Colin Crews

Sarah Hasnip

Laurence Castle

Some factors affecting the formation of furan in heated foods

Keywords:

 (Becalski and Seaman 2005[START_REF] Goldmann | Rapid determination of furan in heated foodstuffs by isotope dilution solid phase micro-extraction-gas chromatographymass spectrometry (SPME-GC-MS)[END_REF][START_REF] Ho | Determination of furan levels in coffee using automated solid-phase microextraction and gas chromatography/mass spectrometry[END_REF]. The method performance can be improved by the use of a deuterium labelled internal standard (d4furan) but care must be taken to ensure a suitable equilibration of the internal standard with the food sample (Becalski and Seaman 2005).

The United States Food and Drug Administration (US FDA) has recently analysed a variety of around 300 food samples for furan and found levels up to approximately 100 ng/g (US FDA 2004). This preliminary survey has been supported by other data presented by EFSA, the European Food Safety Authority [START_REF] Efsa | Report of the Scientific Panel on Contaminants in the Food Chain on provisional findings on furan in food to the European Food Safety Authority[END_REF] and the Swiss Office of Public Health [START_REF] Reinhard | Furan in foods on the Swiss market -method and results[END_REF].

Furan had been identified previously in a small number of heat-treated foods, including coffee, canned meat, bread, cooked chicken, sodium caseinate, hazelnuts, soy protein isolate, hydrolyzed soy protein, rapeseed protein, fish protein concentrate, and caramel [START_REF] Stoffelsma | Volatile components of roasted coffee[END_REF][START_REF] Persson | Aroma of canned beef: Gas chromatographic and mass spectrometric analysis of the volatiles[END_REF][START_REF] Maga | Furans in foods[END_REF]. There is value in expanding the database of information on the occurrence of furan in food as purchased and prepared, and on mechanisms of formation of furan in food. The study on the factors affecting the formation of furan reported here was carried out as part of the EU 6 th framework 'Heatox' project on heat-generated food toxicants [START_REF] Hellenas | The HEATOX project[END_REF].

In 1979, Maga reviewed the formation of furans in food [START_REF] Maga | Furans in foods[END_REF]). The primary source of furans in food was thought to be from thermal degradation and rearrangement of organic compounds, particularly carbohydrates. A variety of experimental systems, including heating of sugars (e.g., glucose, lactose, fructose, xylose, rhamnose), heating sugars in the presence of amino acids or protein (e.g. alanine, cysteine, casein), and thermal degradation of vitamins (ascorbic acid, dehydroascorbic acid, thiamine), have been used to produce, isolate, and identify furan in food. In the studies reviewed by Maga, the furan was found to be formed in the thermal degradation of glucose, glyceraldehydes, D-erythrose, pentosans, hexoses, and polysaccharide; and in a lactose/casein browning system.

More recently Perez Locas and Yaylayan have studied the formation of furan from model systems using pyrolysis-GC-MS analysis and 13 C 3 labelled sugars, amino acids and ascorbic acid (Perez Locas and Yaylayan 2004). They observed that certain amino acids, such as serine and cysteine, can degrade to form furan but that other amino acids, such as aspartic acid, threonine and α-alanine, require the presence of a sugar to form furan. They found from the model systems studied that ascorbic acid had the highest potential to form furan followed by glycoaldehyde/alanine > erythrose > ribose/serine > sucrose/serine > fructose/serine > glucose/cysteine.

In 2005, Becalski and Seaman also reported the formation of furan from ascorbic acid and its derivatives (Becalski and Seaman 2005). In addition they demonstrated that

Materials and methods:

Reagents and samples Furan (99+%) and d 4 -furan >99 atom% D were supplied by Sigma-Aldrich (Poole, Dorset). Food samples were obtained from local retail stores.

Apparatus

Domestic heating was carried out using a Sharp Carousel R-7400 microwave oven, output 600W, frequency 2450 MHz; a Victoriana HPG gas hob; an Electrolux EOB 811 electric oven; a Morphy Richards 4460 toaster; and a Prestige Smartplus 57050 pressure cooker.

Gas chromatography/mass spectrometry was carried out using a ThermoFinnigan

Voyager benchtop system fitted with a CTC CombiPal static headspace autosampler.

The column was of 15 m x 0.32 mm i.d. fused silica, with a 20 µm bonded polystyrenedivinylbenzene phase (HP-Plot Q). The carrier gas was helium at a constant flow of 1.7 mL/min. The split/splitless injector port and mass spectrometer interface were heated to 200 and 250°C respectively. The oven temperature was programmed from 50°C held for 1 min, to 225°C at 10°C min -1 and held 12.5 minutes. Split injections were made at a ratio of 2:1. spike solution (50 uL, 25 µg/ml) was added to one of each pair of samples. Vials were cooled in ice prior to and during the addition of internal standard to avoid loss of furan.

Furan was quantified by comparing the peak area ratios of the response at m/z 68 for furan and m/z 72 for d4-furan for the unspiked sample with that for the standard addition sample. For some experiments quantification was based on an external calibration series. This was necessary since in these experiments the total sample produced on heating constituted one analytical sample and quantification by standard additions could not be used. For both versions of the method the samples were run in batches of up to 30 samples. Each batch included a reagent blank, a low-level tomato sauce reference material (a retail sauce naturally containing furan, which had been characterised by repeated analysis) and a spiked sample fortified at 25 ng/g. The method had a limit of detection of 2 ng/g.

Furan levels in retail foods before and after heating

Levels of furan in retail foods before and after heating under domestic conditions were studied using composite samples of foods purchased in retail stores. The composition of the composites is provided in Table 1. To retain the structure of the food so that a realistic procedure could be carried out samples containing large particulates, such as vegetables, were mixed by selecting equivalent proportions of each sample. This procedure inevitably led to some lack of homogeneity of the composites prior to subsampling for heating. Liquid samples were chilled and stirred together.

Insert Table 1 about here Foods were heated using domestic appliances (electric toaster, microwave oven, gas and electric cookers) using the instructions given on the packets of the individual components. All heating experiments were carried out at least in duplicate. For microwaving, baby foods, stews, sauces, canned vegetables, custard, fish, potatoes, fresh vegetables, soup and baked beans, were heated in either a glass or polyethylene microwave casserole with or without a lid on the high setting for the time indicated by the instructions (between 2 and 4 minutes) with stirring once midway through the total time. Canned vegetables were cooked in their canning liquid. Baby foods were cooked for 30 seconds. Microwaved milk was heated in a cup for 3 minutes on high setting.

To warm baby foods they were placed in a glass basin over water and stirred constantly in for 4 minutes. Infant formulae were prepared in a polyethylene baby feeding bottle by adding boiled water which had been cooled to 60 ° and standing for 10 seconds.

Boiled or saucepan-heated foods (baby foods, stews, sauces, canned vegetables, custard, fish, potatoes, fresh vegetables, soup and baked beans) were heated in a PTFE coated saucepan on a high flame to boiling point and then turned to a low setting for the time indicated by the instructions, which was typically four minutes. Canned vegetables were cooked in their canning liquid. Where appropriate (custard, baked beans, sauces, soup) samples without lids were stirred continually.

Coffees were prepared from ground medium-roasted powder using a cafetiere using quantities recommended on the packet. Instant coffee was prepared in the cup by adding boiling water to a heaped tea-spoon quantity of coffee powder. For both coffees other samples were stood for 5 minutes, or had milk added before cooling prior to analysis.

Bread was toasted in a pre-heated domestic toaster for 4 minutes and left for 30 seconds before being placed in sealed polythene bags for storage. For roasting fish and meats the oven was pre-heated to 190 ºC. Fish were placed in a glass casserole with water and cooked for 30 minutes. Meat samples were purchased as minced meat. Portions of 200 g were formed into thick burger shapes and placed on a PTFE sheet on a baking tray in the oven for 30 minutes.

A representative selection of food types and heating methods was chosen and experiments on these were carried out five times to allow for an estimation of uncertainty on the whole preparation process. Foods were allowed to cool to about 60

°C before bottling to replicate serving time prior to consumption. Samples were then stored in screw cap high-density polyethylene bottles prior to analysis. This would not affect the levels of furan in the samples since the polar furan molecule would have little affinity for the waxy polymer of the storage bottles and the storage period was brief (1-7 days at -20°C). All samples were analysed before and after heating. To allow reasonable comparison for heated and unheated coffee and infant formula samples, cold water was used to prepare the unheated samples.

Circumstances leading to furan formation

This study addressed questions as to whether furan is formed by simply heating foods, whether is it found predominantly in canned and jarred foods because it cannot escape by evaporation, and whether is it produced (in part) by migration from the packaging or by reaction of the food with the packaging.

Retail dried vegetable soup mixtures were used for some experimental work. The mixtures comprised yellow split peas, green split peas, green lentils, red spilt lentils, green beans, onion, pearl barley, pasta and pot barley. The mixtures were milled together to a fine powder and homogenized thoroughly by roller mixing to form a Typically a mixture of 0.5 g dried vegetable mix and 8 ml distilled water was used as the model on which to carry out heating experiments as this resulted in a heated product of suitable mobility for direct analysis, obviating the need for addition of extra water, or homogenisation.

Retail food composites of three types: sweet (rice pudding), savoury (baby food)

and acidic (fruit puree) foods were also used for these experiments. Where possible, retail samples that had not been processed in either jars or cans were used, otherwise food processed and sold in glass jars was used for experiments with coupons of can material (cut from sheets used to manufacture food cans) and food processed and sold in metal cans was used for experiments with sections of gaskets. This ensured that the food used had not previously been exposed to the packaging materials being tested.

