Panu Veikko Rantakokko
email: panu.rantakokko@ktl.fi

Tiina Kuningas

Katri Saastamoinen

Terttu Vartiainen

Market basket study on the dietary intake of organotin compounds in Finland

Keywords: Organotin compounds, tributyltin, triphenyltin, market basket, dietary intake

properties and have been used for many different purposes. The mono-, di-and trisubstituted compounds are the most widely employed of the various OTCs. Monoand disubstituted compounds (e.g. monomethyltin (MMT), monobutyltin (MBT), dimethyltin (DMT), dibutyltin (DBT), mono-n-octyltin (MOT) and di-n-octyltin (DOT)) are used extensively as heat and light stabilizers in the production of polyvinyl chloride (PVC) polymers and as catalysts in the manufacture of polyurethane and silicone elastomers. Therefore they are present in water pipes, food packaging materials, glass coatings, polyurethane foams and many other consumer products. With respect to the trisubstituted OTCs, tributyltin (TBT) and triphenyltin (TPT) compounds have acquired a wide range of uses mostly associated with their strong biocidal activity toward aquatic organisms such as bacteria, fungi, algae, molluscs and crustaceans. [START_REF] Fromme | Occurrence of organotin compounds in house dust in Berlin (Germany)[END_REF][START_REF] Hoch | Organotin compounds in the environment --an overview[END_REF]). Most attention has been paid to TBT pollution in water, sediments, and aquatic biota resulting from its use in antifouling paints in boats and ships. TBT and TPT are extremely hazardous to some aquatic species. Several restrictions on the use of TBT have been imposed, but even after a total ban, TBT deposited in sediments will remain an important exposure source for marine biota for many years to come, because of its high sorption to suspended matter and sediment and its slow degradation to DBT, MBT, and inorganic tin under anoxic sediment conditions (European Union 2003;[START_REF] Hoch | Organotin compounds in the environment --an overview[END_REF].

The toxicity of OTCs has also been demonstrated in a variety of laboratory animals.

They have effects on the immune system, reproductive and developmental effects at 4 levels near to those that are maternally toxic. A European Food Safety Authority (EFSA) scientific panel has assessed the health risks to consumers associated with exposure to OTCs in foodstuffs. The critical toxicological endpoint for risk assessment was considered to be immunotoxicity. The panel focused on the most toxic of the OTCs: TBT, DBT and TPT primarily found in fish and fishery products. DOT was also included in the study because all these tin compounds exert their immunotoxic effects by similar modes of action and with equivalent potency. Because of their toxicological similarities, the panel considered it reasonable to establish a group Tolerable Daily Intake (TDI) for these OTCs. In the absence of specific studies on combined effects, it seemed justified to consider the immunotoxic effects of these compounds as additive.

By applying a safety factor of 100, a group TDI of 250 ng/kg body weight for the sum of TBT, DBT, TPT and DOT was established (European Food Safety Authority 2004).

Fish and fishery products are generally considered to be the most important source for human exposure to OTCs, and for this reason studies on health risks have mainly focused on seafood. Two studies on the TBT health risks from market bought seafood came to the conclusion that either the seafood was unlikely to pose any risk or else the risk was negligible. In the first of these studies, seafood samples were collected solely from US cities, and in the second study samples were collected from Asian, Australian, European, and North American cities [START_REF] Cardwell | Tributyltin in US market-bought seafood and assessment of human health risks[END_REF][START_REF] Keithly | Tributyltin in seafood from Asia, Australia, Europe, and North America: Assessment of human health risks[END_REF]. A European study on sources, consumer exposure and risks of TBT contamination in seafood also came to the conclusion that for a person to be at risk of exceeding the TDI set by WHO (250 ng/kg body weight per day) for TBT as a result of seafood consumption, the person has to be a high consumer of seafood and has to consume 5 seafood with a higher-than-average concentration of TBT [START_REF] Willemsen | Sources, consumer exposure and risks of organotin contamination in seafood. Final report of the European Commission Research Project "OT-SAFE[END_REF]). On the other hand, in another intake study, a tool called the Tolerable Average Residue Level (TARL) was developed to assess TBT exposure [START_REF] Belfroid | Organotin Levels in Seafood[END_REF]. The TARL is defined as the level in seafood that is tolerable for the average consumer with an average body weight of 60 kg. TARLs were calculated using the WHO's TDI value for TBT and average daily seafood consumption in many countries. (CHECK) In 9 of the 22 countries studied, where TBT levels in seafood were available, the TARL was exceeded in one or more samples.. OTCs have also been measured in other foodstuffs, e.g. wines [START_REF] Azenha | Butyltin Compounds in Portuguese Wines[END_REF] and drinking water [START_REF] Sadiki | A study on organotin levels in Canadian drinking water distributed through PVC pipes[END_REF]. Recently twelve types of food from four areas (south 1 and 2, north 1 and 2) in China were analysed for butyltins (MBT, DBT, and TBT). In both southern areas, seven food types contained detectable amounts of at least one butyltin, whereas in only one and three food types in the two northern areas was at least one butyltin was detected [START_REF] Qunfang | Survey of butyltin compounds in 12 types of foods collected in China[END_REF]). However, research on the full dietary intake of OTCs is limited to two Japanese studies. In the early 1990s, daily intakes of TBT and TPT from a market basket were on average 6.8 µg/person, and 3.3 µg/person, respectively. Of the different food groups, TBT and TPT were detected in only two baskets, one of seafood and one of vegetables and seaweeds; however, the seafood accounted for more than 95% of the intake [START_REF] Tsuda | Daily intakes of tributyltin and triphenyltin compounds from meals[END_REF]. In a later study conducted in 1998, daily intakes had decreased, and were 1.7 µg/person for TBT and 0.09 µg/person for TPT. In this study DBT was also measured at a daily intake of 0.45 µg/person. Of the 14 food groups assessed, only the fish and shellfish food group contributed to intakes of TBT and TPT [START_REF] Toyoda | Daily dietary intake of tributyltin, dibutyltin, triphenyltin and diphenyltin compounds according to a total diet study in a Japanese population[END_REF]. The aim of this study was to measure the daily intake of seven OTCs (MBT, DBT, TBT, MPT (monophenyltin), DPT (diphenyltin), TPT, and DOT) by the market basket method in Finland and to compare the intake of OTCs to the group TDI proposed by EFSA. This study can also serve as a model for the population from North Europe.

