E Duffy

A P Hearty

S Mccarthy

M J Gibney

Estimation of exposure to food packaging materials. 3. Development of consumption factors and food type distribution factors from data collected on Irish Children

The estimation of exposure to food packaging materials. 3.

Introduction

Over the past number of years the refinement of exposure assessments for food packaging migratory compounds has been under discussion among regulators and the food packaging industry. One of the main issues is that the current method in the EU is viewed as being overly conservative and does not give any indication of the true or real exposure to a food packaging migrant. To refine the exposure assessment, progress has been made in relation to the use of probabilistic exposure assessment modeling to estimate exposure to a food packaging migrant [START_REF] Holmes | Dietary exposure to chemical migrants from food contact materials: A probabilistic approach[END_REF]. This methodology accounts for variability and uncertainty in the exposure assessment and provides a more realistic exposure based on available data and on expert opinion.

Probabilistic modeling is also used for the exposure assessment of other food chemicals. However, it is only undertaken when there is a need for a highly refined estimate of exposure. The general approach to exposure assessments is a step-wise approach, which begins with crude screening methods based on worst case assumptions and then proceeds to more refined methods, if results from crude methods dictate the need to do so (Nutriscan 1994[START_REF] Gibney | Estimation of food additive intake: methodology overview[END_REF], European Commission 1998[START_REF] Kroes | Assessment of intake from the diet[END_REF].

If the principles of this tiered approach for other food chemicals are borne in mind when examining exposure to food packaging migrants, there is a need for another tier between the current conservative method used by the EU and the highly refined probabilistic exposure assessment for food packaging migrants. An approach similar to the one currently used in the US by the Food and Drug Administration (FDA) to calculate intakes of food packaging migrants could be viewed as being more realistic than the current EU method but less refined than probabilistic exposure modeling. This approach is a deterministic as a single value is chosen for each parameter in the exposure algorithm yielding a single estimate of exposure.

The US FDA estimates probable exposure to food contact substances (FCS) by combining migrant levels in food, often from migration studies, with information on the uses of food-contact articles that may contain the FCS (i.e. on the fraction of a person's diet likely to contact food-contact articles containing the FCS) (FDA 2002).

Both the concentration in the daily diet (i.e. dietary concentration) and the estimated daily intake (EDI) from the FCS and the cumulative EDI (CEDI) from all regulated uses and effective food contact notifications is used by the FDA in the safety evaluation of a FCS. The approach is designed to deal with the majority of FCSs intended for single use. In this calculation, a number of factors are combined to estimate potential exposure. "Consumption factors" describe the fraction of the daily diet expected to be in contact with specific packaging materials. To account for the variable nature of food contacting each food contact article, the FDA has calculated "food-type distribution factors" for each packaging material to reflect the fraction of all food contacting each material that is aqueous, acidic, alcoholic or fatty. This information is then combined with migrant levels in food and a total food intake of 3kg per person per day (total solids and liquids) to calculate exposure to a food migratory compound.

If an approach similar to that of US FDA for estimating exposure to food packaging migrants was adopted in the EU, it could be viewed as the next tier in the exposure assessment of migrants. However the FDA approach has not been adopted in Europe for a number of reasons. A major factor is that the EU and US differ in their approach to regulating chemicals for use in food contact materials. In the US, a petition is submitted to the FDA for approval of, for example, a unique FCS, such as an antioxidant, for use in a polymer in contact with a specific food type. Therefore approval for this FCS is for use in that particular polymer, which in turn is used for a specific food. If this FCS is then used in a different polymer or this polymer is used for a different food group, this new use would then need separate approval. In the EU, once a chemical is permitted for use in plastics it can be used in any plastic for any food type provided that it complies at all times with safety criteria. Therefore, for chemicals with a wide range of end applications, the current EU exposure assessment method may not overestimate intakes by a large margin. In the US, the FDA has collected the data to compute the consumption factors and food-type distribution factors. However, no such data have been collected in the EU. The EU and US also differ in relation to the use of exposure assessment data. In the US, toxicology data requirements are tiered to correspond to the potential dietary exposure to the substance in question [START_REF] Heckman | Food packaging regulation in the United States and the European Union[END_REF]. However in the EU, migration data determines the toxicology requirements and exposure assessments are used mostly to demonstrate adherence with the tolerable daily intake (TDI).

The argument regarding which system (FDA exposure data or EU migration data) is superior for regulatory clearance of a substance has been discussed comprehensively [START_REF] Heckman | Food packaging regulation in the United States and the European Union[END_REF] and is not the topic of the current study. Instead, the aim of the present study was to create consumption factors and food type distribution factors similar to those used by the FDA from data collected as part of an Irish national food consumption survey. These factors, which are for Irish children aged 5-12 years, were derived using data on food consumption, packaging usage and surface area to weight ratios. These factors were calculated to highlight how similar factors could be created for other countries if food consumption surveys collected information on packaging used for foods.

Methodology

In this study, consumption factors and food type distribution factors similar to those used by the FDA were calculated. Due to differences in the underlying data there were small differences in the methodology used to calculate the FDA consumption factors and those presented in this paper. The FDA used data on the types of food consumed, the types of food contacting each packaging surface, the number of food packaging units in each food packaging category, the distribution of container size and the ratio of the weight of all food packaged to the weight of the package. All of this data was obtained from market data analysis (FDA 2002). In the present study, factors were calculated based on (1) food consumption data, (2) packaging use data, and (3) surface area to weight ratios for the area of a packaging material contacting a food. This information was obtained from a number of different sources outlined below. The National Children's Food Consumption Survey (NCFS) was a cross-sectional study of food and nutrient intakes of a representative sample of 594 Irish children aged 5-12 years (IUNA 2005). A 7-day weighed food record was used to measure food and beverage intakes. Respondents recorded the day, time, location and meal type for each eating occasion, along with a detailed description of the food, the weight of the food and the packaging used for the food. The weight of food consumed in the NCFS was corrected, where necessary, to reflect the amount of that food that was actually packaged and not just the amount of food consumed. These correction factors were applied to dried or dehydrated foods that were reconstituted before consumption (e.g. dried soups, sauces, gravies, custards, pasta dishes etc). A complete list of these factors is available in Duffy et al. 2006a. Each food consumed in the NCFS was assigned into one of the food types outlined in European Council Directive 85/572/EEC (i.e. aqueous, acidic, alcoholic or fatty) (European Council 1985). A food type was also created for dried and frozen foods, which are recognised in the EU as having no migration potential, this groups was referred to as the "dry" group. The FDA also uses similar food groupings to create their "food-type distribution factors" with the exception of a "dry" food type. These factors are used to reflect the fraction of all food contacting each material that is aqueous, acidic, alcoholic and fatty. Therefore, by coding each food in the NCFS into one of these food types, a food type distribution factor was calculated. As alcoholic products were not used in this sample age group, there were no foods classified as alcoholic. However, a food type distribution factor was calculated for dry food types.

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2) Packaging usage data

The packaging used for each food consumed in the NCFS was documented in the subjects' diary and the actual packaging used for the food was also collected and forwarded to the coordinating centre where it was used to develop the Irish Food Packaging Database (IFPD) (Duffy et al. 2006b). The IFPD was created to store exact information on the type of packaging used for foods (e.g. clear plastic wrapper), the contact layer of this packaging format (e.g. plastic) and the exact polymer used for the plastic contact layer if available (e.g. polyethylene). For food items with more than one material in contact with the food, this packaging format was also recorded. For example, glass jars with metals lids were categorised into the packaging group "glass and metal and alloys". Other combinations of packaging materials in contact with the food included glass and plastic (e.g. chocolate spread packaged in a glass jar with a plastic lid), metal and alloys and paper (e.g. Chinese takeaway packaged in an aluminium tray with a paper lid), metal and alloys and plastic (e.g. cream cheese packaged in a plastic container with an aluminium foil lid), and plastic and paper and board (e.g. chocolate biscuits packaged in a plastic wrapper with an inner paper tray).

Full details of the analyses of this database are available in Duffy et al. 2006a.

(3) Surface area to weight ratios Before calculating consumption factors and food type distribution factors, the amount of each food consumed was corrected to take into account the amount of contact it had with a specific packaging material. Due to resource constraints, the surface area of the packaging in contact with the food was not calculated in this study. However literature from previous studies was used to assign surface area to weight ratios to the food packaging formats. These ratios and their sources are documented in Table 1. As some foods had more than one type of material in contact with its' surface (e.g. plastic wrapper and paper tray), food surface area to weight ratios were used for the total packaging in contact with the food. Surface area to weight ratios were not available for all foods and hence a conservative surface area to weight ratio of 12 dm 2 /kg was used in the absence of more specific data. The factor of 12 dm 2 /kg was used as recent studies have shown that the currently used default in the EU of 6 dm 2 /kg is not accurate and underestimates the surface area to weight ratio of food in contact with packaging for many foods [START_REF] Bouma | Domestic use of food packaging materials in the Netherlands[END_REF]Grob et al. in press). Closures with large mass-to-surface areas were not accounted for in this study, instead only the fact that package type actually contained that type of closure was documented i.e. glass jars with metal lids were classified as "glass and metal and alloys". Therefore liners (for plastic lids) or sealing gaskets (for metal lids) were not accounted for in the derivation of the packaging factors.

[Insert Table 1

Consumption Factors

Information from the consumption database, packaging database and surface area to weight ratios were merged to create an information source, which included all the relevant details to enable the development of consumption factors. Similar to the FDA approach, the consumption factors developed in the present study represent the ratio of the amount of a specific contact material used to the amount of all contact materials used. In the food consumption survey, there were 2.5% (n=1808) of eating events where no information was recorded on the packaging status of the food consumed (i.e. whether it was packaged or not). These unknown entries were not included in the analysis. As some plastic contact layers had their polymers identified, consumption factors were also calculated for the main polymer types identified in the study. To represent the plastic contact materials that did not have their specific polymer identified, a consumption factor for "unidentified polymers" was created.

By combining the three datasets (food consumption, packaging use and surface area to weight ratio factors) consumption factors (c m ) were calculated for each contact material m. These factors were separately calculated for each specific material in contact with the food (e.g. plastic) and when these materials were used in combination with other materials in contact with the food (e.g. plastic and paper). The following equation was used:

∑ ∑ ∑         = m fm f fm fm f fm m s a s a c
where a fm is the amount consumed of food f packaged in food contact material m. s fm is the surface area to weight ratio for food f packaged in material m.

The product a fm s fm is summed over all foods f, so that the numerator gives the total amount of contact material m used.