To demonstrate whether furan is formed in foods by heating, 0.5 g portions of the ground vegetable mixture (mixed with water) and the other foods were heated in sealed vials in a pressure cooker at 12 p.s.i. and 117°C for 20 minutes. Samples were then analysed in the same vials after chilling, removal of the caps, addition of internal standard and resealing. Prior to heating six 2-4 mm glass beads were placed in the vial with the sample to improve mixing during headspace shaking incubation. This negated the need for sample homogenisation which could have led to the loss of furan.

To assess whether the predominance of furan in canned and jarred foods is due to heat processing in a closed environment, samples of the ground vegetable mix and the other foods were heated as described above in both sealed and open vials. Open vials were sealed immediately after heating. Internal standard was added as before. To assess whether interaction with lacquered packaging plays any part in furan formation, samples of the foods were heated as described above in the presence of coupons of tin-plated steel coated with a variety of food can lacquers. Typical can surface area to food mass ratios were calculated for a variety of canned foods including baby foods, which are sold in particularly small cans. The ratios ranged from 0.8 to 1.2 cm 2 /g. To ensure that a ratio in excess of this was used, eight 1 cm 2 coupons (lacquered on one side) were heated with 5 g of each food.

To assess whether interaction with gasket material plays any part in furan formation similar samples were heated as described above with portions of different gaskets removed from jar lids, and with portions of epoxidised linseed (ELO) and soyabean oils (ESBO) that are commonly used in the formulation of sealing gaskets.

Typical gasket mass to food mass ratios from jars were calculated and an excess (0.03 g) of gasket was heated with 5 g of sample. Quantities of ELO and ESBO, and nonepoxidised linseed and soyabean oils, were heated with ground vegetable mix and water as described above. The oil quantities used were in considerable excess (0.5 g) in order

to amplify any minor effect.

Results and discussion:

Analytical methodology

The headspace gas chromatography mass spectrometry method reported by the US FDA (US FDA 2004) used an incubation period at 80ºC for equilibration of furan between the sample and headspace, which we considered might lead to the formation of furan during this heating step. Using a range of incubation temperatures applied to samples of tomato sauce, baked beans, baby food and heated vegetables we found no apparent formation during incubation if the temperature was less than 60°C, however, furan concentration increased when the incubation temperature was raised to 80°C by between 10-300% depending on the food matrix. We considered that 50°C was a suitable incubation temperature for general use. The use of automated vial shaking removed the need for prolonged equilibration times indicated by Becalski et al (Becalski et al. 2005).

Quality Assurance

All reagent blank samples showed a response equivalent to less than 2 ng/g. All determinations of furan in the tomato sauce reference material fell within two standard deviations of the characterised value (2 ng/g ± 1.8 ng/g). Recoveries for the tomato sauce reference spiked at 25 ng/g ranged from 83 % to 97 %. The correlation coefficients of all calibration standard graphs were higher than 0.99. The analytical measurement uncertainty was calculated from between-batch precision and mean recoveries for in-house reference materials and fortified samples in accordance with Eurachem guidelines [START_REF] Ellison | Quantifying Uncertainty in Analytical Measurement, Eurachem/CITAC Guide[END_REF], was found to be ± 2.0 ng/g at 2 ng/g, ± 9 ng/g at 25 ng/g and ± 33 ng/g at 100 ng/g.

Cooking Variation

Reproducibility experiments on the various heating methods gave an estimate of the variability of results due to cooking variation; a combined measurement uncertainty for both heating and analysis could then be given for each result.

Heating soup over five batches in a saucepan gave a mean furan concentration of 19 ng/g with a standard deviation of 13 ng/g. Heating in a microwave gave a mean of 21 ng/g with a standard deviation of 17 ng/g. Toasting bread over five batches gave a mean furan concentration of 39 ng/g with a standard deviation of 54 ng/g (range: 3-129 ng/g). Coffee prepared in five batches had a mean furan concentration of 4 ng/g with a

Furan levels in raw foods then heated

The levels of furan found before and after heating composites of retail foods which had not been cooked during manufacture were measured.

Furan was detected only in potatoes (2 ng/g) and green vegetables (6 ng/g) of the foods which had not been heated during manufacture. Furan was not detected in these foods after heating but was measured at very low level (2 ng/g) in the roasted root vegetables.

Prior to heating foods that had not been heated in cans or jars during production (fresh vegetables, fish, and meat) furan was either not detected or present at only very low levels.

Furan levels in heat-processed foods then heated

The levels of furan found before and after heating composites of retail foods which had been heated during manufacture are shown in Table 2. Of the foods not processed in cans or jars but subjected to other heat processing during production, i.e., milk (pasteurised), infant formula (spray dried), bread (baked), and coffee (roasted) only coffee contained detectable furan prior to heating. The custard composite was made up of samples purchased both in cans and in foil-lined boxes; it contained no detectable furan prior to heating.

Insert Table 2 purchase formed no significant levels during preparation. No differences resulting from alternative heating methods were greater than the combined measurement uncertainty due to heating and analysis and therefore it is not possible to say whether a particular heating method had an effect on the formation of furan, but if any did then it was not at a significant level.

The only sample that did not contain detectable furan on purchase, but which after heating was bread. A mean level of 39 ng/g was found in the five toasted bread samples, however, the variation was very large (sd = 54 ng/g), as was the variation in toast colour and final temperature (possibly a function of the toaster performance). The link between final temperature and furan level is not clear but generally higher temperatures produced higher levels of furan. Temperatures of about 140-160 °C resulted in 3 ng/g furan whereas temperatures of about 170-200 °C resulted in 12-129 ng/g furan. The fact that no detectable furan was found in baked but untoasted bread might reflect the smaller surface area to volume ratio of the loaf compared with the bread slice. It may also be due to loss of furan through evaporation during open cooling of the loaf, or that the white breads used were baked in steam rather than by traditional 'dry' methods.

Coffee prepared from ground roasted coffee beans contained greater concentrations of furan (based on the brewed product) than that prepared from soluble instant coffee. This is consistent with the much greater concentration of furan in ground coffee beans (about 2000 ng/g) compared to instant coffee granules (about 500 ng/g).

Coffee prepared from ground coffee beans using cold water contained less furan than that prepared using hot water. This may be due to formation in the hot water or it may reflect the greater extractability of furan in hot water, if so it must outweigh any increased losses due to greater evaporation.

Circumstances leading to furan formation

The method performance characteristics were within the range of the previous experiments. The analytical measurement uncertainty was found to be ± 2.1 ng/g at 2 ng/g, ± 4.2 ng/g at 25 ng/g and ± 15 ng/g at 100 ng/g.

Reproducibility experiments on the heating of the dried vegetable model system gave a mean furan concentration of 41 ng/g with a standard deviation of 18 ng/g across 23 batches. Since these experiments concern the effects of additions to the sample rather than the effects of varying heating conditions, and samples for comparison were all prepared in the same batch, between-batch variation due to heating was not included in the measurement uncertainty.

Table 3 shows the concentrations of furan found in samples of retail foods before and after heating at 117 °C under 12 p.s.i. pressure.. An increase in furan on heating was observed in samples of rice pudding, fruit puree and in one of the two baby foods. These retail samples included foods purchased in cans and jars, which had therefore already been subjected to heat treatment. This increase shows that although some furan had already been produced during the manufacture of these foods, there is a potential for further formation. However, both samples of canned foods showed a markedly lower tendency to form further furan under this heating regime than did the other foods and this may indicate that the limit of furan formation had been reached during the heating processes of industrial preparation.

Insert Table 3 suggests that the occurrence of furan in predominantly canned and jarred foods may be due to there being no loss by evaporation, leading to accumulation.

The concentration of furan found in samples of retail foods heated in the presence of coupons of can with various lacquers are shown in Table 4. Comparing the percentage differences of the concentration of furan in samples containing coupons of can to the control samples containing no can it was apparent that the differences are random across the different food types, thus the canning materials played no significant part in the formation of furan.

Insert Table 4 about here

Table 5 shows the concentration of furan found in samples of retail foods heated in the presence of portions of various sealing gaskets. Plotting the percentage differences of the concentration of furan in samples containing portions of sealing gaskets to the control samples containing no gasket showed that the differences were random across the different food types.

Insert Table 5 about hereThe concentration of furan formed on heating ESBO, ELO, and unepoxidised soybean and linseed oils with and without ground vegetable mix is shown in Table 6.

Insert both formed some furan, with the sample containing linseed oil forming appreciably more than that with soybean oil. Similarly when the epoxidised oils were heated with vegetable mix ELO formed considerably more furan than EBSO. There was no cooperative effect observed between ESBO and ground vegetable mix, since the total furan formed was approximately the sum of the furan obtained from each component alone. However, this was not the case for the sample of ELO with ground vegetable mix, which formed more furan than the components alone and more than untreated linseed oil with the same vegetable mix.

Linseed oil contains ~60% linolenic acid (18:3) and ~20% linoleic acid (18:2) whereas soybean oil is less unsaturated and contains ~10% linolenic and ~50 % linoleic.

It has been shown that linolenic acid is a more efficient precursor of furan than linoleic acid (Becalski and Seaman 2005). The greater unsaturation of linolenic acid may provide more potential for formation through auto-oxidation and cleavage. The differences in the compositions of the fatty acids in the oils are, therefore, consistent with the patterns of formation of furan.

Although the experiments show that epoxidised oils do contain and form furan on heating and furthermore, that for ELO this is increased on interaction with food, the concentrations of epoxidised oils used here, 2g/g vegetable, are much greater than result from contact with sealing gaskets in jars of food. Gaskets contain up to 30% epoxidised oil thus the ratio in contact with food is <2mg/g food. The fact that the furan concentration did not increase on the addition of portions of gasket is consistent with this. Furan levels in retail food samples were found to persist during the normal heating practices that precede consumption. If extra furan was formed in these foods during preparation then a concurrent loss by reaction or evaporation also occurred.