Materials and methods

Food consumption data and composition of the market baskets

The methodological principle of the study was to pool a well defined food group (market basket) into a single composite sample to be analysed. Each market basket contained a number of different items, such as wheat, rye, rice, oat and barley in the market basket of cereals (see Table I). The division of food items into different market baskets was adopted from the "Nutrition Report 2003" study of the Finnish National Public Health Institute (Lahti-Koski and Sirén 2004). A minimum of two packages/units of at least 100 g of each food item were purchased from different manufacturers. If possible and practical, packages/units of those domestic or foreign manufacturers were purchased, that have the largest market share in Finnish supermarkets. Fruits and berries and vegetables were an exception, as only one unit was purchased owing to the large number of items in the baskets. In some cases local producers were also used for these baskets. To keep the total number of purchased packages/units within reasonable limits and to simplify their collection, low-processed products that are frequently bought from supermarkets for daily cooking were selected. In many cases, this required some balancing, by buying for example cereal flours rather than whole grains (rice was an exception). Altogether 115 different food packages/units were bought for the thirteen (12) sugar; (13) juices and soft drinks. Alcoholic beverages were omitted from the market baskets. Canned food was only bought for tuna and herring (two packages of both items), since these food items are typically bought as canned. Table I shows the average daily consumption of the market baskets, and the main items within the baskets.

If the Balance Sheets did not contain sufficient details of the consumption of different food items in the baskets, the relevant specialized national authority was consulted. This was especially pertinent with the very important market basket of fish and crustaceans, for which the most recent and accurate consumption figures from the years 2001 -2003 were obtained from the Finnish Game and Fisheries Research Institute.

Preparation of food samples for analysis of organotin compounds

Common household conditions were followed as far as possible in the sample preparation, e.g. fruits and vegetables were washed/peeled/sliced/grated, inedible parts of the fish were removed etc. High quality stainless steel kitchen tools were used throughout and washed after every sample. Next the edible parts of the package/unit from the same manufacturer were mixed (e.g. small fish, small vegetables etc), unless they were homogenous when bought (e.g. liquids, flours, fats etc). Subsequently, identical masses of separate food samples (e.g. apples) from different manufacturers for each food item (apple) were weighed and homogenized with a BÜCHI Mixer B-400.

Finally, all separate food items inside the market basket were mixed and thoroughly homogenized in proportion to their consumption. The proportion of those minor items in a basket that were not bought or were classified as "others" in Balance Sheets, were distributed to the purchased items according to their share inside the collected basket.

Baskets ( 1) -( 8), and (10) were freeze-dried and stored at -20 °C before analysis.

Baskets ( 9) and ( 11) -( 13) were only frozen at -20 °C before analysis.

Analysis of organotin compounds by GC/MS

The compounds to be analysed were MBT, DBT, TBT, MPT, DPT, TPT, and DOT. All weights and concentrations of OTCs are expressed as organotin cations. Individual, pure model OT compounds were bought either from Dr. Ehrenstorfer or from Acros. The purity of model compounds was checked before use. Perdeuterated analogs of MBT, DBT, TBT, MPT, DPT, and TPT were used as internal standards for the respective native 1 H-compounds. These were purchased from the Swiss Federal Institute for Environmental Science and Technology, where they had been synthesized [START_REF] Arnold | Determination of organotin compounds in water, sediments, and sewage sludge using perdeuterated internal standards, accelerated solvent extraction, and large-volumeinjection GC/MS[END_REF]. Perdeuterated DPT was used as an internal standard for DOT. Sample analysis was performed according to the method developed by Ikonomou et al. with slight modifications [START_REF] Ikonomou | Gas chromatography-highresolution mass spectrometry based method for the simultaneous determination of nine organotin compounds in water, sediment and tissue[END_REF]. Baskets (1) -( 11) were analysed according to 8), and (10) (the amount of fresh sample taken for analysis thus varies according to the moisture content). For baskets ( 9) and (11) that were not freeze dried, the sample amount taken for analysis was 2.0 g and 0.1 g of the fresh material respectively. Baskets ( 12) and ( 13) were analysed according to Ikonomou's procedure for water samples, in which 1 g and 20 g of sample materials were dissolved in/diluted to 200 ml with MQ-water, respectively.

A few minor modifications were made to the Ikonomou procedures for tissue and water samples. In the tissue method, NaCl (1 g) used to aid in the extraction was added in a solid form rather than as 30% solution, and the 2 ml of 1 M sodium acetate acetic buffer acid was not added, since the addition of 2 ml of glacial acetic acid undoes the effect of buffer addition. In the water method, sugar/juice samples dissolved in/diluted to 200 ml of MQ water were extracted only once overnight with 20 ml of hexane. In the water method, the derivatization reagent, sodium tetraethylborate, was applied as 2% solution instead of 1%. With both methods, only 3 cm of activated (200ºC overnight, 90 min at 300ºC, and 200 ºC until use) basic alumina was loaded in the Pasteur pipette, instead of 6 cm. The samples were eluted with 10 ml of 4% diethylether in hexane instead of 20%.

Thus more dilute diethylether-hexane solution was sufficient to elute all of the derivatized OTCs from the column and eluted fewer matrix impurities. Tetrabutyltin was used as the syringe standard instead of tetrapentyltin.

The limit of detection (LOD) and the limit of quantitation (LOQ) were calculated using MassLynx software as the concentrations corresponding to 3 times and 8 times the The LODs are recorded in Table III for each compound with a concentration below the LOD. If the concentration was between LOD and LOQ, then the LOQ is added for these compounds. If the concentration was above the LOQ, then the measured concentration is recorded in Table III. The LODs and the LOQs vary considerably between different market baskets mainly because of the different amounts of the fresh sample taken for analysis.

The three level calibration for the tissue method was performed by spiking 0.25 g of freeze dried pike tissue with known amounts of OTCs and treating it in exactly the same way as the true samples. The spiked pike tissue had previously been confirmed to be free of OTCs. The three level calibration for the water method was performed by spiking 200 ml of ultrapure water with OTCs and treating it in the same way as the true samples.

The GC/MS analysis was performed using a HP 6890 Gas Chromatograph connected to a Waters Autospec Ultima high resolution mass spectrometer operating in the selected ion monitoring mode. The two most intensive fragment ions of each ethylated OTC were monitored. Fragment ions are given in Table II. The column used was an HP-1 capillary column (12 m, 0.20 mm I.D., 0.33 µm film). Each sample was extracted in duplicate and the mean was reported. The relative standard deviation of the parallel market basket samples with concentrations over the LOQ had to be less than 20%. The laboratory reagent blank samples were treated and analysed using the same method as the actual samples, one blank for each series of samples. The blank result was subtracted from the sample measurements. All glassware in contact with samples was washed and soaked in 1 M HNO 3 overnight before use. Inhouse-made spiked and freeze dried control fish samples and spiked control water samples were analysed in the sample series for the respective method. The results of the in-house-made control sample had to be within the accepted limits (spiked value ±2*standard deviation of the results in the control chart) for the entire sample series to be acceptable. In the same series in which market basket samples were analysed using the tissue method, the certified reference material CRM 477, freeze dried mussel tissue, was also analysed for organic tin compound. CRM 477 is produced under the Standards, Measurement and Testing Programme of the European Commission, and has certified concentrations for MBT, DBT, and TBT, and indicative concentrations for MPT, DPT, and TPT, respectively [START_REF] Pellegrino | Comparison of twelve selected extraction methods for the determination of butyl-and phenyltin compounds in mussel samples[END_REF]. For butyltins, the recoveries were 125%, 85%, and 108% of the certified values, and for phenyltins the recoveries were 151%, 94%, and 96% of the indicative values, respectively. To confirm the identity of the OTC peak, peak retention times had to match within ±3s of those of calibration samples, and the area ratio of primary and secondary ions in the MS-chromatograms had to be within 20% of the theoretical value. The mean LODs varied from 2.9 pg/g fresh weight (fw) (juices and soft drinks) to 0.31 ng/g fw (fats and oils) and the mean LOQs varied from 7.8 pg/g fw (juices and soft drinks) to 0.82 ng/g fw (fats and oils). These values differ III only for those results where the concentration was either less than the LOD or LOQ, respectively. The Chemistry Laboratory of the National Public Health Institute of Finland is an accredited laboratory according to ISO17025 . The scope of accreditation includes organic tin compounds from tissue, sediment, and water samples.