The denominator is equal to the numerator summed over all contact materials m.

Hence it corresponds to the total amount of all contact materials used. Therefore c m is the ratio of the amount of contact material m used to the amount of all contact materials used.

Food type distributions factors

Food type distributions factors, d mt , correspond to the fraction of all packaging material m that is used with food of type t. The four types of food are: aqueous, acidic, fatty and dry (no alcoholic foods were consumed in this study). The distribution factors were calculated separately for each specific material in contact with the food and when these materials were used in combination with other materials. The following equation was used:

∑ ∑ ∑         = t fmt f fmt fmt f fmt mt s a s a d
where a fmt is the amount consumed of food f, where food f is packaged in food contact material m, and is of type t (where t is either aqueous, acidic, fatty or dry). s fmt is the surface area to weight ratio for that food f (packaged in contact material m and of type t). The product a fmt s fmt is summed over all foods f, so that the numerator gives the total amount of contact material m used with food of type t.

The denominator is equal to the numerator summed over all food types t. Hence it corresponds to the total amount of contact material m used with food of any type. Therefore d mt is the fraction of packaging material m that is used with food of type t.

Exposure calculations

In the FDA document entitled "Guidance for Industry" an example of how to apply food consumption factors and food type distribution factors to estimate exposure to a proposed new antioxidant in polyolefins is provided (FDA 2002). A similar example of such an exposure assessment was completed using data from this study for the addition of a new antioxidant to polyethylene. Four deterministic exposure scenarios were completed for this antioxidant. In the first scenario, some of the EU assumptions for calculating the intake of a chemical migrant (i.e. that 1 kg of packaged food is consumed per day and that the chemical migrates at the maximum permitted level i.e. at the specific migration limit of 15mg/kg in this scenario) and a body weight of 30kg for children were used to calculate exposure to the antioxidant. In the next scenario no migration data were available and hence the SML value of 15mg/kg was used again but the consumption factor for polyethylene (Table 2) was used. This was in addition to children's consumption of packaged food and the mean body weight of children, as derived in the current study. The next two scenarios used consumption factors, food type distribution factors and real migration data in addition to data on children's consumption of packaged food and the mean body weight of children. In the first of these two scenarios, the consumption factor used was a conservative consumption factor, as it was the sum of the consumption factor for "polyethylene" and "unidentified polymers" as some of the unidentified polymers could have been polyethylene and therefore by using the consumption factor for unidentified polymers (in addition to the consumption factor for polyethylene) it was assumed that all of these unidentified polymers were polyethylene. In the other of these exposure scenarios, the consumption factor for polyethylene only was used. Full details of these calculations are described in the appendix.

Food contact area

Another method previously used in the EU to estimate potential exposure to food packaging was to calculate the food contact area exposure of individuals to packaging i.e. dm 2 /person/day (ILSI 1997). The food contact area for all packaging was calculated and again this was a combination of data on food consumption, packaging usage and surface area to weight ratios. The amount of food packaging area used for all foods consumed was initially calculated for each day for each child. This was then averaged over the seven days the children completed the survey to get the average daily food contact area used by each child in the survey. These values were then averaged to get the mean food contact area for packaging used by all children as well as the upper percentiles. The food contact area used for plastics was also calculated in addition to the mean and upper percentiles of food intake and body weight. As some of the food packaging formats (9.5%, n=350) had more than one material in contact with the food (i.e. glass jar and metal lid), results for the consumption factor and food type distribution factor for a specific material were calculated when the material was the sole contact layer and also when it was used in combination with another material that was also a food contact layer.

Results

F

A consumption factor was calculated for the materials in contact with foods. Table 2 lists the consumption factors for each contact layer. "Total plastics" had the highest packaging factor of 0.83, while "total paper & board" had a factor of 0.132 and "total metal & alloys" had a factor of 0.061. Although the exact polymer used for all plastic contact layers was not identified, a consumption factor was calculated for the main polymers identified in the study with polyethylene having the highest factor at 0.332.

Equivalent FDA factors are also provided in Table 2 to allow comparison between the data sets. The packaging factor for "total plastics" was quite similar in the Irish data (0.83) and the FDA data (0.791). However, a large difference existed for the consumption factor for "total metal & alloys" in the Irish data (0.061) and the FDA data (0.2).

[Insert Table 2 about here]

The food-type distribution factors, which represent the fraction of the different types of food in contact with each contact material, are recorded in Table 3. "Total glass" was used mainly for acidic foods (0.670) and "total metals & alloys" were also predominately used for acidic foods (0.551). "Paper & board" was primarily used for dry foodstuffs (0.775). There was no specific food type packaged in plastic as it was used uniformly for all food types in this study (i.e. aqueous, acidic, fatty and dry).

[Insert Table 3 about here]

The results of the exposure scenarios for the new hypothetical antioxidant for use in polyethylene are presented in Table 4. In the first scenario, which used some of the EU assumptions, an exposure of 15mg/day or 0.5mg/kg body weight/day was calculated. The next scenario, which used consumption factors and the SML, resulted in an exposure of 5.95mg/day or 0.18mg/kg body weight/day. For the scenario that used the conservative consumption factor, the estimated exposure was 0.39mg/day or 0.12mg/kg body weight/day, while when the consumption factor for polyethylene only was used the exposure estimate was 0.22mg/day or 0.01mg/kg body weight/day.

[Insert Table 4 about here]

The mean food contact area used for all packaged foods was 13.44 dm 2 /child/day. This corresponded to an intake of packaged food of 1195g/child/day (Table 5). The upper percentiles of food contact area used for all foods were 19.58 dm 2 /child/day (95 th percentile) and 20.73 dm 2 / child /day (97.5 th percentile). When the food contact area for plastic packaging only was calculated, the mean value was 10.67 dm 2 /child/day with upper percentiles of 15.86dm 2 /child/day (95 th percentile) and 17.58dm 2 /child/day (97.5 th percentile).

[Insert Table 5 about here]

Discussion

The tiered approach to food chemical intake has won general acceptance internationally [START_REF] Douglass | Estimation of dietary intake of food chemicals[END_REF] and is used for food additives, pesticides and other food chemicals and contaminants. This methodology prevents unwarranted data collection and ensures that resources for the collection of data are put to the best use [START_REF] Gibney | Estimation of food additive intake: methodology overview[END_REF]. The factors derived in this study can be applied to derive a more realistic deterministic estimate of exposure to food packaging materials for Irish children. As can be seen from Table 4, when assumptions from the EU exposure assessment approach for food packaging migrant were used, the exposure estimate was much greater than the exposure estimates when using the consumption factors and/or the food type distribution factors. In one scenario a conservative consumption factor was used in the assessment as it allowed for the fact that some polymers, which were not identified, could have been polyethylene. The result of this scenario assessment was still much lower than the estimate obtained when using the EU assumptions, even though it was conservative. Further conservatism could be built into the exposure assessment if the upper percentiles of food intake were used to estimate the exposure instead of the mean food intake. Therefore, even by combing more realistic data, conservatism can still be ensured in the overall exposure estimate. However the construction of this dataset demonstrates how consumption factors and food type factors can be derived based on information collected as part of a food consumption survey and form the basis to which more information from other countries could be added in order to achieve more representative factors.

The categories of food contact materials used by the FDA and those used in this study differed to some extent. In this study, the materials types listed in Table 2 and3 are only for materials in contact with the food. For example, in the case where paper had a coating of plastic and it was solely the plastic layer that was in contact with the food (i.e. there was no paper in contact with the food) this material was recorded as plastic in the present study whereas the FDA classified this as paper-polymer coated. Also in this study, all canned foods were classified as packaged in "metal & alloys" with no distinction being made between uncoated and polymer coated cans. Despite these differences when comparisons were made between the factors used by the US FDA and those derived from this study, there were a lot of similarities especially for plastic materials, which are the materials that receive the most attention in terms of exposure assessments and regulatory compliance. The factor derived in this study for "total plastics" was 0.83 while when a similar figure was derived from the FDA data the factor was 0.79. This highlights that in both the US and Ireland plastic packaging dominates as the material that is used most commonly for foods. However, it must be remembered that the FDA data is for the total population and therefore comparisons between the FDA factors and the factors derived in this study are completely assessment process currently used in Europe (ILSI 1997). During this review data from Maurice Palmer Associates (MPA 1995) was processed to estimate the food contact area for all packaging used by individuals across Europe and it was estimated at a mean value of 20.1 dm 2 /person/day, while the mean food contact area for plastics was 12.4 dm 2 /person/day. The figure derived in the present study for the food contact area of all packaging material used by children was 13.44 dm 2 /child/day, while the food contact area for plastics was 10.67 dm 2 /child/day. These value differs greatly from the food contact area value of 6 dm 2 /person/day currently used in the EU exposure assessment. The average plastics food contact area proposed for Europe of 12.4 dm 2 /person/day may seem reasonably close to the figure derived in this study of 10.67 dm 2 /child/day. However, the figure derived in the present study was for children only and adult exposure to food contact material should be higher due to their relatively higher food intake. During the course of the ILSI workshop, it was noted that Ireland and the Benelux countries had a higher use of plastics at 17-19 dm 2 /person/day compared with other countries. This may explain why Irish children have food contact exposure quite similar to the average EU adult consumer as there may be an overall trend for higher food contact area exposure in Ireland compared with other European countries. In light of these results, if the aim of the exposure assessments is to ensure conservatism then the proposed EU average food contact area use may need to be revised upwards to ensure the exposure assessment protects consumers at the upper end of food packaging use and not just the average consumer.

In addition to proposing food consumption factors for the EU, the ILSI workshop focused on how the FDA approach could be applied in the EU for regulatory approval of a substance for use in food packaging. It concluded that this method could have applications for monomers and additives with defined numerical TDIs and specified single polymer use, dossier applications for approval for single polymer use, and applications to the threshold of no regulatory concern for recycling (ILSI 1997).

However even if an approach similar to the FDA was not adopted in Europe for regulatory approval of a substance, these consumption and food type distribution factors could be used to perform more realistic exposure assessments in cases of concern regarding the presence or quantity of a migrant in foods post approval of the substance. This would ultimately lead to a more time and resource efficient exposure assessment as well as being more refined and realistic on which to base opinions regarding the safety of the migrant in question. However if this method of exposure assessment shows that there may still be concerns (i.e. the exposure level approaches or exceeds the TDI) regarding the exposure to a migrant, then more refined exposure assessments could be computed using probabilistic modelling. In a recent safety assessment by the Food Additives, Flavourings, Processing Aids and Materials in Contact Food (AFC) scientific panel of the European Food Safety Authority (EFSA), an opinion was provided on the exposure of adults to epoxidised soybean oil (ESBO) used in food contact materials (EFSA 2006). After completing a conservative exposure scenario, the panel noted that the potential dietary exposure of adults to ESBO was below the TDI and therefore refinement of the exposure estimates was not necessary. However this conservative exposure assessment still required collection of multiple datasets on migration, food consumption and packaging use across Europe and then the merger of all this data in order to perform an exposure assessment. Much of this preliminary data collection could be eliminated with the use of consumption factors and food type distribution factors, making the task of estimating exposure from food contact materials more approachable.