Furan was demonstrated to be formed in foods by heating, although it was shown not to accumulate to a significant degree on heating in an open vessel. Furan appears to accumulate particularly in heat-processed canned and jarred foods, because they are sealed containers that receive a considerable thermal load.

There were no interactions with cans and their coatings nor with gaskets that had a significant influence on furan formation. Studies of the epoxidised oils used in sealing gaskets showed that the epoxidation of oils leads to the formation of furan, particularly in linseed oil which has a high content of linolenic acid. Furthermore heating ELO led to further formation of furan, particularly in the presence of vegetable mix. At the levels of epoxidised oils found in gaskets, however, there should be no effect on the formation of furan in foods.

In agreement with work carried out by Becalski and Seaman (Becalski and Seaman 2005), soybean oil and linseed oil were found to be precursors of furan and their potency as precursors reflected the degree of unsaturation in their fatty acid composition.

  formed from the oxidation of polyunsaturated fatty acids at elevated temperatures and that the addition of commercially available antioxidants (such as tocopherol acetate) reduced the formation of furan by up to 70 %.The objectives of this work were to investigate the comparative levels of furan in retail foods as purchased and after preparation by different domestic cooking practices (microwaving, boiling, and roasting), and to study its formation and accumulation in food contained in cans and jars.

  The headspace incubation oven temperature was 50°C and the syringe needle 80°C. Samples were incubated for 30 minutes with shaking. The injection volume was 1 ml and the injection speed 30 ml/minute.The mass spectrometer was operated in electron ionization mode with a source temperature of 200ºC and an emission current of 150 µA. Furan was detected by selected ion monitoring of the major ion at m/z 68 and confirmed by monitoring of the ion at m/z 39. The internal standard was detected by monitoring the equivalent ion at m/z 72.MethodFor the experiments on the domestic preparation of foods, analyses were carried out using a modification of the USFDA method (US FDA 2004), with a reduced incubation temperature of 50 °C. Stock solutions (c.a. 2.5 mg/mL) of furan and d 4 -furan were prepared by adding 50 µL furan via a chilled gastight syringe through the septum of a weighed 20 ml headspace vial containing 20.0 mL methanol. The mixture was then reweighed to determine the exact concentration of furan. Working solutions were prepared daily by dilution of the stock solutions using the same procedure. External calibration standards equivalent to 5, 25, 50, 125, 250, 500 and 1000 ng/vial were prepared.Foods that were not homogenous were chilled in a refrigerator for a minimum of 4 hours. They were transferred to a beaker immersed in an ice bath and homogenised using a blender for about 20 seconds. Duplicate 5 g portions of each sample were weighed accurately into 20 ml headspace vials and 5 mL chilled water were added quickly. Internal standard solution (50 uL, 25 µg/ml) was added to each vial, and furan

  be stored, handled conveniently, and made into a 'wet' food in the headspace vial by addition of water.

  1 ng/g. Furan was not detected in any meat sample roasted in five batches.

TABLE CAPTIONS Table 1

 CAPTIONS1 Composition of the composite samplesTable 2 Summary of furan levels in heat-processed foods on cooking Table 3 Effects of heating in a closed vessel on furan concentration in food Table 4 Effects of contact with can materials on furan formation Table 5 Effects of contact with jar lid gasket materials on furan formation Table 6 Formation of furan from dried vegetable/epoxidised oil mixtures

	Table 2 Vegetable soup	unheated	17		4
	Experiment	saucepan heated	Furan ng/g 19		MU a 11
	Baby food chicken	uncooked microwaved	23 21		6 15
	Vegetables canned	microwaved unheated	17 4		19 2
		warmed saucepan heated	18 4		17 5
	Baby food vegetable	uncooked microwave glass lid	32 3		8 5
		microwaved microwave open	31 3		35 4
		warmed microwave plastic lid	20 3		19 4
	F o r F o r Baked beans uncooked microwaved saucepan heated White bread uncooked toasted F o r a Total Measurement uncertainty including uncertainty due to both analysis and heating 24 6 31 35 24 23 <2 39 48 b As prepared in cold water F o r
	Coffee instant	P e P e uncooked b as made P P on standing e e	4 4 4		2 1 2
	Ground coffee	e r e r with milk uncooked b e r e r	4 28		2 7
	Custard Infant formula	R e v i e w R e v i e w 54 43 38 R R e e <2 <2 saucepan heated as made on standing with milk uncooked microwaved <2 uncooked v i e v i e <2 heated as directed <2 microwaved <2 w w	13 10 9
	Milk Stew	uncooked saucepan heated microwaved uncooked saucepan heated	<2 <2 <2 25 17	O n l y O n l y O n l O n l 6 16 y y
		microwave glass lid	26		30
		microwave open	17		19
		microwave plastic lid	16		19
	Tomato based sauce	uncooked	12		3
		saucepan heated	6		6
		microwave glass lid	9		10
		microwave open	7		8
		microwave plastic lid	6		7

Some factors affecting the formation of furan in heated foods

	Table 6
	Sample Sarah Hasnip*, Colin Crews and Laurence Castle Type of oil Heated/ Unheated Range ng/vial Introduction:	Mean ng/vial	MU c ng/vial	Mean ng/g vegetable
	None Central Science Laboratory Sand Hutton York YO41 1LZ (UK) Heated 55-59 57 Vegetable mix a Furan is a cyclic ether with a low boiling point (32 °C). It has been reported to occur in 9 114
	ESBO a number of foods that undergo heat treatment, such as canned and jarred foods (US Heated 99-102 101 12 202
	ELO * corresponding author :-s.hasnip@csl.gov.uk Heated 1716-1718 FDA 2004). The presence of furan is a potential concern because it is both carcinogenic 1717 13 3434
	SBO LO ESBO F o r Abstract: Water b Levels of furan in various foods were measured before and after heating under heating Heated 97-103 100 12 200 Heated 321-362 341 33 683 Heated 27-29 28 8 and laboratory conditions. The effect of contact with can coatings, sealing gaskets and and cytotoxic in rodents and is regarded as a possible human carcinogen by the F International Agency for Research on Cancer (IARC 1995). o r Methods for the determination of furan have been based on headspace sampling
	ELO P Heated the epoxidised oils used in gasket manufacture on furan formation was studied. The 986 986 93 P using, syringes (US FDA 2005, Becalski et al. 2005, Becalski and Seaman 2005) or
	ESBO ELO SBO objective was to identify factors affecting furan formation. Furan present in heat-Unheated 27 27 11 Unheated 180 180 26 Unheated 10 10 10 e e r processed food samples persisted during cooking. Furan was shown to form in foods on e solid phase microextraction (Goldmann et al. 2005, Ho et al. 2005). Quantification has e r been achieved by means of standard additions (US FDA 2005) or external calibration
	LO heating, although it did not accumulate to a significant degree on heating in an open Unheated 14 14 10 R R graphs
	Notes:-a vessel. There were no interactions between foods and cans, can coatings or gaskets that e e 8 ml water ± 0.5 g vegetable mix b 8.5 ml water c Analytical measurement uncertainty only since heating experiments were carried out in the same batch and inclusion of between batch heating variation is not appropriate. v i e had a significant influence on furan formation. Furan accumulated particularly in heat-processed canned and jarred foods because they are sealed containers that receive a v i e ESBO Epoxidised soybean oil ELO Epoxidised linseed oil SBO Soybean oil LO w considerable thermal load. Heating epoxidised oils used in sealing gaskets formed furan. w Linseed oil O n l At the levels used in gaskets, however, epoxidised oils should not affect the formation of furan in foods. O n l
			y y
	Keywords: furan, heat processing contaminants, canned food, jarred food

  about hereTable 3 also compares the concentrations of furan found in samples heated in open vials with those of samples heated in sealed vials. Samples heated in open vials accumulated very little furan in comparison to sealed samples. This

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Table 6

 6

	about here
	Unheated soybean and linseed oils contained similarly low levels of furan.
	Furthermore the epoxidised oils contained more furan than the unepoxidised oils,

notably so for linseed oil. Heating the epoxidised oils led to further formation of furan in ELO but not in ESBO. When the unepoxidised oils were heated with vegetable mix

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

Acknowledgements:


Natalia Campillo

Rosa Peñalver

Manuel Hernández-Córdoba

Prof Manuel Hernández-Córdoba

A sensitive procedure based on solid-phase microextraction 2 and gas chromatography for determining pentachlorophenol in 3 different foodstuffs 4 5 6

Keywords: Honey, Gelatine, Pentachlorophenol, Headspace Solid-Phase 15 Microextraction (HS-SPME), Gas Chromatography -Atomic Emission Detection

A sensitive procedure based on solid-phase microextraction

Introduction 19 20

Despite restrictions concerning the use of pentachlorophenol (PCP), its 21 widespread use in the past together with its high resistance to biodegradation 22 has led to its detection in the environment. PCP may be present in honey and 23 related foods, having been transported by bees when travelling to collect nectar 24 or even because of the treatment of wooden beehives. On the other hand, the 25 Bendito and Rubio 1999) and has also been classified by the World Health 6 Organisation (WHO) as a possible carcinogenic agent to humans (Guidelines 7

for drinking-water quality 1998). Although the European Union (EU) considered 8 the need to improve the treatment conditions of beehives (Commission 9