Intake calculations

In the intake calculations, the average daily consumption of the food basket was multiplied by the corresponding concentration of OTCs. When the concentration of an OTC in a basket was less than the limit of quantification, the result was reported as zero. Daily intakes (ng cation/day) for OTCs were calculated on a fresh weight basis as the sum of individual baskets. When calculating the daily intakes per kg body weight (ng cation/kg bw), the average weight (77 kg) of the adult population (25 -74 years) obtained from national FINRISK 2002 study was used (Laatikainen et al. 2003).

Results and discussion

Table III presents the concentrations of OTCs in the different market baskets. Of the thirteen baskets, four contained OTCs at levels higher than the limit of quantitation. As in the Japanese intake studies, in this case, too, TBT and TPT were exclusively present in the fish basket [START_REF] Toyoda | Daily dietary intake of tributyltin, dibutyltin, triphenyltin and diphenyltin compounds according to a total diet study in a Japanese population[END_REF][START_REF] Tsuda | Daily intakes of tributyltin and triphenyltin compounds from meals[END_REF]. The fact that the number of individual fish and crustacean samples pooled to the fish basket was quite limited in this study, consisting of only 22 different packages/units, must be taken into consideration.

The concentrations of TBT and TPT in the fish basket were low, in fact similar to those measured for TBT in US market bought seafood shortly after the peak of TBT usage in [START_REF] Cardwell | Tributyltin in US market-bought seafood and assessment of human health risks[END_REF]). In the current study, there was a large contribution of imported farmed salmon (from Norway), tuna and saithe in the fish basket (see Table I). Imported Norwegian salmon bought in Sweden and France and imported tuna bought in Germany were analysed in the European Commission research project "Sources, consumer exposure and risks of organotin contamination in seafood" [START_REF] Willemsen | Sources, consumer exposure and risks of organotin contamination in seafood. Final report of the European Commission Research Project "OT-SAFE[END_REF]). In salmon bought in Sweden and France, only TBT was found, with the concentrations varying from less than the limit of quantification (4 ng/g fw) to 11 ng/g fw. The concentrations of TBT in tuna bought in Germany were also very low, only a few ng/g fw. These low concentrations in imported salmon and tuna in the European Commission project are in good agreement with the concentrations in the fish basket measured in the current study. In another European Commission project, a report on a Scientific Co-operation (SCOOP) task, eight EU Member States provided data available at the national level on the occurrence of butyltins and phenyltins in different seafood species. The spread of the concentrations in different species in the different countries was large; in some countries, levels were on average an order of magnitude or more higher than those measured in this Finnish seafood market basket (European Commission 2003). Currently very little is known about the levels of OTCs in individual seafood species originating from Finnish water areas. In particular, the OTC concentrations in the Finnish background areas have still not been measured. In a study carried out in the vicinity of twelve cities in Finland, the sum of OTCs (mostly trisubstituted) in pooled pike muscle samples was 1 -33 ng cation/g fw in inland fish and 8 -141 ng cation/g fw in coastal fish [START_REF] Mannio | Screening of organotin compounds in sediments and fish in Finland[END_REF]. However, in those Finnish costal areas with a high density of shipping, sums of OTCs up to several hundred ng/g fw in individual pike perch and burbot samples have been measured (Mannio et In addition to fish, low concentrations of MBT and/or DBT were also detected in the vegetable, potato, and fruit and berries baskets. These compounds have also been observed in similar food types by other research groups. Low concentrations (1 -2 ng cation/g) of butyltins have been detected in French lettuces grown on OTCcontaminated sludged soil [START_REF] Marcic | Pressurised solvent extraction for organotin speciation in vegetable matrices[END_REF]. Higher concentrations (10 -200 ng cation/g) of butyltins were detected in southern areas of China in beans, vegetables, fruits, eggs, milk, sugar, meat, potatoes, and beverages. In the northern Chinese regions these compounds were not detected, but this may be partly attributable to the higher limits of detection, which were from 10 (MBT) -17 (TBT) ng cation/g [START_REF] Qunfang | Survey of butyltin compounds in 12 types of foods collected in China[END_REF]). There are a number of possible sources of MBT and DBT in these food commodities. These include the use of sludge as fertilizer, some pesticides that may contain OTCs, and butyltin stabilized plastic products which may have come into contact with these food products. It has to be emphasized that the market baskets collected in this study did not contain highly processed food, but mainly raw materials intended for further cooking and processing. It is possible that more highly processed foods come more into contact with plastics which may contain MBT and DBT. However, to simplify the sample collection, such foods were omitted from this study.

Table IV presents the daily intakes for OTCs (ng cation/person). Results less than the limit of quantitation were taken as zero. This approach was adopted because only ten marginally higher than the LOD. Of the total intake, 57% is attributable to MBT. This is due to both the higher consumption of those baskets containing lower concentrations of MBT (potato, vegetables, and fruits) and higher concentrations in the less consumed fish, which contribute only 23% to the intake of MBT. The situation is similar for DBT, for which vegetables and fruits dominate, while fish contributes only 21%. The intakes of TBT, DPT and TPT originate solely from fish. For the sum of OTCs of health interest (DBT, TBT, TPT, and DOT), fish contributes 81% to the intake, with the remainder attributable to DBT in vegetables (9%) and fruits (10%). When compared to a Japanese market basket study [START_REF] Toyoda | Daily dietary intake of tributyltin, dibutyltin, triphenyltin and diphenyltin compounds according to a total diet study in a Japanese population[END_REF], the daily intakes in this study (in ng/person) were 10% for DBT, 6% for TBT, and 49% for TPT of those in Japan.

The total daily intake in ng cation/kg bw is presented in the bottom row of Table IV.