The demand for more refined exposure assessments for food packaging migrants has been linked with the concept of the threshold of toxicological concern in Europe (TTC). The TTC is a pragmatic risk assessment tool that is based on the principle of establishing a human exposure threshold value for all chemicals, below which there is a very low probability of an appreciable risk to human health [START_REF] Kroes | Structure-based thresholds of toxicological concnern (TTC): guidance for application to substances present at low levels in the diet[END_REF]). This concept, known as the threshold of regulation (TOR) in the US, has been used by the FDA since 1995 when it was decided that due to their low potential health concern, components of food contact materials present in the diet at a dietary concentration of 0.5 parts per billion (ppb) or less are subjected to an abbreviated regulatory review.

The main objective of this abbreviated regulatory review is to ascertain that the dietary concentration resulting from the intended use is at or below the threshold level and that there is no reason to suspect, based on test data in humans or animals or chemical structure, that the substance may be a carcinogen [START_REF] Cheeseman | A tired approach to threshold of regulation[END_REF]. It is recognised by the FDA that the TOR process is a safety review process based on probabilistic assessment of risk and therefore an estimation of consumer exposure is fundamental to its implementation [START_REF] Cheeseman | Thresholds as a unifying theme in regulatory toxicology[END_REF]). An expert group was convened in Europe to review the TTC principle and they concluded that it could be applied for low concentrations in food of chemicals that lack toxicity data, provided that there is a sound intake estimate [START_REF] Barlow | Threshold of toxicological concern for chemical substances present in the diet[END_REF]. Therefore without a more refined or "sound" exposure assessment for migrants from food contact materials, the TTC principle cannot be used in Europe for food packaging migrants. If representative consumption factors and food type distribution factors were developed for Europe then the TTC concept may have application for the safety assessment of polymerisation production aids, impurities, reaction by products and aids to polymerisation, if the regulators approved its use.

Conclusion

Consumption factors and food type distribution factors may prove useful in the refinement of the current EU method for estimating exposure to food packaging migrants. They could be employed when a more realistic exposure assessment is needed, if there is concern over the presence of a migrant in a food, or for use with the TTC. Their use in exposure assessments could ultimately lead to a more uniform, simple and realistic exposure assessment process. However, only the regulators and the quality of data collected can determine their use for regulatory approval of a substance.

Exposure Scenario

The following hypothetical exposure scenarios are intended to illustrate the calculation of the concentration of a food packaging migrant in the daily diet using some of the EU assumptions and the more refined method of employing consumption factors and food type distribution factors. In the FDA document entitled "Guidance for Industry" an example of how to apply food consumption factors and food type distribution factors to estimate exposure to a hypothetical antioxidant in polyolefins is provided (FDA 2002). In the following exposure scenarios it shall be assumed that the new antioxidant is used in polyethylene materials only and has a specific migration limit (SML) of 15mg/kg in the European union.

Deterministic exposure estimate using EU assumptions.

For an adult, the EU exposure assessment methodology for food packaging migrants assumes that a 60kg person consumes 1kg of food packaged in the material of interest and that migration occurs at the maximum permitted level. There are no set guidelines on how to estimate intakes in children, therefore if we assume that children also consume 1kg of food packaged in the material of interest, which migrates at the SML, but that they have a body weight of 30 kg 1 we can estimate the intake of the antioxidant. The following parameters are used in the exposure calculation: In this scenario the consumption factor (CF) for polyethylene (Table 2) was used in the exposure assessment to represent the fraction of the daily diet likely to contact polyethylene. In this scenario there were no food type distribution factors or real migration data available, therefore the SML of 15mg/kg of food was used as a conservative assumption. However, as data was available on the intake of packaged food for children from this study, in addition to real body weights, these figures were used in computing the intake of the antioxidant. In this scenario the consumption factor (CF) for polyethylene (Table 2) and the food type distribution factors (FT) (Table 3) derived in this study were used to estimate exposure to the antioxidant. Migration values used by the FDA in their "Guidance for Industry" document were used (FDA 2002) instead of assuming maximum migration at the SML. These migration values, which are for the migration of the antioxidant from low density polyethylene (LDPE), are as follows:

CF

The first step in this exposure calculation was to calculate the level of the migrant in the food: As the mean consumption of packaged food in this study (Table 5) was 1.195kg per day, the resulting estimated daily intake (EDI) for children in this study would be: EDI = 0.185 mg antioxidant / kg food * 1.195 kg food/child/day = 0.221mg/child/day 2 In the EU distilled water or water of equivalent quality is used as the aqueous simulant, as opposed to 10% aqueous ethanol used by the US FDA. For the purpose of this exposure scenario the migration value for 10% aqueous ethanol was used for aqueous foods and acidic foods. Therefore, the food type distribution factor (FT) for both aqueous and acidic food is summed and then multiplied by the migration value for 10% aqueous ethanol.

3 No alcoholic foods were consumed in the Irish National Children's Food Survey therefore the migration value for alcoholic foods shall not be needed in this example. If a more conservative exposure assessment scenario of the antioxidant compared with the above example was required, then the consumption factor used in the calculation could be the sum of the "polyethylene" consumption factor (0.33) and the "unidentified polymers" consumption factor (0.248). As not all polymers were identified in the database some of those not identified could be polyethylene and hence it is conservative to assume that all the unidentified polymers could be polyethylene. For this scenario the exposure calculation would be as follows: As the mean consumption of packaged food in this study ( * In this method some of the assumptions used by the EU to estimate exposure to a migrant were employed. It was assumed that 1kg of food packaged in the material of interest was consumed per day and that migration occurred at the maximum level i.e. at a specific migration limit (SML) of 15mg/kg. The EU method uses a 60kg body weight for adults, and as this exposure assessment was for children a body weight of 30kg was used instead. † In this scenario the consumption factor (CF) for polyethylene was used in addition to the SML for the migration level. The intake of packaged food consumed by children and their mean body weight derived from this study were also used in the exposure assessment. ‡ This scenario used a conservative consumption factor (CF) in addition to realistic food type distribution factors (FT) and real migration data. The conservative consumption factor used in this method was conservative as it was the sum of the consumption factor for "polyethylene" and "unidentified polymers". ¶ This scenario used the consumption factors (CF) for polyethylene only and food type distribution factors (FT) derived in this study, in addition to real migration data, food consumption data of packaged foods and body weight data.

Food Type

  The factors derived in this study are representative of consumption factors and food type distribution factors for Irish children aged 5-12 years. Due to the fact that no alcoholic products were consumed, no food type distribution factors could be derived for the fraction of alcoholic products contacting each packaging material. Therefore, these factors are not representative of Europe or Irish adults and differ from the FDA factors as the FDA factors were derived for the entire US population and, as such provide per capita exposure estimates (Komolprasert 2005, personal communication).

  speculative.A workshop organised by the ILSI Europe Packaging Material Task Force discussed food consumption and packaging usage factors to aid the refinement of the exposure

  /kg body weight/day1 The assumption of 30kg body weight for children was used in this exposure scenario as the EPA use this body weight value for children in their "Notice of Filing of Pesticide Petitions"(Federal Register: November 20, 1998 (Volume 63, Number 224)).

Migration

  the antioxidant in the daily diet resulting from the proposed use would be: CF Polyethylene * Migration = 0.332 * 0.556 mg/kg = 0.185 mg antioxidant / kg food

  about here]

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

Deterministic exposure estimate using consumption factors and food type distribution factors.

	3.
	Polyethylene * Migration As the mean consumption of packaged food in this study (Table 5) was 1.195kg per = 0.332 * 15 mg/kg = 4.98 mg antioxidant / kg food F o r
	day, the resulting estimated daily intake (EDI) for children in this study would be: P
	EDI =	4.98 mg antioxidant / kg food * 1.195 kg food/child/day e
	=	5.951 mg/child/day e r
	The mean body weight of children in this study was 33.1kg (Table 5), therefore the R intake per kg body weight for children would be: e
		= =	intake of a child per day (mg/child/day) body weight (kg) v i e 5.951 mg / child / day 33.1kg w
		=	0.180 mg/ kg body weight /day	O n l
				y

exposure estimate using conservative consumption factors and actual food type distribution factors.

  The mean body weight of children in this study was 33.1kg (Table5), therefore the intake per kg body weight for children would be:

	=	intake of a child per day (mg/child/day)
		body weight (kg)
	=	0.221 mg / child / day
		33.1kg
	Simulant 0.007 mg/ kg body weight /day Aqueous & Acidic Foods = 10% aqueous ethanol 2 Alcoholic Foods 50% aqueous ethanol 3 Fatty Foods Miglyol 812 F r o 4. Deterministic	Migration 0.06mg/kg 0.92mg/kg 7.7mg/kg
		P
		e
		e r
		R
		e
		v i e
		w
		O n l
			y

Table 5

 5 Nutriscan 1994. An evaluation of the methodologies for the estimation of intakes of food additives and contaminants in the European Community. Final report. Dublin:

	) was 1.195kg per

Table 1 . Surface area:weight ratios for foods consumed in the Irish National Children's Food Survey.