Directive 97/1221/EC), no maximum residue limit (MRL) for PCP has been 10

established for honey or for edible gelatine. 11 12 Several techniques have been used for PCP analysis, including liquid 13 chromatography (LC) [START_REF] Han | Preparation and evaluation of a molecularly 4 imprinted sol-gel material for on-line solid-phase extraction coupled with 5 high performance liquid chromatography for the determination of trace 6 pentachlorophenol in water samples[END_REF], supercritical fluid chromatography 14 (SFC) [START_REF] Bernal | Determination of phenolic compounds in water samples by online 20 solid-phase extraction-supercritical-fluid chromatography with diode-array 21 detection[END_REF]) and capillary electrophoresis (CE) (Martínez et al. 15 1996[START_REF] Li | High termal-stable sol-gel-coated calyx[4]arene 21 fiber for solid-phase microextraction of chlorophenols[END_REF]); however, gas chromatography (GC) is the most widely 16 used method since it provides the best sensitivity. Mass spectrometry (MS) 17 [START_REF] Crespín | Discrimination of 12 structural isomers of chlorinated phenols in waters using gas 13 chromatography-mass spectrometry in the negative chemical ionization 14 mode[END_REF][START_REF] Diserens | Rapid determination of nineteen chlorophenols in wood, 18 paper, cardboard, fruits, and fruit juices by gas chromatography/mass 19 spectrometry[END_REF]) and electron capture detector (ECD) (Insa 18 et al. 2004[START_REF] Domeño | Development of a solid-phase 21 microextraction method for direct determination of pentachlorophenol in 22 paper and board samples: comparison with conventional extraction[END_REF]) are the most commonly used detectors for 19 pentachlorophenol determination by GC. On the other hand, atomic emission 20 detection (AED) provides selective information which cannot be obtained with 21 other detectors. Compared to GC-MS the instrumentation used in GC-AED is 22 easy to operate, and its chromatograms can be interpreted by a semi-skilled 23 advantages of using the SPME approach with the samples under study in the 17 present work, only one previous report [START_REF] Campillo | Evaluation of solid-8 phase microextraction conditions for the determination of chlorophenols 9 in honey samples using gas chromatography[END_REF]) applies this 18 methodology to PCP analysis in honey. This paper is an extension of that 19 analytical procedure. The sensitivity is here enhanced and the optimized 20 procedure applied to other samples for which the literature describes laborious 21 and time-consuming procedures. If comparing the proposed method with 22 relevant articles involving the use of SPME [START_REF] Ribeiro | Direct 19 determination of chlorophenols in landfill leachates by solid-phase micro-20 extraction-gas chromatography-mass spectrometry[END_REF], Domeño et al. 23 2005) two advantages are achieved by means of the approach here reported, 24 namely a saving of time as well as an enhancement of the sensitivity. 25 Pentachlorophenol (PCP) was purchased from Aldrich (Steinheim, Germany) 4 with 99% purity. A stock solution of 1000 µg ml -1 was prepared in HPLC grade 5 methanol. Aqueous standard solutions of 100 ng ml -1 were freshly prepared for 6 spiking samples in order to optimize the analysis procedure. Acetic anhydride 7 and anhydrous potassium carbonate were purchased from Fluka (Buchs, 8

Switzerland) and sodium chloride of 99.5% purity from Sigma (St. Louis, MO, 9

USA). 10 11

The plasma gas and carrier gas used for GC was helium. The reagent gas for 12 the AED was oxygen. Nitrogen was used for purging the AED system. All the 13 gases were supplied by Air Liquide (Madrid, Spain). 14 15

Instrumentation 16

The SPME device for manual sampling consisted of a holder assembly and 17 several replaceable fibers, obtained from Supelco (Bellefonte, PA, USA). under the conditions recommended by the manufacturer for each fiber coating. 1 Whenever needed, the conditioning step was repeated for fiber cleanup. All 2 analyses were performed in 15 ml clear glass vials and the solutions were 3 stirred using PTFE-coated magnetic stir bars (10 mm x 6 mm o.d.). To prevent 4 analyte evaporation, vials sealed with hole-caps and PTFE/silicone septa were 5 used. A home-made system built in the Central Laboratory Service of the 6

University of Murcia and consisting of a drilled block provided with an electronic 7 temperature control system was used for heating. 8 9 An Agilent 6890 gas chromatograph was directly coupled by a transfer line to a 10 G2350A microwave-induced plasma atomic emission detector (Agilent). 11 Updated G2070AA ChemStation application with the G2360AA GC-AED 12 software was used to control and automate many features of the GC and AED 13 systems, and for data acquisition and treatment. The chromatograph was fitted 14 with a 30 m x 0.32 mm i.d. HP-5, 5% diphenyl 95% dimethyl polysiloxane non-15 polar capillary column from Agilent with a 0.25 µm film thickness. Desorption of 16 the fibers into the injection port was carried out in the splitless mode at 200 °C 17 for 1 min. The oven temperature program simply consisted of rising temperature 18 from 80 °C to 180 °C at 30 °C min -1 and holding for 4 min. Helium was used as 19 the carrier gas and as AED make-up gas, being set at 4 and 40 ml min -1 , 20 respectively. Oxygen was used as the only scavenger gas at 20 psi. Solvent 21 venting was switched on immediately after starting the desorption step and 22

Recovery assays 7

Since no reference materials are available for the validation of the method, 8 spiked samples were prepared. Samples were spiked as follows: 0.1 ml of a 9 working methanolic solution, containing pentachlorophenol at concentration 10 levels ranging from 3.5 to 300 ng ml -1 , were added to 1.5 g of sample (0.5 g of 11 gelatine) placed in a 15 ml SPME-vial, corresponding roughly to fortification 12 levels of 0.25 and 20 ng g -1 , respectively. Fortification levels ranged between 13 0.5 and 5 ng g -1 in the case of the gelatine samples. The spiked samples were 14 set aside for 60 min at room temperature to let the methanol evaporate before 15 being analyzed as described above. The fortification procedure was applied to 16 two different honey samples, to the fresh royal jelly, the palm honey and a 17 gelatine sample at four concentration levels and three replicates corresponding 18 to three aliquots of each sample independently fortified and analyzed, were 19 analyzed in each case. 20 21

Results and discussion 22

Using the previously optimized derivatization conditions [START_REF] Campillo | Evaluation of solid-8 phase microextraction conditions for the determination of chlorophenols 9 in honey samples using gas chromatography[END_REF], 23 the program temperature eluted the acetylated pentachlorophenol at 6.09 min 24 using a carrier gas flow-rate of 4 ml min -1 , while the oven temperature was The extraction efficiency of six different fiber coatings was checked using both 10 the headspace and the immersion mode. These experiments were carried out 11 by exposing the fibers to the sample for 15 min at 90 °C, and desorbing the 12 analyte in the GC injection port for 1 min at a temperature 20 °C below that 13 recommended by the manufacturer for each coating. The carboxen/PDMS fiber 14 showed a marked memory effect, which persisted despite applying desorption 15 times as long as 10 min, for which reason its use was discarded. The results 16 obtained for the other five fiber coatings appear in Figure 1, where the influence 17 The effect of the ionic strength on the extraction efficiency of the acetylated 4 analyte was studied by changing the sodium chloride concentration between 0 5 and 40% (w/v). In the case of the honey sample, the sensitivity increased up to 6 a concentration of 4% (w/v) and then remained practically constant at 7 concentrations up to 15% (w/v), while higher salt concentrations led to a slight 8 decrease in the signal. Probably owing to the high salt content of the products 9 manufactured with animal tissues, the addition of sodium chloride did not 10 change the sensitivity for PCP in the gelatine matrix. A 4% (w/v) sodium salt 11 concentration was finally selected for all the samples. 12 13 Extraction temperature. 14

of
The influence of the sample temperature was examined from 55 to 98 °C. The 15 extraction efficiency was significantly enhanced by increasing the temperature 16 up to 95 °C. While at 98 °C losses of the acetylated PCP were observed, owing 17 to a decomposition process of the analyte, providing a chromatographic peak 18 with a retention time of 5.2 min. Thus, 95 °C was selected as the optimum 19 The stirring speed was varied between 0 to 2000 rpm, although sensitivity 6 practically did not vary between 1000 and 2000 rpm. Therefore, 1400 rpm was 7 adopted because it provided the best repeatability. Moreover, the peak areas 8 The influence of the sample mass was checked by pre-concentrating 4 ml of a 5 20 ng ml -1 PCP solution in the presence of different masses of honey or gelatine in 21 the 0.1-2 g range. The results obtained showed a clear matrix effect in both 22 cases. Sample masses of 0.5 g were found to be suitable for gelatine samples 23 and 1.5 g for honey and the rest of the samples considered. When higher 24 amounts were assayed, the repeatability of the extraction step worsened. 25 The matrix effect was studied by comparing the slopes of aqueous standards 3 and standard additions calibration graphs for two honey samples of different 4 visual aspect and floral origin, two gelatines and the other four samples under 5 study. Different values for the calibration slopes were obtained when standard 6 addition calibration graphs were constructed, the values depending on the 7 sample used. The slopes obtained for the different samples under the optimized 8 conditions appear in Figure 2. In the case of the lyophilized royal jelly, 0.1 g of 9 sample was submitted to analysis, which corresponds roughly to 1.5 g of the 10 fresh royal jelly. In fact, if the total content of the lyophilized sample capsule was 11 submitted to the whole procedure, the matrix effect was so high that the signal 12 did not increase even after adding 100 ng PCP per gram of sample. In all 13 cases, six spiking levels were preconcentrated and measured, peak areas 14 being used for calibration purposes in all cases. The results clearly showed that 15 the standard additions method is required for quantification. Correlation 16 coefficients (r 2 ) higher than 0.9989 were obtained in all cases for the standard 17 additions graphs. Taking into account that the procedure involves derivatization 18 of the analyte and SPME preconcentration, this good correlation is of note. 19 20 [insert Figure 2 about here] 21