The sum of DBT, TBT, TPT, and DOT, is 2.47 ng cation/kg bw, which is only 1.0% of the group TDI of 250 ng/kg body weight established by the EFSA (European Food Safety Authority 2004). If in Table III the results <LOQ had been taken as LOQ and the results <LOD as 0, the recalculated total daily intake in the bottom row of Table IV would only have risen to 3.81 ng cation/kg bw. Furthermore, if also the results <LOD had been taken as LOD, then the daily intake would still only amount to 7.69 ng cation/kg bw. However, especially this latter result bears no quantitative accuracy, and represents only a theoretical maximum intake. Furthermore, even these values are only 1.5% and 3.1% of the EFSA group TDI, respectively. These very low intakes, irrespective of the way they were calculated, mean that the average Finnish consumer need not be alarmed about the intake of organic tin compounds from typical raw 16 materials in their daily meals. However, if more processed foods had been selected for this study, intake of monobutyltin and dibutyltin might have increased slightly, since the foods could have been in greater contact with plastics possibly containing these compounds. More importantly, for those groups of the population who consume larger than average amounts of fish from contaminated or background areas, the daily intakes could be significantly higher. The possible OTC intake from fish from contaminated and background areas of Finland is currently under investigation.

  OTCs) are a large class of compounds with widely varying

  this study. The food and food group consumption data over the years 2000 -2002 used in the composition of the market baskets and in the intake calculations were taken from the report "Balance Sheet for Food Commodities 2001 and 2002" (preliminary) (Finnish Ministry of Agriculture and Forestry 2003). The annual calculations in the Balance Sheets are based on the production of different foodstuffs, changes in stockpiles, export and import figures and domestic consumption. Sample collection was carried out during the period May -August 2005. Seasonal products were collected in the peak season. Samples were collected from local supermarkets and market places. Altogether, thirteen individual market baskets were created: (1) cereals; (2) potato; (3) vegetables; (4) peas and nuts; (5) fruits and berries; (6) meat; (7) eggs; (8) fish and crustaceans; (9) milk and sour milk; (10) milk products; (11) fats and oils;

  25 g of the freeze dried material for the baskets (1) -(

  the channels of the monitored ions, respectively. LODs and LOQs were calculated separately for each compound in each sample on a fresh weight basis.

  compound and matrices, and therefore LODs or LOQs are presented in Table

  paints in the US

  [START_REF] Vatanen | Monitoring of the fish and waterways of the Vuosaari harbour project[END_REF]. A project intended to study the OTC concentrations from the most commonly eaten domestic fish species from background and contaminated areas is currently underway.

Table 1 .

 1 Market baskets, their average daily consumption in g/day (percentage of total daily consumption), and their main items (percentage in the composed basket, minor items not shown for every basket)

	Market basket	Consumption	Main items (%)
		g/day (%)
	(1) Cereals	207 (9.7)	Wheat (65)
			Rye (21)
			Rice (8)
	(2)Potato	174 (8.1)	Potato (100)
	(3)Vegetables	177 (8.2)	Tomato (24)
			Cucumber (17)
			Carrot (18)
			Onion (11)
	(4) Peas and nuts	5.2 (0.2)	Peas (63)
			Nuts (37)
	(5) Fruits and berries	244 (11.4)	Banana (28)
			Apple (28)
			Orange (16)
			Mandarin (13)
			Lingonberry (6)
	(6) Meat	190 (8.9)	Pork (50)
			Beef (28)
			Poultry (22)
	(7) Eggs	26.8(1.25)	Eggs (100)
	(8) Fish and crustaceans	39.5 (1.8)	Salmon and rainbow trout (39)
			Tuna and saithe (20)

1 Sour whole milk or curdled milk is a typical Finnish semisolid milk product that is distinctive and different from ordinary liquid sour milk.

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Table II .

 II Ions monitored in the GC-HRMS analysis of the organic tin compounds. Perdeuterated analogs of organic tin compounds are depicted with "OTC-dx", where x is the number of deuterium atoms in the internal standard molecule.

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	Compound Primary ion	Secondary ion	Compound	Primary ion	Secondary ion
	MBT	233.0504	235.0510	MBT-d9	242.1069	244.1075
	DBT	233.0504	235.0510	DBT-d18	242.1069	244.1075
	TBT	289.1131	291.1137	TBT-d27	316.2826	318.2831
	MPT	253.0192	255.0197	MPT-d5	258.0505	260.0511
	DPT	301.0193	303.0198	DPT-d10	311.0820	313.0826
	TPT	349.0193	351.0199	TPT-d15	364.1135	366.1140
	DOT	373.2072	375.2077	TeBT	289.1131	291.1137

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Table 3 .

 3 Concentrations (ng cation/g fresh weight) of organic tin compounds in different market baskets. Results less than the LOQ are depicted with "< x.x", and results less than the LOD are depicted with nd (x.x), where x.x is either the LOQ or the LOD.

			Food Additives and Contaminants
	Market basket	MBT	DBT	TBT	MPT	DPT	TPT	DOT
	(1) Cereals	nd (0.053)	nd (0.086)	nd (0.16)	nd (0.55)	nd (0.086)	nd (0.032)	nd (0.16)
	(2) Potato	0.57	< 0.054	nd (0.031)	nd (0.11)	nd (0.012)	nd (0.008)	nd (0.042)
	(3) Vegetables	0.35	0.10	< 0.030	nd (0.031)	nd (0.005)	nd (0.002)	< 0.061
	(4) Peas and nuts	< 0.13	< 0.19	nd (0.20)	nd (0.22)	nd (0.049)	nd (0.022)	< 0.64
	(5) Fruits and berries	0.17	0.080	nd (0.024)	nd (0.049)	nd (0.005)	nd (0.007)	nd (0.019)
	(6) Meat	nd (0.021)	nd (0.030)	nd (0.060)	nd (0.14)	nd (0.023)	nd (0.011)	nd (0.075)
	(7) Eggs	nd (0.041)	nd (0.075)	nd (0.13)	nd (0.17)	nd (0.044)	nd (0.041)	nd (0.22)
	(8) Fish and crustaceans	1.52	0.25	2.53	nd (0.076)	0.14	1.11	nd (0.091)
	(9) Milk and sour milk	nd (0.011)	< 0.054	nd (0.024)	nd (0.045)	nd (0.015)	nd (0.007)	nd (0.059)
	(10) Milk products	< 0.059	<0.10	nd (0.052)	nd (0.10)	nd (0.014)	nd (0.010)	< 0.20
	(11) Fats and oils	nd (0.15)	nd (0.34)	nd (0.23)	nd (0.50)	nd (0.15)	nd (0.35)	nd (0.43)
	(12) Sugar	nd (0.033)	nd (0.026)	nd (0.043)	nd (0.20)	nd (0.009)	nd (0.006)	nd (0.016)
	(13) Juices and soft drinks nd (0.0014) nd (0.0014) nd (0.0017) nd (0.013)	nd (0.0011)	nd (0.0008)	nd (0.0010)

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Table 4 .

 4 Daily intakes of organic tin compounds (ng cation/person) from different market baskets. Only those compounds that were detected in at least one basket, and only those baskets that contained at least one compound at a higher level than the LOQ, are tabulated. Weight of the average person in the intake calculations is 77 kg.