 1

	Food Groups	dm 2 /kg	Reference
	Soft drinks bottle	5.2 Bouma et al 2003
	Juice	6.5 Bouma et al 2003
	Milk	6.7 Bouma et al 2003
	Cream Cheese	7	Bouma et al 2003
	Custard	7.04 Bouma et al 2003
	Margarine	7.1 Bouma et al 2003
	Soup (general)	8	Bouma et al 2003
	Yoghurt Drink	9	Bouma et al 2003
	Convenience Meals	12	Bouma et al 2003
	Icecream Container	12.1 Bouma et al 2003
	Confectionary (includes chocolate & gum)	17	Bouma et al 2003
	Meat (includes Quorn products)	18	Bouma et al 2003
	Sauce container (plastic)	18.4 Bouma et al 2003
	Yoghurt	18.4 Bouma et al 2003
	Fish	19	Bouma et al 2003
	Biscuits	21	Bouma et al 2003
	Bread	21	Bouma et al 2003
	Cakes	21	Bouma et al 2003
	Fruit & Veg	21	Bouma et al 2003
	Nuts	27	Bouma et al 2003
	Dessert Mousse	28.02 Bouma et al 2003
	Soup dried sachet	54	Bouma et al 2003
	Herbs	95	Bouma et al 2003
	Sauce dried sachet	96	Bouma et al 2003
	Crisps	60	Assumptions
	All other foods	12	Assumptions
	Canned Foods
	Fruit	7.48 Dionisi et al 2002
	Meat	11.06 Dionisi et al 2002
	Fish	16.63 Dionisi et al 2002
	Ready meals	7.25 Dionisi et al 2002
	Vegetable	6.96 Dionisi et al 2002
	Soup	8.36 Dionisi et al 2002
	Milk/cream	9.07 Dionisi et al 2002
	Other Foods	7.84 Dionisi et al 2002

Table 2 : Consumption factors of packaging materials derived from the Irish National Children's Food Survey for children aged 5-12 years.

 2

		Irish Consumption	Equivalent FDA
	Material	Factor	Consumption Factor
	Glass	0.002
	Glass & metal & alloys	0.013
	Glass & plastic	0.003
	Total Glass	0.017	0.1
	Metal & alloys	0.029
	Metal & alloys & paper & board	0.001
	Metal & alloys & plastic	0.018
	Metal & alloys & glass	0.013
	Total metal & alloys	0.061	0.2 *
	Paper & board	0.125
	Paper & board & metal & alloys	0.001
	Paper & board plastic	0.005
	Total paper & board	0.132	0.1 †
	Plastic	0.805
	Plastic & metal & alloys	0.018
	Plastic & paper & board	0.005
	Plastic & glass	0.003
	Total Plastic	0.830	0.791 ‡
	Wax	0.0001
	Polymers:
	Polyethylene	0.332	0.31 ¶
	Polypropylene	0.129	0.04
	Polystyrene	0.084	0.1
	Polyethylene terephthlate	0.033	0.16
	PVC & PVDC	0.003	0.1
	Unidentified polymers	0.248

Table 3 : Food Type Distribution Factor derived from the Irish National Children's Food Survey for children aged 5-12 years.

 3

	Material

Table 4 . Exposure assessment of a hypothetical antioxidant in polyethylene using different methodologies (full details of these assessment scenarios are documented in the appendix).

 4

					Exposure	Exposure
	Exposure Method				mg/day	mg /kg body weight / day
	Deterministic (EU Assumptions)*			15	0.500
	Deterministic (CF and SML)	†			5.951	0.180
	Deterministic (Conservative CF, FT and migration data)	‡	0.386	0.117
	Determinisitc (CF, FT and migration data)	¶		0.221	0.007

Table 5 : Food contact area, food intakes and body weights derived from the Irish National Children's Food Survey for children aged 5-12 years.

 5

	Page 29 of 29
		Food contact area for	Food contact area for	Intake of	Body
		all packaging	plastic packaging	packaged food	Weight
	Parameter	(dm2/person/day)	(dm2/person/day)	(g/day)	(Kg)
	Mean	13.44	10.67	1195	33.1
	Percentile 90th	17.29	14.38	1602	48.6
	Percentile 95th	19.58	15.86	1735	54.3
	Percentile 97.5th	20.73	17.58	1959	63.3
	Percentile 99th	22.95	18.78	2202	70.1

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Food Additives and Contaminants

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

Acknowledgements

The work was funded by the Department of Agriculture, Food and Rural Development who provided support through the Food Institutional Research Measure and also Enterprise Ireland who provided funding through the CREMe project.


Jan Velisek

Zuzana Zelinkova

Blanka Svejkovska

Marek Dolezal

Z Zelinková

B Svejkovská

J Velíšek

M Doležal

Fatty acids esters of 3-chloropropane-1,2-diol in edible oils

Keywords: Chromatographic analysis, Chromatography -GC/MS, Health significance Additives/Contaminants: 3-MCPD, Process contaminants -3-MCPD Food Types: Oils and fats, Olive oil, Processed foods 3-Chloropropane-1, 2-diol, 3-MCPD, bound 3-MCPD, 3-MCPD fatty acid esters, edible oils, contaminants

A series of 25 virgin and refined edible oils, obtained from the retail market was analyzed for their content of free 3-MCPD and 3-MCPD released from its esters with higher fatty acids (bound 3-MCPD). The oils contained free 3-MCPD ranging from < 3 µg/kg (LOD) to 24 µg/kg. Surprisingly, the bound 3-MCPD level was much higher and varied between < 100 µg/kg (LOD) and 2462 µg/kg. On average, virgin oils had relatively low levels of bound 3-MCPD ranging from < 100 µg/kg (LOD) to < 300 µg/kg (LOQ). Higher levels of bound 3-MCPD were found in oils obtained from roasted oilseeds (337 µg/kg) and in the majority of refined oils (< 300 µg/kg to 2462 µg/kg), including refined olive oils. In general, it appears that the formation of bound 3-MCPD in oils is linked with the preliminary heat treatment of oilseeds and with the process of oil refining. The analysis of unrefined, de-gummed, bleached, and deodorized rapeseed oil showed that the level of bound MCPD decreased during the refining process. However, additional heating of seed oils for 30 min at temperatures ranging from 100 o C to 280 o C, and heating at 230 o C (260 o C) for up to 8 hours led to an increase of the bound 3-MCPD level. On the contrary, heating of olive oil resulted in a decrease of the bound 3-MCPD level. For comparison, the fat isolated from salami was analyzed for the intact fatty acid esters of 3-MCPD. This fat contained the bound 3-MCPD at the level of 1670 µg/kg and the fatty acid esters of 3-MCPD mainly consisted of 3-MCPD diesters, monoesters of 3-MCPD were present in smaller amounts. The major types of 3-MCPD diesters (about 85%) were mixed diesters of palmitic acid with C18 fatty acids (stearic, oleic, linoleic acids). These diesters were followed by 3-MCPD distearate (11%) and 3-MCPD dipalmitate (4%).

Generally, very little 3-MCPD existed as the free compound (31 µg/kg).

Introduction

3-Chloropropane-1,2-diol (also known as 3-MCPD) is a representative of the so called food borne or food processing contaminants. It was first identified in acid-hydrolyzed vegetable protein (acid-HVP) in 1981 [START_REF] Davídek | New chlorine-containing compounds in protein hydrolysates[END_REF] as a reaction product of either acylglycerols or glycerol with hydrochloric acid and quite recently shown to be a racemic mixture of the corresponding enantiomers, (R)-3-MCPD and (S)-3-MCPD [START_REF] Velíšek | Optical isomers of chloropropanediols: mechanisms of their formation and decomposition in protein hydrolysates[END_REF]. 3-MCPD has been extensively monitored in acid-HVP because of its suspected carcinogenicity. In view of its toxicity, a provisional maximum tolerable daily intake of 2 µg per kg of body mass has been set (European Commission 2001) and a regulatory limit of 20 µg/kg, based on a 40% dry matter content, has been adopted for 3-MCPD in acid-HVP and soy sauce and came into force in the European Union in 2002(European Commission 2001).

Recent studies (Crews 2002;Hamlet et al. 2002a;Hamlet et al. 2002b;Divinová 2004) have shown that elevated levels of 3-MCPD (< 5 to 134 µg/kg) can occur not even in acid-HVP, soy sauces and related products but also in a wide range of retail processed foods such as meat, dairy and cereal products. Cereal products, particularly biscuits, crackers, doughnuts and bread, accounted for the highest levels of 3-MCPD reported (Hamlet et al. 2002b).

Furthermore, 3-MCPD was found in malt products comprising brewing malts and malt extracts. The typical levels of 3-MCPD for specialist malts (having typical EBC colour units > 20) ranged from < 30 to < 500 µg/kg (Hamlet et al. 2002a), which is well over the maximum level for acid-HVP.

While levels in retail foods have not been found to exceed 134 µg/kg (Crews 2002;Hamlet et al. 2002a;Hamlet et al. 2002b;Divinová et al. 2004a), the potential for additional generation of 3-MCPD from < 5 to 275 µg/kg, resulting from domestic cooking procedures, has been also reported [START_REF] Crews | Effect of domestic cooking on the levels of 3monochloropropane-1,2-diol in food[END_REF][START_REF] Breitling-Utzmann | 3-MCPD-occurrence in bread crust and various food groups as well as formation in toast[END_REF][START_REF] Breitling-Utzmann | Influence of dough ingredients on 3-MCPD formation in toast[END_REF]. Significant levels of 3-MCPD were found notably in the high temperature crust region of grilled cheeses [START_REF] Crews | Effect of domestic cooking on the levels of 3monochloropropane-1,2-diol in food[END_REF]) and toasted breads [START_REF] Crews | Effect of domestic cooking on the levels of 3monochloropropane-1,2-diol in food[END_REF][START_REF] Breitling-Utzmann | 3-MCPD-occurrence in bread crust and various food groups as well as formation in toast[END_REF][START_REF] Breitling-Utzmann | Influence of dough ingredients on 3-MCPD formation in toast[END_REF]. More than 500 µg/kg 3-MCPD can be found in well-toasted breads [START_REF] Breitling-Utzmann | 3-MCPD-occurrence in bread crust and various food groups as well as formation in toast[END_REF][START_REF] Breitling-Utzmann | Influence of dough ingredients on 3-MCPD formation in toast[END_REF]. However, at the moment there are no limits for any food product or food ingredient other than acid-HVP and soy sauce. Model experiments simulating processed foods [START_REF] Velíšek | 3-Chloropropane-1,2-diol in models simulating processed foods: precursors and agents causing its decomposition[END_REF][START_REF] Calta | Formation of 3chloropropane-1,2-diol in systems simulating processed foods[END_REF] showed that 3-MCPD evidently arised from either acylglycerols or glycerol in a reaction with naturally present or intentionally added sodium chloride. The 3-MCPD level strongly depends on temperature and the content of lipids, glycerol, salt and water. It is believed that the precursors (acylglycerols and glycerol) react with negatively charged nucleophiles, hydroxyl and chloride anions, which leads first to 3-MCPD esters and finally to free 3-MCPD.

Generation of 3-MCPD in model dough systems showed that free glycerol is the key precursor of 3-MCPD in leavened doughs (Hamlet et al. 2004a). Furthermore, monoacylglycerols, lysophospholipids, and phosphatidylglycerols present in flour improvers were important precursors of 3-MCPD in leavened doughs (Hamlet et al. 2004b).