22

The detection limit for PCP using a signal-to-noise ratio of three and considering 23 the slope of the calibration graph obtained for 1.5 g of honey sample 4 was 24 calculated to be 0.075 ng g -1 . The quantification limit for the same honey 25 sample using a signal-to-noise ratio of ten was calculated to be 0. 25 ng g -1 . 1

These values should be considered as orientative as they are dependent on the 2 sample analyzed. Thus, the detection limits obtained for palm honey and 3 lyophylized royal jelly (which correspond to the samples with the lowest and the 4 highest matrix effect, respectively) were 0.03 and 6.0 ng g -1 , respectively. The 5 repeatability was calculated using the relative standard deviation for ten 6 successive injections of the honey sample 4 fortified at two concentration levels, 7 the obtained values being 9.8 and 11.8% for 3.5 and 10 ng g -1 , respectively. 8 9

Real samples and validation of the method 10

The optimized procedure was applied to ten different honey samples, to four 11 different related food samples, as well as to seven gelatine samples. Table I 12 shows the results obtained. All the honeys and one of the gelatine samples 13 analyzed provided signals for PCP which corresponded to very low 14 concentration levels in the 0.3-1.5 ng g -1 range. PCP was not detected in the 15 other samples analyzed. Note that a 25 g-daily intake of the samples appearing 16 in Table II is far from what considered as dangerous for human health. It should 17 be noted that, taking into consideration the maximum admissible concentration 18 in drinking water of 1.0 ng ml -1 established by the US EPA (http://www.epa.gov), 19 a daily intake of 1.4 kg of honey sample 2, would provide the same dose of PCP 20 contained in 2 l of a drinking water containing the maximum residue limit 21 permitted (1.0 ng ml -1 ). Obviously, such a honey intake is not viable, so that the 22 low PCP value found does not represent a risk for the consumer. As no reference materials were available, recovery studies were carried out in 2 order to check the accuracy of the proposed method, fortifying honey samples 2 3 and 6, gelatine sample 6, as well as the fresh royal jelly and the palm honey at 4 four concentration levels with PCP as described above. The data of PCP found 5 for honey samples 2 and 6 have been calculated by subtracting the 6 corresponding analyte contents. The data obtained appear in Table II

Conclusion 13

The SPME-GC-AED combination provides a rapid and sensitive procedure that 14 can be useful in the routine control of PCP residues in honey as well as in 15 several other foodstuffs. The high pre-concentration power of solid phase 16 microextraction under the selected conditions and the excellent sensitivity of the 17 atomic emission detector provide nearly specific chromatograms, and detection 18 limits for the samples analyzed never before attained, applying low extraction 19 times and disposable commercial fibers. Nevertheless, the complexity of the 20 matrix of the samples under study makes it necessary to quantify the analyte by 21 means of the standard additions method.

3 4

 3 in sap syrups and gelatines can be attributed to its use in 1 agriculture or to prevent mould and fungal growth in animal hides used for 2 gelatine manufacture.Pentachlorophenol is included in the most relevant priority pollutant lists (Pérez-5

2 5 ( 8 9

 258 Solid-phase microextraction (SPME) (Buchholz and Pawliszyn 1994, Bianchi et 1 al. 2002, Llompart et al. 2002, Ribeiro et al. 2002, Li et al. 2004, Martínez-Uruñuela et al. 2004, Bagheri et al. 2005, Domeño et al. 2005) appears as an 3 interesting alternative to conventional liquid-liquid extraction (LLE) 4 (Hoogerbrugge et al. 1999, Diserens 2001) and solid-phase extraction (SPE) Muiño and Lozano 1991, Rodríguez et al. 1996, Crespín et al. 1999, Lacorte et 6 al. 2000, Oliveira et al. 2002) because extraction can be made rapidly and 7 directly, without organic solvent, and can easily be automated. In spite of the importance of controlling the PCP content of foods, the number of 10 articles dealing with its determination is relatively low. As far as we know, only 11 three publications deal with honey samples (Muiño et al.1991, Sherma and 12 McGinnis 1995, Campillo et al. 2006) and another three with gelatine samples 13 (Borsetti and Thurston 1984, Yip 1985, AOAC Official Methods of Analysis 14 1990). The procedures proposed for gelatines are based on tedious sample 15 treatment involving acid-hydrolisis, base partition, cleanup and LLE. Despite the 16

switched off 3 .Food 5 6 16 17

 3516 5 min later. The transfer line and the cavity temperatures were 23 set at the same value as recommended by the manufacturer, 325 °C. Filter and 24 backamount adjustment in the AED were set according to Agilent default 25 Page 6 of 25 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk spectrometer was purged with a nitrogen gas flow rate of 2.5 1 l min -1 . The element monitorized was chlorine at the 479.45 nm line. Taking 2 into account the time of 15 min adopted for the SPME adsorption step, and 3 since the analyte eluted with a retention time of 6.09 min, the analysis of each 4 sample lasts about 22 min.Samples. SPME and in situ derivatization procedure 7Ten different honey samples labelled as eucalypus (samples 1-3), ericaceous 8 (sample 4), rosemary (samples 5-7), orange blossom (samples 8-9) and 9 heather (sample 10) were obtained from a local supermarket. No indication of 10 the country of origin was provided on the label. Two royal jelly samples (fresh 11 and lyophilized), palm honey and a maple syrup samples were obtained from a 12 local dietetic shop. Three powdered mixtures containing approximately 15% 13 (w/w) of gelatine of different flavours (samples 1-3) were obtained from a local 14 supermarket and four pure gelatines (samples 4-7) used for sweets production 15 were provided by a local manufacturer.To carry out the extraction, samples of 1.5 g (0.5 g for gelatine samples) were 18 weighed into a 15 ml SPME glass vial and 4 ml of a solution containing 4 and 19 0.75% (w/v) of sodium chloride and potassium carbonate, respectively, were 20 added. Then 160 µl of acetic anhydride were added, the vial was immediately 21 sealed with the cap after introducing the magnetic stir bar and the mixture was 22 homogenized by inserting the vial in the home-made heating block previously 23 programmed at 95 °C and maintaining the stirring at maximum power for 1 min.

  for 15 min to the headspace over the aqueous mixture, which was 1 continuously stirred at 1400 rpm and thermostated at 95 °C. Subsequently the 2 PDMS-DVB fiber was retracted into the needle and transferred to the injection 3 port of the GC with the split valve closed at 200 °C for 1 min. Each sampling 4 was performed in triplicate. 5 6

2 3 7 8

 27 °C. Higher flow-rates are not recommended because 1 problems in the stationary phase appear.Optimization of the SPME stage 4All the optimization experiments were carried out in parallel for honey and 5 gelatine, in both cases dissolving 0.5 g of fortified samples in 7 ml of aqueous 6 solution containing the appropriate chemicals for the derivatization step.Selection of the extraction mode and the type of fiber. 9

Food

  this parameter is expressed by reference to the maximum extraction 18 efficiency, which was obtained with the PDMS/DVB fiber in the headspace 19 mode. Except for the PDMS coating, the headspace mode provided better 20 extraction efficiencies than the immersion mode. Since the HS extraction mode 21 is also advisable for prolonging the life time of the fiber coatings, this extraction 22 mode was adopted. The extraction efficiency did not exceed 50% for any 23 coating except in the case of the PDMS/DVB fiber. 24 25 Page 9 of 25 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

22 Food 3 4

 223 The extraction time was studied in the 5-60 min range with 95 °C as the 23 sample-vial temperature, equilibrium being reached in 15 min. Of note is the 24 fact that the signal decreased at 60 min, probably as a consequence of the 25 Page 10 of 25 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk analyte. An adsorption time of 15 min was adopted, for the 1 maximum sensitivity. Desorption parameters of 200 °C and 1 min were used, as 2 has been previously recommended[START_REF] Campillo | Evaluation of solid-8 phase microextraction conditions for the determination of chlorophenols 9 in honey samples using gas chromatography[END_REF]).Agitation speed. 5

11 12

 11 obtained in the selected stirring conditions and without stirring indicated that 9 stirring resulted in a 1.5 fold increase in the extraction efficiency of the 10 acetylated pentachlorophenol.Extraction solution volume and sample mass. 13Extraction volumes of 4, 5, 6 and 7 ml were assayed, resulting that the lower 14 the extraction solution volume the higher the extraction efficiency. A solution 15 volume of 4 ml was finally selected, the headspace volume being 11 ml.[START_REF] Lacorte | Broad spectrum analysis of 17 109 prioriy compounds listed in the 76/464/CEE council directive using 18 solid-phase extraction and GC/EI/MS[END_REF] Volumes below 4 ml are not recommended because of poor homogenization of 17 the samples. 18 19

1

  Analytical characteristics of the method 2

23 24[Food

 23 insert Table I about here] 25Page 13 of 25 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

22

 22

Figure 1 . 7 Figure 2 .*

 172 Figure 1. Influence of the type of fiber coating and the extraction mode on the 3

Table II

 II Determination results of the recovery study in several samples for the optimized procedure

	Honey 2	Honey 6	Fresh royal jelly	Palm honey	Gelatine 6
	Added*	Found * , †	Added*	Found* , †	Added*	Found* , †	Added*	Found* , †	Added*	Found* , †
	0.98	0.80 ± 0.35	0.62	0.56 ± 0.13	2.02	1.74 ± 0.91	0.25	0.23 ± 0.09	0.50	0.42 ± 0.10
	1.5	1.61 ± 0.22	1.04	0.96 ± 0.19	5.10	4.87 ± 0.35	1.04	0.91 ± 0.21	2.20	1.60 ± 0.11
	5.02	5.22 ± 0.15	2.37	2.25 ± 0.15	10.25	11.09 ± 0.22	2.51	2.63 ± 0.14	2.98	2.09 ± 0.20
	10.4	10.7 ± 0.30	5.02	4.85 ± 0.13	18.39	19.41 ± 0.24	5.09	4.74 ± 0.29	4.98	4.89 ± 0.24

µm, divinylbenzene/carboxen/polydimethylsiloxane (DBV/CAR/PDMS) of 50/30

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

Journal of Chromatography A 1095:8-15.