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	Market basket	MBT	DBT	TBT	DPT	TPT	Σ OTCs	Σ DBT, TBT, TPT, DOT
	(2) Potato	98.8					98.8
	(3) Vegetables	61.4	17.1				78.5	17.1
	(5) Fruits and berries	40.7	19.5				60.2	19.5
	(8) Fish and crustaceans	60.1	9.9	99.9	5.5	43.9	219	154
	Total intake	261	46.6	99.9	5.5	43.9	457	190
	Total intake (ng cation/kg body	3.39	0.61	1.30	0.07	0.57	5.93	2.47
	weight) a

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José Fernandes

Determination of acrylamide in coffee and coffee products by GC-MS 1 using an improved SPE clean-up 2 3

Keywords: Food Science Cunha, Sara; FFUP, Food Science Contaminants: Acrylamide, Process contaminants -acrylamide Food Types: Coffee, Processed foods Coffee, SPE clean up, acrylamide, GC-MS 21 22

published or not. The documents may come

Determination of acrylamide in coffee and coffee products by GC-MS using an improved SPE clean-up

The occurrence of acrylamide (AA) in foodstuffs particularly in certain baked and fried 25 products such as potato chips and French fries was first reported by [START_REF] Tareke | Analysis of 428 Acrylamide, a Carcinogen Formed in Heated Foodstuffs[END_REF]. 26

AA is now known to be formed by the Maillard reaction during industrial food 27 processing, retail, catering and home food preparation ( [START_REF] Mottram | Food chemistry: Acrylamide is formed 395 in the Maillard reaction[END_REF]Stadler et 28 al., 2002;[START_REF] Becalski | Acrylamide in Foods: Occurrence, 366 Sources, and Modeling[END_REF].. The foodstuffs that contribute most to AA exposure 29 vary depending upon the population's nutritional habits and the way the food is 30 prepared and processed [START_REF] Dybing | Human exposure and internal dose assessments of acrylamide in 385 food[END_REF]. Generally, the most important sources of 31 AA appear to be potato products (potato chips, French fries, and potato snacks), cereals 32 (breakfast cereals, roasted cereals), baked goods (bread, cookies, biscuits), and brewed 33 coffee. 34 35 Recent reports showed that coffee is among the highest contributors to the AA intake in 36 some countries in Europe. In Sweden the main dietary intake of AA from coffee was 37 estimated to be 12 µg/day, representing ~39% of the total dietary intake of 31 µg/day 38 [START_REF] Svensson | 425 Dietary intake of acrylamide in Sweden[END_REF]. These results were based on ready-to-drink coffee samples that 39 contained around 25 µg kg -1 AA. [START_REF] Dybing | Risk assessment of acrylamide in foods[END_REF] reported that a mean intake 40 of AA from coffee was estimated to contribute to 28% of the 38 µg/day for males and 41 29% of the 29 µg/day for females in Norway. Their results were based on a study 42 (Norwegian Food Control Authority, 2002) that reported concentrations of AA in filter 43 coffee of 25 µg kg -1 and 10 µg kg -1 in instant coffee. A Swiss total diet study of AA in 44 the diet of 14 men and 13 women showed that coffee contributed 36% of the total intake 45 [START_REF] Brunner | Assessment of acrylamide intake by duplicate diet study. 375[END_REF]. [START_REF] Granby | Analysis of acrylamide in coffee and dietary exposure to 389 acrylamide from coffee[END_REF] found in their study 2-16 µg L -1 of AA 46 in brewed coffee and 10 µg L -1 AA in instant coffee. They concluded that the AA intake 47 from coffee consumption in Denmark was 10 µg/day for males and 9 µg/day for 48 Assuming that the total exposure of Danish consumers is comparable to those 49 of Sweden and Norway, the mean exposure to AA from coffee contributes to 20% of 50 the total AA exposure. In Netherlands, [START_REF] Boon | Calculations of dietary exposure to acrylamide[END_REF] accounted an AA 51 concentration in coffee in the range 4 -45 µg kg -1 that contributes with 13% of the total 52 AA intake in adults. The US Food and Drug Administration (FDA) reported levels of 53 AA in brewed coffee ranging from 6 to 16 µg L -1 [START_REF] Andrzejewski | Analysis of Coffee for the 363 presence of Acrylamide by LC-MS/MS[END_REF]. 54 55In Portugal, coffee is highly consumed as "espresso" which has a peculiar brewing 56 technique: a small amount of hot water (± 30 mL) is percolated in a very short time at 57 high pressure through a layer of ground roasted coffee (± 6-7 g), the coffee cake, to 58 produce efficiently a very concentrated brew (≈200 g of ground coffee per litre) 59 comparing with the 20 -60 g of ground coffee per litre related in the above mentioned 60 countries (Nunes & Coimbra, 1987). 61 62 Several methods to determine AA in food have been developed and they are based on 63 either GC-MS with derivatisation with bromine [START_REF] Andrawes | Chemistry of acrylamide bromination for 360 trace analysis by Gas Chromatography and Gas Chromatography-Mass 361 Spectrometry[END_REF]Nemoto et al., 64 2002;[START_REF] Ono | Analysis of acrylamide by LC-MS/MS and GC-MS in processed Japanese 409 foods[END_REF][START_REF] Pittet | Trace level determination of acrylamide in 411 cereal-based foods by gas chromatography-mass spectrometry[END_REF] and without derivatisation (Jezussek & 65 Schieberle, 2003;[START_REF] Biedermann | Two 368 GC-MS Methods for the Analysis of Acrylamide in Foodstuffs[END_REF][START_REF] Amrein | Acrylamide in 357 Roasted Almonds and Hazelnuts[END_REF] or LC-MS with 66 derivatisation with mercaptobenzoic acid [START_REF] Jezussek | A New LC/MS-Method for the Quantification of 391 Acrylamide Based on a Stable Isolute Dilution Assay and Derivatization with 2-392 Mercaptobenzoic Acid. Comparison with Two GC/MS Methods[END_REF] and without 67 derivatisation (Roach et al, 2003;[START_REF] Becalski | Acrylamide in Foods: Occurrence, 366 Sources, and Modeling[END_REF][START_REF] Ahn | Verification of the 354 findings of acrylamide in heated foods[END_REF]Rosén et al., 68 2002;[START_REF] Şenyuva | Study of acrylamide in coffee using an improved liquid 416 chromatography mass spectrometry method: Investigation of colour changes and 417 acrylamide formation in coffee during roasting[END_REF][START_REF] Şenyuva | Interference-free determination of acrylamide in potato 420 and cereal-based foods by a laboratory validated liquid chromatography-mass 421 spectrometry method[END_REF][START_REF] Delatour | Improved Sample 379 Preparation to Determine Acrylamide in Difficult Matrixes Such as Chocolate 380 Powder, Cocoa, and Coffee by Liquid Chromatography Tandem Mass 381 Spectrometry[END_REF]Andrzejewski et al., 69 2004;[START_REF] Yusà | Determination of acrylamide in 431 foods by pressurized fluid extraction and liquid chromatography-tandem mass 432 spectrometry used for a survey of Spanish cereal-based foods[END_REF]. Independently of the chromatographic technique adopted, the 70 success of the methods is very dependent of the extraction and clean-up steps. The most 71 used procedures usually fail when dealing with coffee products, due to their inability to 72 4 avoid the presence of interferences that co-elute with the analyte thus preventing its 73 correct quantification [START_REF] Delatour | Improved Sample 379 Preparation to Determine Acrylamide in Difficult Matrixes Such as Chocolate 380 Powder, Cocoa, and Coffee by Liquid Chromatography Tandem Mass 381 Spectrometry[END_REF]. 74