Recent findings (Rétho and Blanchard 2005) indicate that also bromine anions react with acylglycerols and glycerol yielding 3-bromopropane-1,2-diol and 2-bromo-1,3-propanediol that were found in vegetable oils at the level ranging from 7 to 45 µg.kg -1 .

The mechanisms of 3-MCPD formation in processed foods are still very little understood.

However, there have been recent studies of mechanism of its formation in cereal products e.g. from glycerol (Hamlet et al. 2004a), mono-and diacylglycerols and glycerol phosphate (Hamlet et al. 2004b).

Fatty acid esters of 3-MCPD were the known precursors in the formation of 3-MCPD in model mixtures consisting of hydrochloric acid and triacylglycerols, phospholipids, soybean oil, soybean meal, wheat and maize lipids [START_REF] Velíšek | New chlorinecontaining organic compounds in protein hydrolysates[END_REF][START_REF] Davídek | New chlorine-containing compounds in protein hydrolysates[END_REF]. They were also found as the constituents of raw acid-HVP [START_REF] Velíšek | New chlorinecontaining organic compounds in protein hydrolysates[END_REF] and reported in the neutral fraction of goat's milk lipids [START_REF] Cerbulis | O ccurrence of diesters of 3-chloro-1,2-propanediol in the neutral lipid fraction of goat´s milk[END_REF], where their occurrence was tentatively ascribed to the use of chlorine-based sanitizers.

Our recent findings indicate that the formation of 3-MCPD esters (monoesters and diesters with higher fatty acids) is characteristic of a variety of processed foods (Divinová et al. 2004b;[START_REF] Svejkovská | Esters of 3chloropropane-1,2-diol in foodstuffs[END_REF][START_REF] Doležal | Occurrence of 3chloropropane-1,2-diol and its esters in coffee[END_REF]. These compounds represent a new class of food contaminants, the bound form of 3-MCPD, from which free 3-MCPD could be released by a lipase-catalyzed hydrolysis reaction. In many cases the amount of bound 3-MCPD exceeded that of the free 3-MCPD. We have analyzed 20 samples of selected retail food products for their bound 3-MCPD content. The levels of bound 3-MCPD found in the analyzed foods varied between traces (140 µg/kg) and 6100 µg/kg and were 10 to 396 times higher than the free 3-MCPD levels. Five foods of plant origin that were processed at high temperatures (particularly crisp bread, salty crackers, doughnuts, French fries and dark malt) contained elevated levels of bound 3-MCPD.

The highest level of bound 3-MCPD (6100 µg/kg) was found in a sample of French fries [START_REF] Svejkovská | Esters of 3chloropropane-1,2-diol in foodstuffs[END_REF]). The level of bound 3-MCPD in roast coffee was relatively low and varied between 6 µg/kg (soluble coffees) and 390 µg/kg (decaffeinated coffee) and exceeded the free 3-MCPD level 8 to 33 times [START_REF] Doležal | Occurrence of 3chloropropane-1,2-diol and its esters in coffee[END_REF]. The presence of bound 3-MCPD in pickled olives and herrings suggests that these compounds can also form at relatively low temperatures and even in acid media [START_REF] Svejkovská | Esters of 3chloropropane-1,2-diol in foodstuffs[END_REF]).

Analysis of white bread showed (Hamlet and Sadd 2004) that the highest levels of bound 3-MCPD were found in the crust (547 µg/kg) and toast (160 µg/kg) while in crumb they were found in much lower amount (26 µg/kg). The corresponding levels of the free 3-MCPD were 477 µg/kg, 93 µg/kg and < 0.5 µg/kg, respectively, i.e. 6 to 52 times lower. It was also showed that free 3-MCPD can be released from its esters by a commercial lipase (from Aspergillus oryzae) hydrolysis.

The vegetable oils recently analyzed by Rétho and Blanchard (2005) contained very low levels of 3-MCPD, between 3 and 7 µg/kg. The possible formation of 3-MCPD in oils by a lipase-catalyzed transesterification of triacylglycerols has recently been reported [START_REF] Robert | Model studies on the formation of monochloropropanediols in the presence of lipase[END_REF]). It is not yet known whether this phenomenon is due to hydrolysis of 3-MCPD esters or lipase catalyzed formation from triacylglycerols and chlorides.

Due to the occurrence of 3-MCPD esters in a variety of retail and homemade foods, we assumed that these lipophilic compounds could also arise in edible oils during the refining process. Described herein are our investigations into the determination of 3-MCPD esters in a variety of oils and fats. Our attention was mainly focused on the role of various factors that can influence the level of 3-MCPD esters during industrial processing of oils and their culinary use.

Materials and methods

Chemicals and samples

3-Chloropropane-1,2-diol (> 98%, 3-MCPD) was purchased from E. Merck (Darmstadt, Germany), 3-MCPD-d 5 (99.4%) from Dr. Ehrenstorfer (Augsburg, Germany), phenylboronic acid (PBA, ≥ 97%) from Fluka Chemie (Buchs, Switzerland). 1,2-Diacyl-3-chloro-1,2propanediols were synthesised according to [START_REF] Kraft | Massenspektrometrische Strukturuntersuchung stellungsisomerer Fettsäureester der Halogenpropandiole (Desoxyglyceride)[END_REF] and purified on a silica gel column using light petroleum ether/diethyl ether mixtures. All other reagents and solvents were of analytical purity.

Vegetable oils as well as roasted almonds and peanuts were obtained from retail outlets in Prague. The sample of salami was provided by Central Science Laboratory, York, UK.

Unrefined (mixture of pressed and extracted oil), degummed, bleached, deodorized rapeseed oils were supplied by Setuza a.s. (Ústí n. L., Czechia).

Instrumentation

GC/MS analysis of 4-chloromethyl-2-phenyl 1,3,2-dioxaborolane, obtained by derivatization of 3-MCPD using phenylboronic acid, was carried out on an Agilent Technologies 6890N gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) equipped with a Series 5973 quadrupole mass selective detector Agilent 5973 MSD (70 eV) and data processing system (MSD ChemStation, G1701CA version C.00.00). Gas chromatography was performed on a capillary column Equity™-1 (30 m x 0.25 mm i.d., thickness of 1 µm, Supelco, PA, USA).

The injector was held at 250 o C (splittles), the column temperature was programmed from The helium carrier gas was supplied at a constant rate of 0.7 ml/min. The analytes were ionized by EI at 70 eV and the obtained mass spectra of 3-MCPD esters with fatty acids were consistent with the spectra of the synthesized standards [START_REF] Kraft | Massenspektrometrische Strukturuntersuchung stellungsisomerer Fettsäureester der Halogenpropandiole (Desoxyglyceride)[END_REF], Velíšek et al. 1980). The individual esters were quantified using 1,2-dipalmitoyl-3chloro-1,2-propanediol (purity of 96.9) as the external standard.

Isolation of fat from nuts and salami

To obtain fat from the roasted almonds, roasted peanuts, and salami the material (20 g) was extracted with diethyl ether (80 ml) using Ultra Turrax T25 (Janke and Kunkel, IKA-Labortechnik, Switzerland). The obtained extract was filtered off and the residue again extracted with two portions of diethyl ether (20 ml each). The combined extracts were washed with water (20 ml), dried with anhydrous sodium sulfate and the solvent evaporated to dryness. The oil content was determined using the Soxhlet method (extraction with either diethyl ether -nuts or hexane -salami).

Heating of oils

The oil (20 g) was placed to a beaker and heated in an oven at the desiderative temperature

Free 3-MCPD

The oil (5 g) placed in a 100 ml Erlenmeyer flask was dissolved in a hexane : acetone mixture (1:1, v/v, 30 ml) and 50 µl of an aqueous solution of 3-MCPD-d 5 (internal standard, 0.5 µg) were added. The solution was transferred to a 100 ml separatory funnel and the Erlenmeyer flask was washed again with two 10 ml portions of the hexane/acetone mixture. The combined extracts was transferred to a separatory funnel and extracted with water (10 ml).

The aqueous phase was evaporated to dryness using a vacuum rotary evaporator at 55 o C. The residue was dissolved in 20% sodium chloride (2 ml), derivatised with phenylboronic acid (0.4 ml) and used for the determination of free 3-MCPD according to the procedure of Divinová et al. (2004b). Three parallel determinations of each sample were made.

Bound 3-MCPD

The oil (100 mg) placed in a 10 ml distillation flask was dissolved in tetrahydrofurane (1 ml).

To this solution 1.8 ml sulphuric acid solution (98%, 1.8 ml in 100 ml methanol) was added and the mixture was heated at 40 o C for 16 hours. The cooled mixture was then neutralised with saturated NaHCO 3 solution (0.5 ml), 1 ml of 3-MCPD-d 5 (internal standard, 1 µg) was added and the solution was evaporated to dryness using a vacuum rotary evaporator at 55 o C.

The residue dissolved in 20% sodium chloride (2 ml), extracted with two portions of hexane (2 ml), derivatised with phenylboronic acid (0.2 ml) and used for the determination of bound 3-MCPD according to the above procedure (Divinová et al. 2004b). Three parallel determinations of each sample were made.

Results and discussion

Our recent experiments simulating processed foods have revealed that the models consisting of the recognized 3-MCPD precursors (i.e. glycerol, triolein, lecithin) and heated (230°C, 30 min) with sodium chloride at a level typically present in foods yielded both enantiomers of 3-MCPD, i.e. (R)-3-MCPD and (S)-3-MCPD, in equimolar amounts [START_REF] Velíšek | Optical isomers of chloropropanediols: mechanisms of their formation and decomposition in protein hydrolysates[END_REF].

Stereospecific analysis of diesters of 3-MCPD found in goat's milk also showed that they were a racemic mixture [START_REF] Myher | Stereospecific analysis of fatty acid esters of chloropropanediol isolated from fresh goat milk[END_REF]. In total, six different compounds, four monoesters and two diesters, can be derived from racemic 3-MCPD and a fatty acid (Figure 1).