Rodríguez I, Turnes MI, Mejuto MC, Cela R. 1996. Determination of

Acknowledgements

24

Food Additives

The authors are grateful to the Spanish Ministerio de Educacion y Ciencia, MEC 1 (Project CTQ2006-08037/BQU) for financial support. R. Peñalver acknowledges 2 a fellowship from MEC.


Francisco J Morales

Gema Arribas-Lorenzo

Acrylamide content of selected Spanish foods: A survey of biscuits and 1 bread derivatives 2 3

Keywords: Biscuits, Bread, Cereals Acrylamide, biscuits, crisp bread, crackers, bread sticks, food 23 contaminants

An overview of the acrylamide content in commercial biscuits and bread derivatives 11 (bread sticks, bread crust, crackers) marketed in Spain is presented. Acrylamide was 12 determined by stable isotope dilution LC-MS with an LOQ of 30 µg kg -1 . Acrylamide 13 content ranged from <LOQ -2085 µg kg -1 , <LOQ -151 µg kg -1 , <LOQ -296 µg kg -1 , 14 <LOQ -23 µg kg -1 for biscuits, crisp bread, crackers and bread sticks. Acrylamide was 15 significantly higher in samples when ammonium hydrogen carbonate had been used as a 16 rising agent and high fibre content (> 5%) used in the formulation, but lower when 17 functional ingredients such as polyols were used. An estimation of the acrylamide 18 dietary exposure related to biscuits and bread-derivatives was calculated as 0.082 µg kg - 19 1 day -1 . Estimated dietary intake ranged from 0.002 to 0.058 µg kg -1 day -1 for crackers 20 and biscuits respectively. 21 22

The detection of acrylamide, which has been classified as a probable carcinogen by the 3 International Agency for Research on Cancer (IRAC, 1994), in a large variety of heated 4 foods [START_REF] Tareke | Analysis of 5 acrylamide, a carcinogen formed in heated foodstuffs[END_REF] has created much concern among regulating authorities, the 5 food industry and the public. Consequently, worldwide monitoring of acrylamide in 6 different food types was started [START_REF] Lineback | Overview of 18 acrylamide monitoring database[END_REF]. Extensive data on levels of 7 acrylamide have been collected by the European Commission (2005) and by the US 8 Food and Drug Administration (2004). Among the most important matrices included in 9 the European monitoring database are potato chips, crispbread, breakfast cereals and 10 biscuits. 11 12 Acrylamide is formed in the Maillard reaction, reducing sugars such as glucose and 13 fructose, and the amino acid asparagines being the major reactants (Stadler et al. 2002, 14 2004; [START_REF] Zyzak | Acrylamide 19 formation mechanism in heated foods[END_REF]. Two mechanisms, varying in details, have been proposed 15 [START_REF] Becalski | Acrylamide in foods: occurrence, 9 sources, and modelling[END_REF][START_REF] Zyzak | Acrylamide 19 formation mechanism in heated foods[END_REF]). However, evidence clearly points to the 16 reaction between asparagine and reducing sugars as the main culprit. 17 18 It is now well-established that processing conditions such as time, temperature, and 19 matrix influence acrylamide formation and degradation [START_REF] Friedman | Chemistry, biochemistry, and safety of acrylamide. A review[END_REF]. Acrylamide 20 formation was found to occur during the browning process at temperatures above 120 21 °C [START_REF] Mottram | Acrylamide is formed in the Maillard 22 reaction[END_REF][START_REF] Yaylayan | Why asparagine needs carbohydrates to 12 generate acrylamide[END_REF]). In the case of starchy products (mainly 22 bakery) acrylamide contents up to 2000 µg/kg were observed [START_REF] Croft | Australian survey of acrylamide in 16 carbohydrate-based foods[END_REF]). The 23 highest contents were often found in products, such as gingerbread, prepared with the 24 baking agent ammonium hydrogen carbonate [START_REF] Amrein | Acrylamide in gingerbread: 6 Critical factors for formation and possible ways for reduction[END_REF] which has been shown that strongly promotes the acrylamide formation in sweet bakery 1 [START_REF] Biedermann | Model studies on acrylamide formation in potato, wheat 11 flour and corn starch; ways to reduce acrylamide contents in bakery ware[END_REF]; [START_REF] Graf | Reducing the 8 acrylamide content of a semi-finished biscuit on industrial scale[END_REF]). 2 3 As stated above investigations about acrylamide formation has been focused on food 4 commodities with high acrylamide formation, such as potato-derived foodstuffs, or with 5 significant impact on the dietary population habits, like cereal-based foodstuffs. Biscuits 6 and bread-derivatives comprise a huge family of products obtained by baking a variety 7 of cereal crops. Generally, manufacture of both kinds of products involves different 8 steps such as mixture of ingredients and kneading, dough fermentation (mainly in bread 9 crust and bread sticks), baking -up to 250ºC for 15-30 min and in some cases a final 10 toasting step (Ramirez-Jimenez et al. 2000). This last step favour the main chemical 11 reactions involved during biscuits or bread derivatives manufacture, the Maillard 12 reaction and caramelisation, which are responsible for the colour and flavour of these 13 kinds of products (Ramirez-Jimenez et al. 2000). 14

15

The study presented here should give an overview of the acrylamide content of 16 commercial biscuits and bread derivatives marketed in Spain. These products, because 17 of their relatively widespread consumption, could be a key source of acrylamide in the 18 Spanish diet. Therefore, the relationship between acrylamide intake and population 19 factors is also presented. In addition the correlation among levels of acrylamide and 20 compositional parameters of the samples (i.e. presence of ammonium hydrogen 21 carbonate, dietary fibre content and biscuit speciality) was investigated. Experiments were conducted with a series of commercial biscuits (62 samples), bread 1 crust (24 samples), bread sticks (10 samples) and crackers (11 samples) randomly 2 purchased on different supermarkets from the Autonomous Community of Madrid 3 (Spain). The distribution of samples was representative of the proportions of these food 4 commodities in the Spanish market. Samples (300-500 g) were mixed and finely ground 5 to ensure homogeneous distribution. Sub-samples (200 g) were divided into 4 6 containers and stored under vacuum and light protected at 4ºC until analysis. 7 8 Chemicals and materials 9

[ 13 C 3 ]-acrylamide (isotopic purity 99%) was from Cambridge Isotope Labs (Andover, 10 MA, USA). Acrylamide (99%), potassium ferrocianide (Carrez I), zinc acetate (Carrez 11 II) were from Sigma-Aldrich (St-Louis, MO, USA). Acetic acid (ultrapure grade) and 12 Pronase E were from Merck (Darmstadt, Germany). Methanol and acetonitrile (HPLC 13 grade) were from Scharlau (Barcelona, Spain). The solid-phase extraction (SPE) 14 cartridges Isolute ® Multimode (500 mg, 3 ml) were from IST (Hewgoed, Mid-15 Glamorgan, UK) 16 17

Acrylamide standard and reagents 18

Stock solutions of acrylamide (0.01 mg ml -1 ) and [ 13 C 3 ]-acrylamide (5 µg ml -1 ) were 19 prepared by dissolving the compounds in Milli-Q water and methanol respectively. 20

These solutions were then appropriately diluted with Milli-Q water (Millipore Corp., 21 Madrid, Spain) to prepare working standards at 1.0 µg ml -1 . All stock solutions and 22 working standards were stored light-protected in a refrigerator at 4ºC up to 3 months. 23 Carrez I solution was prepared by dissolving 15 g of potassium ferrocianide in 100 ml 24 of water and Carrez II solution by dissolving 30 g of zinc acetate in 100 ml of water. 25 Sample extraction. Sample powder (0.75 g) was weighed with a precision of 0.1 mg and 5 suspended with 8 ml of MilliQ water in polypropylene centrifugal tubes. Mixture was 6 spiked with 200 µl of a 5 µg ml -1 [ 13 C 3 ]-acrylamide methanolic solution as internal 7 standard and later homogenized. Acrylamide extraction was performed at room 8 temperature for 20 minutes, and 10 seconds shaking every 10 minutes. In order to 9 clarify the solution, 0.5 ml of each Carrez I and Carrez II solutions were added and 10 finally the mixture was centrifuged (9000g; 10 min; 4ºC). 11 12 Sample clean up. Isolute ® Multimode SPE cartridges were preconditioned with 2 ml 13 methanol, 2 ml water and 2 ml air to remove excessive water. An aliquot of the clear 14 supernatant (1 ml) was loaded onto the cartridge at a flow rate of 2 ml min -1 . Then 2 ml 15 of air were passed and finally acrylamide was eluted with 1 ml of water at the same 16 flow rate. The solution was filtered through a 0.45 µm filter into an amberlite LC-MS 17 Electrospray ionization in the positive ionization mode was used. The MS detector 1 operated in selected ion monitoring (SIM) mode at m/z ratios of 72.1 and 75.1 for 2 acrylamide and [ 13 C 3 ]-acrylamide respectively. Under these chromatographic 3 conditions, acrylamide eluted at 6.8 min. A delay time of 3 min was selected to avoid 4 the introduction of coextracted matrix components into the MS instrument prior 5 acrylamide elution. The needle and cone voltages were set at 3.0 kV and 100 V 6 respectively. Nitrogen was used as nebulizer gas (12.0 l h -1 ) and the source temperature 7 was set at 300 ºC. 8 9 Quantitation. Acrylamide was quantified using a linear calibration function that was 10 established with standard solutions of acrylamide and [ 13 C 3 ]-acrylamide dissolved in 11