75

The methods developed so far to determine AA in coffee and coffee products, are based 76 mainly in LC-MS/MS with variations in the clean-up conditions employed. Roach et al. 77 (2003) used a combination of Oasis HLB and Bond Elut-Accucat SPE cartridges before 78 the LC analysis but found coffee to be a troublesome matrix. The method still presents 79 some problems regarding contaminations, as it requires frequently reconditioning the 80 HPLC column [START_REF] Andrzejewski | Analysis of Coffee for the 363 presence of Acrylamide by LC-MS/MS[END_REF]. [START_REF] Granby | Analysis of acrylamide in coffee and dietary exposure to 389 acrylamide from coffee[END_REF] performed a single-81 step clean-up on 300 mg Isolute Multimode SPE columns of five hundreds microlitres 82 of sample to make the clean-up more efficient and eliminate almost completely the 83 colour from the coffee. However, they referred that the loading of the SPE cartridge 84 with the sample exceeding 0.5 mL resulted in a suppression of the MS response. 85 [START_REF] Şenyuva | Study of acrylamide in coffee using an improved liquid 416 chromatography mass spectrometry method: Investigation of colour changes and 417 acrylamide formation in coffee during roasting[END_REF] extracted AA from ground coffee using methanol instead 86 of water and the clean-up was made with Oasis HLB SPE cartridges prior to LC 87 analysis. [START_REF] Delatour | Improved Sample 379 Preparation to Determine Acrylamide in Difficult Matrixes Such as Chocolate 380 Powder, Cocoa, and Coffee by Liquid Chromatography Tandem Mass 381 Spectrometry[END_REF] used 500 mg of Isolute Multimode SPE cartridges to 88 clean-up 2 mL of samples but realized that a single step was not effective and developed 89 a combination of liquid-liquid and solid phase extraction methods. Ono et al. (2003) 90 determined acrylamide in several food items including roasted coffee by GC-MS. 91 However no reference was made about the efficiency of their method to coffee analysis. 92

The set of methods developed so far demonstrate a lack in the methodologies employing 93 GC-MS with the objective to specifically determine AA in coffee samples. 94 The objective of this work was to develop a method to determine AA in several coffee 96 products using GC-MS as analytical technique. The method was successfully applied to 97 determine acrylamide in ready-to-drink products and ground coffee, that was analysed 98 as "espresso". 99 100 Experimental 101

Sampling and coffee preparation 102

Eighteen samples of roasted coffee beans, five samples of instant coffee, two of them 103 decaffeinated, two samples of coffee blends with cereals and one soluble "cappuccino" 104 were collected from several supermarkets and stored at room temperature. The most 105 common brands were selected. The roasted coffee beans were ground in a coffee grinder 106 incorporated in a Philips coffee machine, model Espresso Professional selected for a 107 fine grain size typical for espresso coffee preparation. Each espresso was prepared in 108 duplicate according to the typical conditions used in Portugal for an "espresso" 109 preparation, i.e.: 7 g of ground coffee extracted with hot water during 30 ± 5 s (about 30 110 to 35 mL of water) at an inlet pressure of 9 ± 2 bar. Two grams of instant coffee, coffee 111 blends and cappuccino were dissolved in 10 mL of deionised water. 112 mL, filter pore size 10 µm (Symta, Madrid, Spain). The n-hexane and ethyl-acetate were 121 of pesticide residue analysis grade and methanol and acetonitrile were of ultrapure 122 grade, all from Fluka. The potassium bromide for IR spectroscopy grade and the 123 bromine analytical grade were from Merck (Darmstad, Germany). Sodium chloride 124 analytical grade was from J.T. Baker (Deventer, Holland). Potassium hexacyanoferrate 125 (II) trihydrate, zinc sulphate heptahydrate, acetic acid (glacial) and absolute ethanol 126 were analytical grade obtained from Panreac (Barcelone, Spain). Hydrobromic acid 127 48% and sodium tiosulphate volumetric solution 1 mol L -1 were from Riedel-de Häen 128 (Seelze, Germany). 129 130

Standards and reagents 131

A stock solution of AA (2 g L -1 ) was prepared by dissolving the compound in 132 acetonitrile, and appropriately diluted to prepare a working standard solution at 4 mg L - 133 1 . A stock solution of internal standard, 13 C 3 -AA, was prepared by the same way with 134 the difference that the working standard solution was made at 40 mg L -1 . All stock and 135 working solutions were stored at 4ºC. Saturated bromine water solution and Carrez 136 reagents were prepared according to [START_REF] Pittet | Trace level determination of acrylamide in 411 cereal-based foods by gas chromatography-mass spectrometry[END_REF]. 137 138

Sample preparation 139

To 10 mL of "espresso" coffee or 2 g of other coffee product dissolved in 10 mL of 140 water, 15 mL of absolute ethanol were added and the solution shaken for ~ 1 min. 0.5 141 µg of IS was added and the solution was shaken again and then kept at -20ºC during 15 142 minutes. It was centrifuged at 15000 g (5 minutes, 4ºC) and the resulting solution was 143 To the collected extract 1 g of calcinated KBr was added. The solution was then 169 acidified with HBr until pH 1-3 (100-150 µL) and 2 mL of saturated bromine solution 170 were added. The solution was allowed to stand in an ice bath and kept from the light at 171 least during 1 h. The excess bromine in the solution was decomposed by the addition of 172 1 mol L -1 sodium tiossulphate until the yellow colour disappeared (50-150 µL). The 173 extract was saturated with 4 g of NaCl and extracted twice with 10 mL and 5 mL 174 portions of ethyl acetate/n-hexane 4:1 (v/v). The volume was reduced to 3 mL under a 175 stream of nitrogen and a small quantity of anhydrous Na 2 SO 4 was added. Finally, the 176 solution was centrifuged at 3000 rpm during 3 min, the upper layer was transferred to 177 another vial and evaporated to 0.5 mL under a gentle stream of nitrogen. The solutions 178 were then injected in the gas-chromatograph. 179 180