The method used throughout our research and focused on the occurrence of 3-MCPD esters in foods (Divinová et al. 2004b) was based on the determination of 3-MCPD released from the esters by methanolysis and thus did not have the potential to differentiate between the monoesters and diesters of 3-MCPD or the esters derived from its individual enantiomers. As a part of the present work, the above method has been optimized to increase its sensitivity so that the limit of detection (LOD) and limit of quantification (LOQ) of 3-MCPD released from the esters, when expressed as 3-MCPD (so called bound 3-MCPD), was 100 µg/kg oil and 300 µg/kg oil, respectively. The LOD and LOQ for the method used to determine the free 3-MCPD was 3 µg/kg oil and 9 µg/kg oil, respectively. Using these two methods, a series of retail market oils, oils isolated from nuts, and oils obtained from our industrial partner was analyses for the levels of free and bound 3-MCPD. Groups of retail market oils were selected to focus on virgin oils produced by different producers and from different raw materials, as well as on various grades of oils that might contain 3-MCPD esters resulting from heat processing during the refining process (Table I). It can be seen that the analyzed oils, with only three exceptions, contained free 3-MCPD at the levels of either LOD or LOQ (3 replications). The levels of 3-MCPD higher than the LOQ were only found in virgin hazel nut oil (10 µg/kg), virgin soybean oil (12 µg/kg), and virgin wheat germ oil (24 µg/kg) and they did not exceed the level of 50 µg/kg dry matter adopted for 3-MCPD in acid-HVP and soy sauce (European Commission 2001).

The bound 3-MCPD was found in oils at levels ranging from < 100 µg/kg (LOD) to 2462 µg/kg. As it can be seen (Figure 2), the average bound 3-MCPD level of oils (half of the value of either LOD or LOQ was used to calculate the average values) is low in virgin seed oils obtained from unroasted seeds (63 µg/kg), and increases in the following sequence: virgin olive oils (75 µg/kg), virgin germ oils (100 µg/kg), virgin seed oils from gained roasted seeds (337 µg/kg), refined seed oils (524 µg/kg), and refined olive oils (1464 µg/kg).

Within the sets of virgin oils, levels exceeding 300 µg/kg (LOQ) were only found in sesame oil obtained from roasted seeds (337 µg/kg). Refined seed oils (< 300-1234 µg/kg) and refined olive oils (< 300-2462 µg/kg) had higher levels of bound 3-MCPD. The amount of 2462 µg of bound 3-MCPD/kg found in refined olive oil then corresponds e.g. to 7780 µg/kg of 3-MCPD monooleate or to 13110 µg/kg of 3-MCPD dioleate and exceeds by 500% the level adopted for 3-MCPD in acid-HVP and soy sauce (European Commission 2001).

The average ratio of free 3-MCPD to bound 3-MCPD was 19 within the set of virgin seed oils and even 12 if the roasted sesame seed oil was not included. Virgin olive oils (extra virgin olive oils have a free acidity, expressed as oleic acid, of not more than 1 gram per 100 grams, virgin olive oils have a free acidity of less than 2 grams per 100 grams) had their average free 3-MCPD/bound 3-MCPD ratio 22, while this ratio increased to 150 within the set of refined seed oils and even to 325 within the set of refined olive oils.

It appears that the formation of 3-MCPD esters in oils is linked either with the preliminary heat treatment of seeds (e.g. roasting as in the case of roasted sesame seed oil) or with the process of oil refining (refined seed and olive oils). Moreover, there is certain dissimilarity between seed oils and olive oils as the latter have been obtained from the pulp. The pulp oils have high water content and active lipases yielding by hydrolysis partial acylglycerols during handling of fruits and their subsequent storage prior processing. Partial acylglycerols may then become the precursors of 3-MCPD esters [START_REF] Calta | Formation of 3chloropropane-1,2-diol in systems simulating processed foods[END_REF]. Virgin olive oils are produced by thrifty procedures where temperature does not exceed 40 o C while refined olive oils are commonly obtained by physical refination (degumming and bleaching). Moreover, the diatomaceous earth used for bleaching is sometimes purified by hydrochloric acid, which results in the formation of metal chlorides that may become the precursors of MCPD esters.

Olive oil is a blend of virgin oil with refined oil and olive pomace oil is a blend of refined pomace olive oil and possibly some virgin oil [START_REF] Firestone | Olive Oil[END_REF].

To check the influence of heat treatment on oilseeds, we have analyzed the level of free and bound 3-MCPD in retail roasted almonds and roasted peanuts, both unsalted and salted. The obtained results are summarized in Table II. For example, the amount of bound 3-MCPD in the oil extracted from roasted salted almonds was 1035 µg/kg while the level of bound 3-MCPD in virgin almond oil was < 100 µg/kg. Similarly, roasted salted peanuts contained bound 3-MCPD at the level of 883 µg/kg, which is much higher than the level (< 100 µg/kg) found in virgin peanut oil (Table I). The presence of salt did not markedly influence the level of 3-MCPD esters as it is usually added to the cooled roasted product.

We have also analyzed unrefined, degummed, bleached and deodorized rapeseed oil obtained from our industrial partner and, surprisingly, the level of bound MCPD esters decreased during the refining process (Table III). Bleaching was done at about 90-100 o C for 30 min and deodorization at 230-250°C for approximately 30 min. Therefore, it seems that the chemical composition of the oil given by its origin (pulp versus seed) plays a crucial role in the formation of MCPD esters.

We were further interested how different batches of oil from the same producer differed in their bound 3-MCPD level. Therefore, 6 batches of rapeseed oil produced in 2005 and 2006 were analyzed and it was found that the bound 3-MCPD levels ranged from 381 to 484 µg/kg (Table I).

We were also interested whether the thermal treatment of commercial oils exerts influence upon the level of bound 3-MCPD. Therefore, samples of refined rapeseed oil (with the original amount of 3-MCPD esters of 381 µg/kg), virgin wheat germ oil (<100 µg/kg), and refined olive oil (2462 µg/kg) were heated for 2 hrs at temperatures ranging from 100 o C to Our findings indicate that the level of bound 3-MCPD in rapeseed and wheat germ oils increased with temperature and time of heating while the level of bound 3-MCPD in olive oil decreased(Figure 3 and Figure 4). The increase in bound 3-MCPD level is probably due to the formation of 3-MCPD esters de novo while its decrease in olive oil is mainly caused by rapid decomposition, oxidation, and polymerization of 3-MCPD esters [START_REF] Svejkovská | Formation and decomposition of 3chloropropane-1,2-diol esters in models simulating processed foods[END_REF].

Heating of oils at 260 o C (Figure 5 and Figure 6) first leads to an increase of the bound 3-MCPD level in olive and rapeseed oils (achieved in about one hour), which is followed by a decrease with prolonged heating. Again, the bound forms of 3-MCPD in olive oil are more susceptible to decomposition, oxidation, and polymerization. The amount of free 3-MCPD did not change under the experimental conditions and remained at the level of < 9 µg/kg.

Since the 3-MCPD fatty acid esters derived from fats and oils seem to be universally distributed in processed foods (Divinová et al. 2004, Hamlet and Sadd 2004[START_REF] Doležal | Occurrence of 3chloropropane-1,2-diol and its esters in coffee[END_REF], the individual naturally occurring esters of 3-MCPD were investigated in the last stage of this study. We have analyzed the fat isolated from salami (fermented for 3 days at 62 o C, water 28.0%, protein 19.3%, fat 45.2%, salt 4.31%, carbohydrates 1.9%) that contained free and bound 3-MCPD at the level of 31 µg/kg and 1670 µg/kg, respectively. The bound 3-MCPD mainly consisted of 3-MCPD diesters, monoesters of 3-MCPD were present in smaller amounts. The major types of 3-MCPD diesters (about 85%) were mixed diesters of palmitic acid with C18 fatty acids (stearic, oleic, linoleic acids). These diesters were followed by 3-MCPD distearate (11%) and 3-MCPD dipalmitate (4%). Analogous results were obtained analyzing bakery products.

Further research aimed at the reaction pathways leading to the formation of 3-MCPD esters (that seem to be universally distributed in processed foods) and conditions under which these compounds form is ongoing.

Acknowledgement

O Cl CO R C O H CH 2 CH 2 OH Cl CO R C H OH CH 2 CH 2 O Cl CO R C H O CH 2 CH 2 OH Cl CO R C O H CH 2 CH 2 O Cl CO R CO R C H O CH 2 CH 2 O Cl CO R CO R (R)-1-acyl-3-chloropropane-1,2-diol (R)-2-acyl-3-chloropropane-1,2-diol (S)-1-acyl-3-chloropropane-1,2-diol (S)-2-acyl-3-chloropropane-1,2-diol (R)-1,2-diacyl-3-chloropropane-1,2-diol (S)-1,2-diacyl-3-chloropropane-1,2-diol

  80 o C (1 min) to 300 o C (37 min) at a rate of 10 o C/min. Helium at a flow rate of 0.8 ml/min was used as the carrier gas, 1 µl sample was injected. For quantification purposes, single ion monitoring was used to monitor ions at m/z 147 (3-MCPD) and at m/z 150 (3-MCPD-d 5 ). Ions at m/z 91and 196 (3-MCPD) and at m/z 93 and 201 (3-MCPD-d 5 ) were used as qualifiers.The fat isolated from salami was separated by preparative TLC[START_REF] Davídek | Glycerol chlorohydrins and their esters as products of the hydrolysis of tripalmitin, tristearin and triolein with hydrochloric acid[END_REF]) into the individual fraction and the fractions containing diesters and monoesters of 3-MCPD were analyzed by GC using the same instrument as above and a DB-1HT fused silica capillary column (15 m x 0.25 mm i.d., thickness of 1 µm, J&W Scientific, CA, USA). One 1 µl sample was injected into the split injection port (1:4) held at 300 o C with a solvent delay of 2 oven was initially set to 100 o C, then programmed to 340 o C at a rate of 10 o C/min and kept at 340 o C for 26 min.

(

  100 o C, 150 o C, 200 o C, 230 o C, 260, and 280 o C) for 30 minutes. Other portions of the oil were heated at either 260 o C or 280 o C for 0.5, 1, 2, 4, and 8 hours. The oils were let to cool to room temperature and analyzed for the content of free and bound 3-MCPD.

  , then at 230 o C and 260 o C for 0.5, 1, 2, 4 and 8 hrs and analyzed for the content of bound 3-MCPD. The extreme temperatures of 260 o C and 280 o C and very long times of heating were done for comparison. The achieved results can be seen in Figure 3 and Figure 4, respectively.

Figure 1 .

 1 Figure 1. Structure of fatty acid monoesters and diesters of 3-MCPD.

Figure 2 .

 2 Figure 2. Average amount of bound 3-MCPD in oils.

Figure 3 .

 3 Figure 3. Changes in bound 3-MCPD level in rapeseed, wheat germ, and olive oils heated to various temperatures for 30 min.

Figure 4 .

 4 Figure 4. Changes in bound 3-MCPD level in rapeseed, wheat germ, and olive oils with time of heating at 230 o C.

Figure 5 .

