether the reproducibility of analysis results is sufficiently high to allow the detection of any relation between the frying or baking temperature and the acrylamide content of potato crisps or biscuits from the database.

However, the access to information on the processing temperature is different if frying is applied in the laboratory, e.g. for the processing of prefabricated potato chips. In that case information on temperature could be useful for the assessment of the comparability of data.

Regarding the workload of putting data into the database, electronic means that could facilitate data collection are deemed to be necessary. There is indeed a lack of an interface between laboratory data management systems and such monitoring databases that would allow transferring Retrospectively it has also to be stated that the different set-up of databases from which data were fed into the EU database led to differences in the information content of the respective data sets. This was caused by both a different number of fields that were filled and a difference in the level of detail of information that was given e.g. in text fields. For example, a lot of data was reported without information on production/expiry date. For the monitoring of any time dependent mitigation of the acrylamide content, exactly this type of information is required.

Practical details also need to be considered. An important question is related to the software that is used for data collation. Applying standard office software has the advantage of widespread accessibility but has the disadvantage of potential compatibility problems depending on firmware or language version. This happened with the EU database. Disparate date formats were reported.

As a consequence they were not recognised, preventing the consideration of the data in trend analysis. The differences of date formats had to be cleared manually. This is not only time consuming, but of course also prone to error. In that sense any electronic tool that is not sensitive to such influences shall be preferred for data collection to standard office software.

Information on the performance of laboratories in proficiency tests (PTs) was very valuable for data assessment. Within the EU, official food control laboratories need to be accredited according to ISO 17025, meaning that they have to participate regularly in PTs. About 6800 data in the database were produced in accredited laboratories. This does not necessarily mean that the laboratories were accredited for acrylamide analysis. However, in the course of a PT organised by the JRC in 2004, 12 of 42 laboratories reporting results stated explicitly that they were accredited for the analysis of acrylamide in food. It can be expected that the number has been increased meanwhile. [START_REF] Owen | Acrylamide analysis: assessment of six rounds of Food Analysis Performance Assessment Scheme (FAPAS®) proficiency testing[END_REF] reported the participation of between 30 and 50 laboratories in each of 6 consecutive PT rounds organised by the Food Analysis Performance Assessment Scheme (FAPAS®). Nevertheless, obtaining information on the performance of the laboratories was not easy. In most cases data was requested directly from the laboratories. The reason for this was probably a lack of specificity in the instructions on how to fill the respective field of the data submission form.

Another problem is that excel sheets are very valuable for housing static information. Dynamic information such as the performance in different PT rounds, which change with time, is difficult to integrate in a way that ambiguity is avoided. For future databases, it is recommended to develop prior to collection appropriate software solutions that consider all these aspects and make them available to the public.

Conclusions

Despite the problems in data collection that were highlighted in this paper, it shall be stated that the database as a whole has found much appreciation especially for exposure and risk assessment studies. The number of data fed into the database is very high and is publicly accessible. An evaluation of the distribution of acrylamide contents over the data collection period was performed for food categories that are well represented in the database. A trend towards lower acrylamide content levels is indicated for at least some food categories. Regarding the data collection in general, the significance of the data could be increased if data collection systems were harmonised and data transfer simplified. 2002-2004 2004-2006 2002-2004 2004-2006 2002-2004 2004-2006 2002-2004 2004-2006 2002-2004 2004-2006 2002-2004 2004-2006 Total

  . The performance of laboratories in proficiency tests (PTs) provided a valuable insight..

  substitutes.Data for many food classes were produced regularly, while data for potato chips come nearly exclusively from 2002 and 2003. Only about 60 of 1410 samples were analysed after 2003. This

  amount makes the estimation of a trend in the acrylamide content of potato chips meaningless.

  simultaneously respecting all requirements on confidentiality and security of data.

  Figure captions

Figure 1 :

 1 Figure 1: Kernel density plots of the acrylamide contents of potato crisp samples in dependence of the calendar year of sampling respectively analysis

Figure 3 :

 3 Figure 3: Plot of the acrylamide content of gingerbread samples against the expiry date of the product. The solid line represents the regression curve, the medium dashed line the 95% confidence interval of the regression curve and the dotted line the 95% prediction level

Table 1 :

 1 Overview on most abundant food items/food categories in the EU database on AA levels in food:

			Food Additives and Contaminants				Page 18 of 20
	Number of records: number of data received; Number of accepted records: data that passed data assessment; Min: minimum AA
	content, 1 st Quartile: 25% of data are below that content value; Median: 50% of data are below that content value; 3 rd Quartile: 75% of
	data are below that content value; Max: maximum acrylamide content that was reported for that food category
	Food	Number of records	Number of accepted records	Min	1 st Quartile Median 3 rd Quartile	Max
				µg/kg	µg/kg	µg/kg	µg/kg	µg/kg
	Potato chips (French fries)	1666	1408	5	85	186	363	4653
	Bakery ware, biscuits	1459	1074	4	53	145	350	3324
	Gingerbread	1131	1007	5	138	303	668	7834
	Potato crisps	920	843	5	314	528	938	4215
	Crispbread	546	455	5	79	244	505	2838
	Diabetics cakes and biscuits	475	402	5	102	230	620	3044
	Snacks, Popcorn, Salt crackers	442	112	5	25	145	428	3100
	Cereals	410	274	5	30	70	153	1649
	Coffee beans roasted	383	291	79	240	320	578	1188
	Bread and toast	313	151	5	15	50	133	1987
	Baby food	211	191	5	32	67	143	432
	Potato pancake	133	118	10	177	385	730	3072
	Infant biscuits	173	157	5	35	79	166	910
	Coffee substitutes	122	108	116	497	773	1226	2955

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Table 2 :

 2 Data exclusion criteria and number of data that were excluded according to the

	Page 19 of 20
	respective criterion
	No.	Assessment criterion	Data excluded
	1	The reported AA content was below the reported LOQ.	181
	2	The reported AA content was reported as "below LOQ",
		with an LOQ ≥ 100 µg/kg.	53
	3	The LOQ was not reported.	583
	4	The reported values for LOD and LOQ were the same.	662
	5	Information on LOD and LOQ was missing.	332
	6	The calculated running z-score (RZ) was greater than |2|.	12
	7	The reported value was greater than the LOQ, but not
		exactly specified,	93
		e.g. < 100 µg/kg with a LOQ of 30 µg/kg.
	8	Not reasonable values were reported instead of the LOQ,
		e.g. a date instead of a numeric value.	73

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Table 3 :

 3 Comparison of distribution parameter of the AA content of selected food categories that were analysed either between April 2002 and April 2004 or May 2004 and May 2006. The data were categorised according to the data of sampling or analysis of the product.

	Bakery products, biscuits	Potato crisps	Gingerbread	Crispbread	Coffee	Diabetics cakes and biscuits

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Acknowledgement

The authors would like to thank all data contributing laboratories from the EU Member States and food industry.

, the data were assessed for their reliability. This paper presents an update of the database as well as results of the evaluation of data for selected food commodities in order to