Calibration standard 181

Aliquots of the working standard AA solution (equivalent to 0 to 3 µg of AA 182 corresponding to 0 to 300 µg L -1 in the samples) were taken into 50 mL centrifugal 183 tubes and 0.5 µg of internal standard was added to each tube and made up to 10 mL 184 with water. These solutions were treated according to the overall procedure (clean-up 185 included) described for the samples. 186 As reported by other authors [START_REF] Delatour | Improved Sample 379 Preparation to Determine Acrylamide in Difficult Matrixes Such as Chocolate 380 Powder, Cocoa, and Coffee by Liquid Chromatography Tandem Mass 381 Spectrometry[END_REF][START_REF] Andrzejewski | Analysis of Coffee for the 363 presence of Acrylamide by LC-MS/MS[END_REF] that used 227 similar methods, some drawback occurred when difficult matrices were analysed, 228 namely coffee products. Taking this into account some modifications were introduced 229 in our previous developed method. 230 231 The addition of absolute ethanol to coffee samples intended to control the great quantity 232 of foam that was constantly being formed during almost all steps of the sample 233 preparation and resulting in severe losses of the analyte and contamination of the used 234 instruments. The problem was particularly serious during the evaporation step on the 235 rotary evaporator because the foam was always being projected. The adopted solution 236 was an adaptation from the work of Nunes and Coimbra (1998), where the precipitation 237 of polymeric polysaccharides, responsible for the "foam stability" in espresso coffees, 238 was achieved by adding 55 and 75 % ethanol solution at 4ºC during one hour. After 239 The SPE clean-up was a challenge in the sense that it was difficult to achieve the correct 244 ratio between the two used sorbents. The use of a single purification step with C 18 245 revealed to be useful to eliminate the coffee colour but it was not effective to remove 246 other compounds that overlapped the 2,3-DBPA peak no matter what quantity of 247 sorbent was used. When using Isolute Multimode cartridges (0.5 or 1 g) it was verified 248 that the sorbent layer was completely saturated with very small quantities of coffee 249 samples (0.5-1.0 ml). Because it was difficult to achieve such small sample volumes 250 during the evaporation step, the rejection of an important portion of the extract was 251 necessary, with the inherent loss of sensitivity of the overall method. The solution found 252 was to increase the quantity of sorbent material instead of decreasing the sample 253 volume. The use of higher quantities of Isolute Multimode (3g) resulted in an increase 254 of the analytical signal but it was not effective to eliminate the coloured compounds and 255 suppression of the MS response was observed. The addition of both sorbents in a single 256 step was the solution found to surpass both problems. It was found that a ratio of 1:3 of 257 C 18 : Isolute was ideal to eliminate the most relevant contaminants present in "espresso" 258 coffee. 259 260 Soluble coffees, revealed to be a troublesome matrix due to its high percentage of 261 concentrated polysaccharides. The addition of ethanol was not enough to eliminate the 262 majority of the polysaccharides that increased the formation of foam and the 1:3 ratio 263

Method application 315

The method developed was applied to 26 coffee samples (roasted beans, instant and 316 coffee blends). Samples were randomly selected from supermarkets, and therefore may 317 not be representative of coffee supply. Nevertheless they represent a general guide to 318 AA concentration in a selected segment of coffee supply. The results obtained are 319 presented in Table I. Accordingly AA levels ranged from 11.4 to 36.2 µg L -1 in ground 320 roasted coffee analysed as "espresso", 47.4 to 95.2 µg L -1 for instant coffee and 200.8 to 321 229.4 µg L -1 for coffee blends with cereals. Soluble "cappuccino" contained 6.4 µg L -1 . 322

Considering that 6 g of ground coffee extracted with ~30 mL water are used to prepare 323 an "espresso", the concentration of AA per cup is therefore 0.32 to 1.46 µg/30 mL. To 324 prepare a cup of instant coffee the usual measure is 2 g per individual portion making 325 0.47 to 0.95 µg per 30 mL. Considering that coffee blends with cereals were prepared as 326 soluble coffee (2 g/ 30 mL cup) the concentration of AA per cup is therefore 2.01 to 327 2.09 µg per 30 mL cup. "Cappuccino" as analysed is a soluble mixture of coffee, milk 328 powder, cocoa and sugar and each individual dose correspond to 14 g, resulting in an 329 AA concentration of 0.45 µg per 30 mL cup. These results highlight the fact that the 330 addition of cereal products to coffee increased the amount of AA. This in agreement 331 with studies [START_REF] Yusà | Determination of acrylamide in 431 foods by pressurized fluid extraction and liquid chromatography-tandem mass 432 spectrometry used for a survey of Spanish cereal-based foods[END_REF] To date, LC/MS has been the most widely used analytical approach to assay AA in 338 coffee. GC/MS methods usually suffer from the presence of co-extractives which 339 rapidly contaminate the chromatographic system, degrading the respective performance 340 and avoiding the correct quantification of AA. This paper describes a GC/MS method 341 based on an improved sample preparation procedure that enables the correct 342 quantification of AA in coffee extracts without the problems generally associated with 343 those kind of samples. The high levels of sensitivity, and reproducibility achieved 344 recommend it when dealing with this type of food matrices. The method was applied to 345 several coffee products and the quantity of AA was found to be dependent on the coffee 346 processing and preparation, namely the addition of cereals. 347

  Determination of acrylamide in coffee and coffee products by GC-MS using an improved SPE clean-up

  ) 99% purity grade was acquired from Aldrich (Steinheim, Germany). 115 Acrylamide 1,2,3-C13 ( 13 C 3 -AA) 99% in methanol was purchased from Cambridge 116 Isotope Laboratories (Andover, MA, USA). The solid-phase extraction (SPE) columns, 117 Isolute Multimode, 3 g custom made, were from International Sorbent Technology 118 (Hengoed, Mid Glamorgan, UK). The preparative C 18 sorbent 125 Å, 55-105 µm was 119 Milford, USA). The filters were polyethylene single fritted reservoir 25 120

  to a centrifugal flask. Afterwards, it was acidified with acetic acid until pH 144 4-5 (0.1-0.2 mL). 1 mL of Carrez I and 1 mL of Carrez II solutions were added and then 145 the flask shaken and kept at 4 ºC for 15 minutes. The solution was centrifuged at 15000 146 g (15 minutes, 4ºC) and filtrated to a 40 mL vial through the polyethylene filters. The 147 volume of the solution was reduced to 2-3 mL in a rotary evaporator at 55ºC and 148 transferred to a 20 mL vial. The rotary evaporator vessel was washed with 2 mL of 149 water that was added to the solution in the vial. If necessary a centrifugation at 3000 g 150 was performed to deposit suspended solids in the solution prior to the SPE clean-up. 151 152SPE clean-up 153The SPE columns were prepared by adding a 1g layer of C18 sorbent to the Isolute 154 Multimode 3 g column. This layer was covered with a polyethylene frit (pore size 10 155 µm). The columns were conditioned first with 20 mL of methanol and then with 20 mL 156 of water. The sample solution (~5 mL) was loaded on the column and the first 2 mL of 157 the eluate were discarded. The remaining solution (~3 mL) was collected in a 40 mL 158 vial and the column was washed with 10 mL of water that was collected to the same 159 vial. 160 161 For instant coffee it was necessary to perform a previous SPE cleaning with a 3g C18 162 columns home made prior to the Isolute cleaning. The column was conditioned under 163 the same conditions as the Isolute column and the sample was loaded and eluted with 164 just 1 mL of water. This solution was collected and loaded in the prepared