 5 Figure 5. Changes in bound 3-MCPD level in olive oils with time of heating at 230 o C and 260 o C.

Figure 6 .

 6 Figure 6. Changes in bound 3-MCPD level in rapeseed oils with time of heating at 230 o C and 260 o C.

Figure 3 .Figure 4 .Figure 5 .Figure 6 .

 3456 Figure 2. Average bound 3-MCPD level in oils.

Table II .

 II The content of oil, free and bound 3-MCPD in roasted almonds and peanuts.

	Roasted nuts	Oil	Free	Bound	Bound	Ratio
		%	µ µ µ µg/kg	µ µ µ µg/kg	µ µ µ µg/kg oil bound/free
	Almonds (salted)	48.35	31	500	1035	16
	Peanuts (unsalted)	49.55	21	437	882	21
	Peanuts (salted)	49.02	20	433	883	22
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Table III .

 III Changes in the content of free and bound 3-MCPD in rapeseed oil during physical Figure 1. Fatty acid monoesters and diesters of 3-MCPD.

	refining	CH 2
	Rapeseed oil C HO	H	Free	Bound	Ratio
		CH 2	µg/kg	µg/kg	bound/free*
	Unrefined			20	990	50
	Degummed		11	< 300	14
	Bleached			< 9	< 300	33
	Deodorized		< 9	< 300	33
	* Half of the value of LOQ was used.
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Wolfgang Vautz
email: vautz@ansci.de

Dunja Zimmermann

Jörg Michelle Hartmann

Ingo Baumbach

Jürgen Nolte

Johannes Jung

Jörg I Baumbach

Ingo

ION MOBILITY SPECTROMETRY FOR FOOD QUALITY AND SAFETY

Keywords: Metabolomics Nolte, Jürgen, ISAS, Metabolomics Jung, Johannes, DAB food process control, food quality control, food safety control, ion mobility spectrometry Additives/Contaminants: contaminants, flavour, metabolites, Volatiles Food Types: Beer, Beverages, Cheese, Wine

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

• Detection of metabolites from bacteria for identification and control of their growth.

• Process control in food production -beer fermentation as an example.

• Detection of the metabolites of mold for process control during cheese production, for quality control of raw materials or for the control of storage conditions.

• Quality control of packaging materials during the production of polymeric materials.

• Characterisation of products -wine as an example.

The challenges of such applications were the operation in humid air, fast on-line analyses of complex mixtures, high sensitivity -detection limits have to be e.g. in the range of the odour limits -and in some cases the necessity of mobile instrumentation. It could be shown that ion mobility spectrometry is optimally capable of fulfilling those challenges for many applications.

Keywords: ion mobility spectrometry, quality control, process control, food production, food quality, food safety, metabolomics

INTRODUCTION

Food quality and safety is an important issue not only for the comfort of the consumer but also related to human health [e.g. [START_REF] Peshin | Potential food contaminants and associated health risks[END_REF][START_REF] Bordajandi | Survey of persistent organochlorine contaminants (PCBs, PCDD/Fs, and PAHs), heavy metals (Cu, Cd, Zn, Pb, and Hg), and arsenic in food samples from Huelva (Spain): Levels and health implications[END_REF]] and for the benefit of the producer. The quality and origin of raw materials, the development of the production process as well as packaging and storage are relevant in this context. The growth of bacteria and mould can deteriorate the quality of raw materials and final products and may disturb the production process [e.g. Mbgua et al. 2004[START_REF] Annan | Volatile compounds produced by Lactobacillus fermentum, Saccharomyces cerevisiae and Candida krusei in single starter culture fermentations of Ghanaian maize dough[END_REF]. Furthermore, their metabolites may affect human health [e.g. [START_REF] Chung | Dose-dependent allergic responses to an extract of Penicillium chrysogenum in BALB/c mice[END_REF]]. The origin and quality of raw materials as well as storage conditions will influence the characteristics of the final product. The development of the production process is responsible for the quality of the product on the one hand and has to be controlled properly and terminated in good time in the economic interest of the producer [START_REF] Conte | The advancement of the assessment of food quality control as stressed by recent developments in analytical chemistry[END_REF][START_REF] Tarkiainen | On-line monitoring of continuous beer fermentation process using automatic membrane inlet mass spectrometric system[END_REF]]. In addition, many efforts are made to characterise the final product of a food production process for it's quality and sensory characteristics [e.g. Coghe et al. 2004, Fritsch and[START_REF] Fritsch | Identification based on quantitative measurements and aroma recombination of the character impact odorants in a Bavarian Pilsner-type beer[END_REF]. Finally, suitable packing and storage is required to keep the quality of the final product [START_REF] Sanders | Identification of 8-nonenal as an important contributor to "plastic" off-odor in polyethylene packaging[END_REF].

Taking samples for analysis during all stages of food production is obviously a direct way to control quality and safety. But the procedure is elaborate because the implementation of an automatic sampling system is required and samples have to prepared for the following analyses in the laboratory. On the other hand, many volatile compounds carry significant information about the characteristics of their source. Therefore, the analyses of volatiles in the headspace of a sample could be an indirect but -in the experimental point of view -much easier and faster method to control a process or the quality of foodstuff.

Traditionally, selective and sensitive headspace analyses during food production or of raw materials requires in general enrichment of the sample air and later analyses in the laboratory [START_REF] Conte | The advancement of the assessment of food quality control as stressed by recent developments in analytical chemistry[END_REF]. Standard analytical techniques like atomic absorption spectroscopy, gas chromatography and mass spectroscopy have in common that they are time consuming and expensive as well and that they require pre-concentration of the sample [e.g. [START_REF] Wang | Rapid Analysis of Flavour Volatiles in Apple Wine Using Headspace Solid-Phase Microextraction[END_REF]]. Therefore, on-line or on-site measurements are rather elaborate. During recent years olfactometry as well as so-called electronic noses and tongues are increasingly in the use for quality control of or characterisation foodstuff [Bendedetti et al. 2004, Pardo and Silverbergi 2004[START_REF] Hallier | New gas chromatography-olfactometric investigative method, and its application to cooked Silurus glanis (European catfish) odor characterization[END_REF][START_REF] Legin | Application of electronic tongue for quantitative analysis of mineral water and wine[END_REF].

A fast and sensitive low-cost on-line and on-site detection of the relevant volatiles would be a powerful tool in the field of food quality and safety control. Ion mobility spectrometry -which was developed first for the detection of narcotics, explosives and chemical warfare agents [Eiceman andKarpas 2005, Baumbach and[START_REF] Baumbach | Ion mobility spectrometry: Arriving on-site and moving beyond a low profile[END_REF]] -offers such characteristics.

Depending on the ionisation energy necessary to ionise relevant analytes, ion mobility spectrometry enables the detection of a huge number of volatiles including e.g. alcohols, aldehydes, alkanes, cyclo-alkanes, amines, aromatic amine, aromatics, esters, ketones, pyridines, organo-phosphorus compounds, polycyclic aromatic hydrocarbons and volatile acids as concluded by [START_REF] Eiceman | Ion mobility spectrometry[END_REF] for more than 220 detected substances. Many of them are also relevant for food quality and safety as they are e.g. known as metabolites from mold and bacteria or are related to other characteristics of their sources. Typical detection limits using ion mobility spectrometry are found in the lower ng/g and often in the ng/kg range and by that are in the range of odour thresholds in many cases.

At ISAS -Institute for Analytical Sciences, ion mobility spectrometry was applied successfully on various examples in the field of food quality and safety in recent years. Most recently investigations were carried out especially related to chracterisation, food safety and process control. After an introduction in ion mobility spectrometry a number of representative studies will be presented in detail, including process control of beer fermentation and cheese production as well as quality control with regard to contamination by mold or bacteria. Finally the possibility of characterisation of foodstuff and the control of the quality of packaging materials will be discussed.

MATERIALS AND METHODS

Ion Mobility Spectrometry

In an ion mobility spectrometer (IMS) the sample air is ionised using ß-radiation, UV light or partial discharges. Radioactive sources as well as partial discharges ionise the present gas and the resulting so-called reactant ions transfer charges to the analyte molecules. The reactant ions cause the "reactant ion peak" (RIP) in the IMS spectra. Using UV-light the analyte molecules are ionised directly -no reactant ions are formed [START_REF] Eiceman | Ion mobility spectrometry[END_REF].

In a second step, the ions will move in a weak electric field towards a Faraday plate used as detector. A shutter grid initially avoids the drift of ions in the field and only when it is opened for short time intervals (30 -1,000 µs), an ion swarm enters the so-called drift region of an IMS and moves towards the detector plate. In the opposite direction a drift gas flow is applied which avoids neutral analyte molecules from entering the drift region. Furthermore, the ions collide with the drift gas molecules, thus being slowed down depending on their size, shape and charge. Finally, a rather constant drift velocity of the ions will be realised due to energy gain from the electric field and loss by various collisions with other gas molecules.

The time the ions need to cover the drift distance from the shutter grid to the detector -the socalled drift time -is characteristic for the analyte ion. The number of detected ions is a measure for the concentration of the analyte. From the drift time and the instrumental parameters the l -length of the drift region in cm

E -electric field strength in V/cm t -drift time in s, p -atmospheric pressure in hPa (p o = 1,013.2 hPa) T -temperature of the drift gas in K (T o = 273.2 K)
The acquisition of a complete ion mobility spectrum needs less the 100 ms. The use of ambient air as drift and carrier gas avoids the use of expensive and unhandy gases of high purity. The instruments are operated under ambient temperature and pressure and furthermore can be miniaturised. Therefore, ion mobility spectrometry is optimally capable for fast on-line and onsite detection of volatiles also in humid air under ambient conditions without expansive sampling, enrichment and later analyses in the laboratory as it is the case using traditional methods [e.g. [START_REF] Hallier | New gas chromatography-olfactometric investigative method, and its application to cooked Silurus glanis (European catfish) odor characterization[END_REF]].

Pre-Separation

If complex mixtures are under investigation, several analytes which are present may have similar drift times. To enable their identification anyhow, pre-separation can be used to obtain additional information for identification from the retention times of the analytes in a gaschromatographic (GC) or multi-capillary column (MCC) [START_REF] Xie | Determination of Acetone, 2-Butanone and Diethylketone Using HSCC-UV-IMS[END_REF], Baumbach et al. 2003].