Milli-Q water (25 to 1000 µg l -1 ). Acrylamide contents in sample extracts were 12 calculated from the calibration slope and intercept value, taking into account the 13 recovery calculated by means of [ 13 C 3 ]-acrylamide slope. Limit of quantification (LOQ) 14 was 30 µg kg -1 on the basis of signal-to-noise ratio of 3:1. 15 16

Statistical. Statistical significance of data was tested by one-way analysis of the 17 variance (ANOVA) (Table 5), followed by Duncan Test to compare means that showed 18 significant variation (P < 0.05). Analyses were performed using Statgraphics Plus, 19 a box-and-whisker plot was used since this graphical presentation uses a non-parametric 9 test (Figure 1A). Acrylamide ranged from < LOQ -151 µg kg -1 for crispbread, with a 10 mean value of 87 µg kg -1 and a median of 100 µg kg -1 . In the case of crackers 11 acrylamide ranged from < LOQ -296 µg kg -1 , being the mean value 140 µg kg -1 and the 12 median 155 µg kg -1 . Finally, in the case of bread sticks acrylamide ranged from <LOQ -13 323 µg kg -1 with a mean value of 157 µg kg -1 and a median of 139 µg kg -1 . 14 15 Similar values where reported by other authors (i.e. [START_REF] Murkovic | Acrylamide in Austrian foods[END_REF] showed that the acrylamide levels of crisp bread are significantly lower (p<0.05) than 20 that of bread sticks and crackers. The composition of both crispbread and bread sticks is 21 similar (mainly wheat flour, baking powder, salt and water) whereas in the case of 22 crackers sodium hidrogencarbonate is used instead of baking powder. Taking into 23 account these similarities, the differences in the acrylamide content could come from 24 the different baking conditions (time and temperature) applied to the products. 25 ALARA principle (as low as reasonably achievable). In this sense the German Federal 7

Office of Consumer Protection and Food Safety (BVL, 2004) stated a signal value of 8 560 µg kg -1 for bread derivatives, as a concept of minimisation. Signal value is defined 9 as lowest level of the 10% containing the highest level of acrylamide. Then, if 10 acrylamide contents are above this signal value, the food producers are aware to take 11 adequate actions to lower the contents. As stated if Figure 2A, acrylamide values ranged 12 from < LOQ -323 µg kg -1 for bread derivatives Then, if the same concept is applied in 13 our survey none of the samples levels was higher than 560 µg kg -1 . In this sense, 14 because of the lower values of the bread derivatives marketed in Spain, a signal value of 15 222 µg kg -1 was established for the whole group. However, if the samples are divided 16 into the three original groups, the obtained signal values for crisp bread, bread sticks 17 and crackers are 129, 293 and 232 µg kg -1 respectively. This is an important issue taking 18 Biscuits. 62 samples from 15 different producers where studied. In the case of biscuits 1 the acrylamide content ranged from < LOQ -2085 µg kg -1 , with an average value of 2 423 µg kg -1 and a median of 268 µg kg -1 . In addition, as shown in the box-and-whisker 3 plot (Figure 1B), eight outliners where found, all of them with acrylamide contents 4 higher than 1000 µg kg -1 . Similar values have been reported by other authors (i.e. 5 [START_REF] Murkovic | Acrylamide in Austrian foods[END_REF]; Senyuva and Gokmen. 2006; Yusa et al. 2006) who found acrylamide 6 levels up to 1060 µg kg -1 . When the statistical treatment was applied to the biscuits and 7 bread derivatives groups, it was found that biscuits presents a statistically significant 8 (p<0.05) higher acrylamide content. Contrary to the group of bread derivatives, the 9 composition of biscuits is quite different and complex because different ingredients 10 such as reducing sugars are usually added (glucose and fructose mainly added as corn 11 syrups), fats (including ω 3 -PUFAs), technological agents (ammonium and sodium 12 hydrogencarbonate), etc. It is well established that one of the components that promote 13 the generation of acrylamide are reducing sugars [START_REF] Friedman | Chemistry, biochemistry, and safety of acrylamide. A review[END_REF][START_REF] Stadler | In-depth mechanistic study on the formation of acrylamide and other 2 vinylogous compounds by the Maillard reaction[END_REF]; 14 [START_REF] Zyzak | Acrylamide 19 formation mechanism in heated foods[END_REF]). In addition it has been shown that the baking agent ammonium 15 hydrogen carbonate strongly promotes the acrylamide formation [START_REF] Amrein | Acrylamide in gingerbread: 6 Critical factors for formation and possible ways for reduction[END_REF]16 Biedermann et al. 2003;[START_REF] Graf | Reducing the 8 acrylamide content of a semi-finished biscuit on industrial scale[END_REF][START_REF] Konings | Acrylamide exposure from foods of the Dutch 15 population and an assessment of the consequent risk[END_REF]. Then, because of the 17 presence of a higher content of acrylamide precursors in biscuits, their higher 18 acrylamide content is a logical consequence. 19 20 As described previously, biscuits comprise a heterogeneous food commodities group 21 because of they are composed by a great variety of ingredients. Then, in order to find 22 out the possible differences in the acrylamide content, biscuits were grouped according 23 to the biscuit speciality, presence of ammonium hydrogen carbonate and dietary fibre 24 content, The results of the statistical treatment are depicted in Table I. At the beginning, 25 the first analysis showed no statistical differences (data not shown) but it was found a 1 wide distribution among groups of the outliners of Figure 2B. Then, because of their 2 high acrylamide content the mean values of the different groups was slanted. According 3 to the table of composition -supplied by the manufacturers-it was found that the 4 outliners were just addressed to samples with ammonium hydrogencarbonate as the sole 5 raising agent, in contrast with the other 54 samples where different proportions of 6 sodium and ammonium hydrogencarbonate was used. In this sense it could be thought 7 that a higher amount of ammonium hydrogencarbonate was used for the manufacture of 8 the outliners and, because this raising agent enhance the acrylamide generation ( wheat-bran and/or whole wheat flour, dietary fibre increases up to 10 -30 %. As 9 illustrated in Table 1, there were statistically significant differences in the acrylamide 10 content between the dietary fibre added biscuits (283 ± 38.1 µg kg -1 ; n = 18; 11 fibre content > 5%) and not dietary fibre-added (166 ± 31.0 µg kg -1 ; n = 44; fibre 12 content < 5%), results which are in accordance to those previously reported for 13 breakfast cereals (Rufian-Henares et al. 2006). Similar behaviour was reported by 14 [START_REF] Senyuva | Interference-free determination of acrylamide in 21 potato and cereal-based foods by a laboratory validated liquid chromatography-mass 22 spectrometry method[END_REF] who found that the acrylamide content of biscuits with 15 dietary fibre was higher than that of the usual biscuits (486 and 261 µg kg -1 16 respectively). This could be explained by the fact that asparagine, the main precursor of 17 acrylamide in cereal products (RHM Technologies 2005) is concentrated in the bran, 18 particularly in wheat-bran (CIAA. 2005). 19 20Finally, the relationship was studied between the acrylamide content of biscuits and the 21 signal value stated by the German Federal Office of Consumer Protection and Food 22 Safety (BVL, 2004). This organization stated a signal value of 575 µg kg -1 for adult 23 population and 360 µg kg -1 for children. As depicted in Figure 2B, when applying the 24 same concept most of the samples (80%) are under the 575 µg kg -1 level. Taking into 25 account the results in this reported survey, a signal level of 1283 µg kg -1 is found 1 although, if the eight outliners are omitted (because of the explanation given above 2 concerning to the addition of ammonium hydrogen carbonate), a corrected level of 519 3 µg kg -1 is obtained, quite similar to that of the German Office one. [START_REF] Tareke | Analysis of 5 acrylamide, a carcinogen formed in heated foodstuffs[END_REF] 5

Acrylamide intake mediated by biscuits and bread derivatives 6

Estimating consumer dietetic exposure to acrylamide is a high priority for governments 7 and industry alike. Extensive research is underway to determine the extent to which 8 acrylamide found in food is bioavailable and to identify methods to reduce levels in 9 order to decrease consumer exposures. Consumer exposure assessments will need to be 10 conducted in order to gauge the utility of various control options. It is particularly 11

important to be able to assess the impact of proposed changes to the food supply 12 including the impact of modifications in processing and cooking procedures on 13 consumer exposures. 14 15 In order to estimate the dietary exposure of Spanish consumers to acrylamide from 16 biscuits and bread derivatives, the intake of these food supplies was used ( MAPA 17 2005). The results, collected in Table II, have been expressed as µg kg -1 day -1 taking 18 into account a mean body weight of 70 kg. For total population the intake of acrylamide 19 ranged from 0.002 -0.058 µg kg -1 day -1 for crackers and biscuits respectively. In the 20 case of biscuits, their contribution to the total daily intake of acrylamide is intermediate 21 to that of breakfast cereals (0.038 µg kg -1 day -1 ) and potato chips (0.093 µg kg -1 day - 22 There are considerable differences even between many of the European countries, due 1 to the differences in food consumption patterns and cooking traditions. Irrespective of 2 the cultural differences in nutritional habits, the overall daily intake is about 0.4 µg kg -1 3 day -1 according to the most recent FDA estimates, basically confirming earlier 4 estimations from the US and several European authorities [START_REF] Dybing | Human exposure and internal dose assessments of acrylamide in 20 food[END_REF]. 5

Assuming a mean Spanish intake of acrylamide similar to that of other European 6 countries, a mean daily intake of acrylamide of 0.2 µg kg -1 day -1 can be calculated 7 assuming only the intake of the above reported foods (bread-derivatives, biscuits, 8 breakfast cereals, potato chips). Estimations of WHO of the average intake of 9 acrylamide for the general population ranged between 0.3 -0.8 µg kg -1 day -1 . It was 10 supposed that children and adolescents would generally have exposures up to two to 11 three times those of adult consumers when expressed on a body weight basis (WHO, 12