Furthermore this avoids negative effects from clustering in the ionisation chamber when humid air is analysed: Using pre-separation the water molecules and the analyte molecules enter the ionisation chamber successive. Therefore relevant substances can be detected even in humid air up to 100% relative humidity without negative effects on the detection limits [Vautz et al. 2004a]. Using pre-separation techniques, the analyte is introduced discontinuously by a 6-way valve into the column (variable time steps depending on the retention time of the analyte in the column, introducing 1 mL volume each time).

Calibration

For calibration of the IMS, permeation cells [Vautz et al. 2004b] were filled with standard analyte (puriss. p.a., Sigma-Aldrich) using a 1 mL tube closed with a membrane. This permeation cell was placed into a 150 mL bottle and exposed to a carrier gas flow of 100 mL/min. The concentration of the resulting calibration gas could be determined from the flow rate and from the weight of the permeation cell before and after the experiment. For the analytes investigated here, the initial concentrations for the exponential dilution were in the range of 1-100 mg/L. A 1 L bottle was flown through by the calibration gas for 2 hours to guarantee complete exchange of the air to the sample. Then this bottle was rinsed with a constant flow of synthetic air, thus leading to an exponential dilution of the initial concentration. The calibration curves can be used for the quantification of a detected signal. Detection limits were obtained from a signal/noise ratio of 1:3 and were found in the range of µg/L down to ng/L depending on the analyte and on the ionisation method.

Experimental

For the presented investigations ion mobility spectrometers with UV light (UV-IMS) as ionisation source were used as well as ß-radiation ( 63 Ni-IMS). For pre-separation gas-chromatographic (GC) and multi-capillary columns (MCC) were used in the combination GC-UV-IMS and MCC- pg/L (ppb V -ppt V range). Applications were e.g. the analyses of human breath or the detection of the metabolites of mold [START_REF] Ruzsanyi | Detection of the mold markers using ion mobility spectrometry[END_REF]]. Further details of the IMS and its application have been described elsewhere [START_REF] Xie | Determination of Acetone, 2-Butanone and Diethylketone Using HSCC-UV-IMS[END_REF][START_REF] Sielemann | Detection of trans-1,2dichloroethene, trichloroethene and tetrachloroethene using multi-capillary columns coupled to ion mobility spectrometers with UV-Ionisation sources[END_REF]]. In all cases, dry synthetic air was used as drift and carrier gas. The determined drift time as well as the calculated ion mobility were corrected for temperature and pressure [Vautz et al. 2004a, Vautz et al. 2004b]. For details of the experimental setup, see Figure 1 and Table 1.

PROCESS CONTROL Beer Fermentation

The fermentation process of beer is a time-consuming part of the complete beer production process and takes 5 to 6 days. The end of the process is determined by concentration thresholds for diacetyl and 2,3-pentandione [START_REF] Hough | The Biotechnology of Malting and Brewing[END_REF]] because of their smell like rancid butter. Both substances are formed in the beginning of the fermentation process and later are degraded while the process is going on. Their concentrations have to be decreased consequently below the human odour threshold. Furthermore the ratio of both substances is a measure for potential micro-biotic contamination which can occur during the fermentation process.

In a brewery, the concentrations are determined by taking and preparing samples manually before a gas-chromatographic headspace analysis in the laboratory. The full procedure takes about 3 hours. Therefore, this kind of analysis is made only once a day and no continuous concentration data are available. On-line methods are not available at present. In small breweries where no laboratory is available, the fermentation is extended with up to 5 days to assure sufficient degradation of both analytes without analyses. Using a GC-UV-IMS the detection of both relevant analytes in the matrix of different beer types was possible within less than 10 min [Vautz 2004d]. The variation of the total ion current with the retention time of the GC-columns is shown in Figure 2, left. After a baseline correction the quantification of both concentrations was possible by calculation of the peak area. A comparison with the results of the breweries routine analyses of beer samples after different fermentation time showed good agreement (see Figure 2, right). Both analytes can be detected and quantified below the odour threshold -even in beer ready for consumption in some cases traces of diacetyle and 2,3.-pentandione could be detected. Therefore using GC-UV-IMS a continuous on-line control of the concentration of the analytes responsible for the termination of the process is possible.

Cheese Production

For this study, the headspace of a complete camembert cheese and of parts of the cheese without the rind was introduced into the GC-UV-IMS. The detection of the metabolites of the camembert cheese with a GC-UV-IMS and of the cheese after complete removal of the rind showed that the metabolites from the camembert fungi can be differentiated from the volatiles of the cheese itself. The signal height enables quantification, especially of those peaks identified as metabolites from the mould fungi. Therefore, the method developed can be used for control of the growth of mould.

QUALITY CONTROL

Detection of the Metabolites from Mould

The detection of metabolites from mould as shown above enables not only the control of the growth of wanted mould but also of unwanted moulds as it could appear e.g. during storage of raw materials or even final products. In this case, not only one mould type will contribute to the culture but various types as they are available in ambient air. Therefore, the IMS spectra and chromatograms are expected to be more complex -various analytes can be expected as metabolites [Ruszanyi et al. 2003].

Figure 3 shows the variation of the IMS chromatogram of a bread mould culture which was investigated over a period of one month weekly. Without a detailed discussion of the contributing mould types, it is obvious that the shape of the chromatogram changes significantly during this period. Especially the signal at about 530 s retention time is growing strongly during the first 3 weeks. This indicates that not only the detection and identification of mould but also information about the state of the mould culture can be obtained using GC-UV-IMS.

As 63 Ni-IMS are in general more sensitive, identification of different mould types from the complete 3-dimensional IMS spectra (drift time, retention time and signal height) should be possible. Such investigations are planned for the near future. The growth of a mould culture then can be described from the changes a 3-dimensional plot (drift time, retention time and peak height) as shown e.g. in Figure 5. Quantification is possible using the peak volume.

Identification will be possible when using peak pattern as for the identification of bacteria as shown in the next section.

Detection of Metabolites from Bacteria

In the frame of investigations of human breath [Ruszanyi et al. 2005a, Ruszanyi et al. 2005b], the metabolites of various bacteria were investigated in detail not only in breath but also growing on controlled bacteria cultures. The metabolites of those bacteria were detected using a MCC-63 Ni-IMS. With statistical methods specific pattern of signals (retention time/drift time) could be determined for 10 different bacteria and a fungi which are relevant for pulmonary diseases (see Figure 4). Those pattern were used successfully to detect the bacteria in the breath of patients directly.

For sure related to food quality and safety different bacteria should be taken into account. But the procedure of definition of typical peak pattern for each bacteria or mold type and later identification e.g. in the headspace of a sample or in the ambient air in a storage location will be similar.

CHARACTERISATION

Many volatiles emitted from foodstuff are relevant for the character of the source as they are related to flavour, taste or quality [e.g. [START_REF] Wang | Rapid Analysis of Flavour Volatiles in Apple Wine Using Headspace Solid-Phase Microextraction[END_REF]]. Similar to the use of the metabolites of mould or bacteria for their identification, the volatiles emitted from any sample could also be used to characterise the sample. Figure 5, left shows the GC-UV-IMS chromatogram of a Brazilian sugar cane liquor, a French red wine and a German white wine. In this case no efforts were made to quantify the signals. But however it is obvious that the liquor causes a higher ethanol signal at about 1 min retention time than the other beverages while the further slope is relatively flat. Both wines show more clear signals and moreover there are significant differences between both wines. This demonstrates that not only liquor from wine but also red and white wine could be differentiated. Further investigations with various other wines showed that even between different red wines or between different white wines significant different signals can be observed. The full 3-dimensional IMS spectra -as shown in Figure 5 right for a German white wine -can be used to define characteristic peak pattern for different types of wine for differentiation from other types and for quality control as well.

PACKAGING MATERIALS

Finally, any foodstuff has to be packed before vending. Polymeric materials as commonly used for packaging of foodstuff are the product of polymerisation of up to 3 monomers. Those monomers play an important role as allergens and may be responsible for various lung diseases. Therefore an accurate control of the monomere concentration in the final polymer is strongly recommended in the interest of the consumer as well as in the interest of the producer.

Using a 63 Ni-IMS without pre-separation, the remaining traces of monomers in the polymer after complete polymerisation could be detected as shown in Figure 6. As already mentioned before, those traces can be quantified by help of a calibration carried out earlier. Therefore IMS can be used to control the compliance of threshold concentrations of monomers in packaging materials.

It is obvious, that such a sensitive method can also be used for control of the polymerisation process itself as shown in Vautz et al. 2004d. The concentration of monomers in the mixture during the polymerisation process was measured using a UV-IMS without pre-separation which analysed the headspace over a continuous droplet flow from the reaction tank. The process can be described with sufficient sensitivity and with a fast response of the headspace concentration on the concentration in the mixture.

However, the monomer concentration in the final polymer does not directly reflect the migration of the monomere into the packaged foodstuff. But it provides information about the potential for contamination of the foodstuff and after investigations of the uptake mechanisms of particular foodstuffs, the possible contamination can be estimated.

CONCLUSION

The investigations described above demonstrate the qualification of ion mobility spectrometry as a powerful tool for food process, quality and safety control. For each particular problem first of all the relevant volatiles have to be defined as they are correlated to the characteristics of the investigated foodstuff to be described. Then the suitable ionisation source and optionally a reasonable pre-separation have to be chosen. Finally the ion mobility spectrometer can be used

  investigated in recent years for a number of representative examples which are summarized in the following including new studies as well:

  K 0 can be calculated, considering the influence of ambient pressure and temperature as: with:

  63Ni-IMS. The instruments were operated at ambient pressure. They were built at ISAS and have been applied successfully for the detection of various trace gases in the range of ng/L to

  Figure 1.Scheme of an ion mobility spectrometer including pre-separation and headspace

  Figure 2.GC-UV-IMS chromatogram of beer (left) -the relevant diacetyle and 2,3-

  Figure 4.Identification of 9 bacteria and a fungi using their specific peak pattern.

  Figure 6.Traces of the initial monomers can be detected in the final polymer even after

Table 1 .

 1 -even when necessary in a miniaturised version -for fast, sensitive and selective on-line control of food quality, process development, packaging materials or storage conditions. Experimental parameters of the applied ion mobility spectrometers.

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ACKNOWLEDGEMENTS

Financial support has been given by the German Federal Ministry of Education, Science, Research and Technology and the Ministry of Science and Research of the State of Northrhine-Westfalia. The dedicated and accurate work of Luzia Seifert and Stefanie Güssgen during the experiments was a precedent condition for the successful performance of the presented investigations. The polymeric materials were generously provided by Sebastian Engell, Department of Biological and Chemical Engineering at the University of Dortmund, Germany.