2002). 13 14

The foods that contribute most to acrylamide exposure vary depending upon the 15 population's eating habits and the way the foods are processed and prepared. Generally, 16 the most important categories of food appear to be: fried potato products such as French 17 fries and chips, ready-to-eat breakfast cereals, baked goods such as cookies, pies and 18 cakes, brewed coffee and breads, according to the most recent results of the FDA/ 19 CFSAN [START_REF] Dybing | Human exposure and internal dose assessments of acrylamide in 20 food[END_REF]. In this survey representative from the Spanish market 20 contribution of biscuits, crisp bread, crackers and bread sticks will be of 14.5, 4.75, 0.5 21 and 0.75% respectively of the estimated dietary intake of acrylamide. In addition the 22 contribution of potato chips and breakfast cereals will be of a 23 % and a 9. In consumer exposure assessments it is also important to know whether there are 1 significant differences in exposures in different sub-groups of the population. In this 2 sense the exposure to acrylamide related to the those foodstuffs studied was analysed in 3 more detail taking into account socio-economic aspects (MAPA 2005). It was observed 4 that there are great differences in the intake of bread derivatives depending on the socio-5 economic community studied, almost doubling their intake. When the number of family 6 members was analysed it could be stated that the higher intakes was in families 7 composed of 1 or 2 independent adults whereas the lower intakes where found in young 8 couples with children. In the higher cases the biscuits contribution turned from 14.5 to 9 20 % of the overall acrylamide intake. In the case of the socio-economic status the 10 exposure to acrylamide is higher in upper class due to their higher biscuit intake. 11

Contrary lower class showed higher bread derivatives intake whereas biscuits 12 consumption decreased. Finally, taking into account the population size it was found 13 that the lower acrylamide intake was recorded in cities with more than 500,000 14 inhabitants whereas the maximum intake was in localities with low-intermediate 15 number of inhabitants. 16 17

Conclusions 18

This work describes the determination of acrylamide in biscuits and bread derivatives 19 cereals marketed in Spain, as a previous survey of foodstuffs had indicated these to 20 contain acrylamide. The data was used to determine the dietary intake of acrylamide for 21 the Spanish population for risk assessment purposes, it is estimated a mean daily dietary 22 intake of acrylamide of 0.082 µg kg -1 day -1 for which the contribution of biscuits was 23 higher than that of bread derivatives. It was also shown that in the case of biscuits, 24 higher acrylamide content was found in those manufactured with ammonium hydrogen 25

  ; Konings et al. 2003) 25

  as described by Rufian-Henares et al. (2006) with some minor 3 modifications. 4

  analysis. Sample extracts and calibration standards were analyzed on an Agilent 20 1100 liquid chromatograph coupled to an Agilent Quadrupole MS detector (Agilent 21Technologies, Palo Alto, CA, USA). Analytical separation was achieved with a Luna 22 ODS2 (25 x 0.46 cm, 5 µm; Phenomenex, Torrance, CA, USA) at 32ºC. Isocratic 23 elution was achieved with a mobile phase of acetic acid-methanol-Milli-Q water 24 (0.1:2.5:97.4) at a flow rate of 0.8 ml min -1 . The injection volume was 80 µl.25

version 5 . 4 the targeted food commodities 5 6

 545 1, 2001. At least, two independent analyses were carried out per sample. The analysis of acrylamide was carried out by means of an analytical methodology 1 previously established for potato chips (Rufian-Henares and Morales 2006) and 2 subsequently adjusted for breakfast cereals (Rufian-Henares et al. 2006). The procedure 3 was accurate, precise and had adequate sensitivity to detect the acrylamide content of Bread derivatives. A total of 45 bread types were studied: 24 crispbreads, 10 bread 7 sticks and 11 crackers. A detailed study of acrylamide distribution was carried out, and 8

  been accomplished to find strategies to minimize the levels of 2 acrylamide. This objective can be achieved either by modifying the processing 3 parameters such as pH, temperature/time of heating or acting on precursors or key 4 intermediates. WHO and the Scientific Committee for Food of the European Union 5 called for strategies to reduce acrylamide formation to a minimum by implementing the 6

  into account that the California Environmental Protection Agency (Office of 19Environmental Health Hazard Assessment) has proposed recently an alternative cancer 20 risk level, calculated to result in one excess case of cancer in an exposed population of 21 10 000 and assuming lifetime exposure, when consuming bread and cereals with an 22 acrylamide concentration lower than 200 µg kg -1 (OEHHA. 2005

6 7

 6 Amrein 9 et al. 2004;[START_REF] Biedermann | Model studies on acrylamide formation in potato, wheat 11 flour and corn starch; ways to reduce acrylamide contents in bakery ware[END_REF][START_REF] Graf | Reducing the 8 acrylamide content of a semi-finished biscuit on industrial scale[END_REF][START_REF] Konings | Acrylamide exposure from foods of the Dutch 15 population and an assessment of the consequent risk[END_REF]) a larger 10 amount of acrylamide was formed. This hypothesis explains the statistically significant 11 differences (p<0.05) obtained between the group where ammonium hydrogen carbonate 12 is the unique raising agent and those with other agents added (mean acrylamide content 13 of 1549 and 230.4 µg kg -1 respectively). 14 15 Taking into account the explanation given above, the outliers were removed for the rest 16 of the statistical treatment. Biscuits were classified into three groups based on the health 17 orientation, which is closely related to their composition: those mainly used for 18 breakfast (n = 21), those enriched with functional ingredients (n = 15) such as 19 conjugated linoleic acid, ω 3 -PUFA, polyols, soy extracts, or L-carnitine, and finally 20 those fibre enriched (n = 18). Enriched biscuits showed significantly (p<0.05) lower 21 values of acrylamide (83 vs. 247 and 284 µg kg -1 for breakfast and fibre enriched 22 biscuits respectively). It was found that most of the enriched biscuits had polyols (such 23 as maltitol or lactitol) and potato starch replacing the addition of glucose and/or 24 fructose. This replacement give rise to a decrease on the reducing sugar content, then 25 generation of acrylamide. Moreover the enriched biscuits are 1 elaborated by adding other kind of flours such as rice or malt. Because of, in the case of 2 breakfast cereals it has been shown that a higher potential in acrylamide formation in 3 wheat-based cereals followed by corn, oat and rice (CIAA. 2005; Rufian-Henares et al. 4 2006) it is plausible that the decrease on the wheat flour content reduce the acrylamide 5 generation in biscuits.Biscuits usually have dietary fibre contents of about 3 -4 %, but when enriched with 8

1 23 24Food

 23 ) previously reported (Rufian-Henares et al. 2006; Rufian-Henares and Morales 2006). Page 12 of 24 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

25 F

 25 5 % 23 respectively (Rufian-Henares and Morales, 2006; Rufian-Henares et al. 2006). 24

4 PageFigure 1 . 5 Figure 2 .

 4152 Figure Captions

Table I .

 I Statistical treatment for acrylamide levels in commercial biscuits grouped 3

	1 1	Food	and	Drug	Administration Tables	(FDA)	(available	at:
	2
	7 6 4 5	F o r P e e r R e v i e F o r P e e r R e v i e F o r P e e r R e v i e F o r P e e r R e v i e F o r P e e r R way ANOVA and Duncan test, p < 0.05) e i a Different letters within the same factor indicate statistical differences (One-v > 5% 166 ± 31.0 b 36 e according to the biscuit speciality, presence of ammonium hydrogencarbonate as solely raising agent and dietary fibre content. Factor Acrylamide (µ µ µ µg kg -1 ) No. of samples Total Mean 423 ± 65.6 62 Median 268 Minimum 15 Maximum 2085 Biscuit speciality Breakfast 247 ± 41.8 a 21 Enriched 83 ± 37.4 b 15 Fibre added 284 ± 39.9 a 18 Ammonium hydrogencarbonate Yes 1549 ± 117 a 8 No 230 ± 36.2 b 54 Dietary fibre content < 5% 283 ± 38.1 a 18 F o r P e e r R e v i e w	O n l y
	8					w w w w w
						O n l O n l O n l O n l l n O 2
							y y y y y
					1

Table II .

 II Contribution of commercial bread derivatives and biscuits to the acrylamide intake a of the Spanish population. 1 Results expressed as µg kg -1 day -1 taking into account a mean body weight of 70 kg. 3

	2
					Food
				Crisp		Bread
	Factor	Interval	Example	bread	Crackers	sticks	Biscuits
	Total population			0.0190	0.0020	0.0030	0.0580
	Autonomic	Maximum Extremadura for bread derivatives and Balear Islands for biscuits	0.0200	0.0030	0.0040	0.0670
	community	Minimum Canary Islands for bread derivatives and Andalusia for biscuits	0.0110	0.0017	0.0020	0.0410
	Family	Maximum Independent adults for biscuits and couples without children for bread	0.0230	0.0034	0.0042	0.0801
	components	Minimum Young couples with children	0.0100	0.0018	0.0018	0.0418
	Socio-economic	Maximum Upper class for biscuits and middle/lower class for bread derivatives	0.0235	0.0027	0.0042	0.0621
	status	Minimum Upper class for bread derivatives and lower class for biscuits	0.0116	0.0016	0.0024	0.0481
	Population	Maximum >2000 Inhabitants for bread derivatives and 10 000-100 000 for biscuits	0.0217	0.0027	0.0039	0.0630
	size	Minimum <500 000 Inhabitants	0.0131	0.0020	0.0024	0.0480

a

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