  analysis were performed in a gas-chromatograph, model HP GC-6890, split-189 splitless injector, coupled to a Mass Selective Detector model Agilent MSD-5973N 190 (Agilent, Palo Alto, CA, USA). The analytical separation was performed in a capillary 191 (30 m x 0.25 µm, 0.25 mm i.d.) from J&W Scientific (Folsom, CA, 192 USA). The centrifugations were made in an ultra-centrifuge from Eppendorf, model 193 5810 R (Hamburg, Germany) at 15000 g and in a Heraeus Sepatek, model Labofuge Ae 194 (Osterode, Germany) at 3000 g. The SPE clean-up was made in a Visiprep Solid Phase 195 Extraction Manifold from Supelco (Taufkirchen, Germany) with capacity for 12 196 columns. Evaporation of the solvents was performed in a Büchi Rotavapor model RE 197 111 and 461 water bath (Flawil, Switzerland). The evaporation under a stream of 198 nitrogen was carried out on a Pierce, model Reacti-therm 18790 (Rockford, IL, USA) 199 with capacity for 9 vials. 200 201 GC-MS Operating conditions 202 Gas-chromatography. Carrier gas: helium (constant flow at 1 mL min -1 ). Sample 203 injection volume: 1 µL (splitless, pulsed pressure 32 psi, 60 sec). Injector temperature: 204 280 ºC. Oven temperature: 85ºC (1 min), 15ºC min -1 to 280ºC, hold 10 min (24 min)., 205 transfer line, 240 ºC. 206 Mass-spectrometry. Electron energy, 70 eV (EI mode). Mode of acquisition: selected 207 ion monitoring (SIM), m/z 106, 108, 150 and 152 for 2,3-dibromopropionamide (2,3-208 DBPA) and m/z 110, 153, 155 for 2,3-13 C 3 -dibromopropionamide (2,3-13 C 3 -DBPA). 209The ions m/z 150 for 2,3-DBPA, m/z 155 for 2,3-DBPA( 13 C 3 ) were used for 210 quantification and the others for confirmation. AA was determined with the internal 211 standard using the ratio of peak area of 2,3-DBPA to 2,3-13 C 3 -DBPA. The identity of 212 the peak was confirmed by retention time and by comparing the relative abundance 213 ratios of the confirmatory ions with those of standard solution.

  10, 15, 20, 25 mL of ethanol, 15 mL were found to give the best results. 240The precipitation time was decreased to 15 minutes by changing the precipitation 241 temperature from 4ºC to -20ºC. 242 243

  was incapable of eliminate all the interferences observed in the 264 analytical signal. Besides, these samples provoked contamination of the injector, what 265 demanded constant changing of the liner sometimes just after few injections. The 266 addition of a bigger top layer of C 18 was impracticable due to the dimensions of the 267 used cartridges. The use of a previous clean-up step with a C 18 sorbent, in which the 268 majority of the coloured compounds were eliminated, followed by a second clean-up 269 step with the "C 18 layered" Isolute column was the best solution found. Notwithstanding 270 the slight decrease observed in the analytical signal, the use of a "clean" extract 271 provided a much better quality of the chromatographic results. 272273The analytical separation of acrylamide was performed in a DB 1301 capillary column. 274In Figure1is presented a chromatogram for an "espresso" coffee sample with 20,8 µg 275 L -1 AA and 0.5 µg 13 C 3 -AA corresponding 50 µg L -1 in the sample. The linearity of the method was tested several times using standard 282 (calibrating) solutions performed in water and treated using the same method developed 283 for the samples. Six standards were simultaneously prepared and treated in parallel with 284 the sample. The range of concentrations varied according to the expected levels of AA 285 in coffee samples. Typical ranges were 0-300 µg L -1 AA in the sample. The quantity of 286 this level of concentration the values obtained registered an R.S.

Figure 1 .

 1 Figure1. Chromatogram of a ground coffee sample analysed as "espresso" coffee with 20.8 µg L -1 AA and addition of 0.5 µg of 13 C 3 -AA corresponding to a concentration of 50 µg L -1 AA. m/z 150 is the quantification ion for AA and ion m/z 155 is the quantification ion for 13 C 3 -AA.

Table I .

 I Acrylamide levels measured in 26 coffee products. The minimum and maximum values are presented and also the mean level of AA in the different coffee products analysed.

	Page 21 of 21
	Samples	n	AA found µg L -1	µg AA / 30 mL cup
			Min	Mean	Max	Min	Mean	Max
	"Espresso"	18	11.4	21	36.2	0.32	0.62	1.46
	Soluble coffee	5	47.4	72.4	95.2	0.47	0.72	0.95
	Coffee blends with cereals	2	200.8	215.2	229.4	2.01	2.15	2.29
	Cappuccino	1	-	6.4	-	-	0.45	-

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Food Additives and Contaminants

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Acknowledgements 349

The authors are thankful to FCT for financial support in the framework of the project 350 POCI/AGR/61543/2004. 351


Tina ; Zuidema

P P J Mulder

J J P Lasaroms

S Stappers

J A Van Rhijn

Can high-performance liquid chromatography coupled with fluorescence detection under all conditions be regarded as a sufficiently conclusive confirmatory method for B-group substances?

Keywords: Sodium salicylate, B-Group substances, fluorescence detection, confirmatory method, tandem mass spectrometry, avermectines

Can high-performance liquid chromatography coupled with fluorescence detection under all conditions be regarded as a sufficiently conclusive confirmatory method for B-group substances?

Introduction

Sodium salicylate (2-hydroxybenzoic acid mono-sodium salt) is a non-steroidal antiinflammatory drug belonging to the group of salicylates that are commonly used in veterinary practice. It is the sodium salt of salicylic acid and it is used in ointments, creams or solutions for disinfecting wounds. Typically, the active concentration correlates to a dose of 2 to 400 µg kg -1 bodyweight/day [START_REF] Emea | Salicylic acid, sodium salicylate, aluminium salicylate, basic and methyl salicylate, Summary Report[END_REF]. For topical applications this substance is authorised in the EU for cattle, horses, sheep, goats and poultry. Sodium salicylate is included in Annex II of Council Regulation 2377/90/EC for topical use on all food-producing animals except fish and hence it has no maximum residue limit (MRL) associated to it [START_REF] Ec | Commission Regulation (EC) 1286/2000 of 19 June 2000 amending Annexes I, II and III of Council Regulation (EEC) No 2377/90 laying down a Community procedure for the establishment of maximum residue limits of veterinary medicinal product in foodstuffs of animal origin[END_REF]. Recently oral application in porcine and bovine (except milk producing) animals has been included in Annex II [START_REF] Ec | Commission Regulation (EC) 1875/2004 of 28 October 2004 amending Annexes I, II and III of Council Regulation (EEC) No 2377/90 laying down a Community procedure for the establishment of maximum residue limits of veterinary medicinal product in foodstuffs of animal origin as regards sodium salicylate and fenvalerate[END_REF]. Sodium salicylate is generally applied via the drinking water at a dose of 40 mg kg -1 bodyweight/day [START_REF] Emea | porcine). Summary Report The European Agency for the Evaluation of Medicinal products[END_REF]. No MRL was deemed necessary as depletion after oral administration was very rapid and residues remaining in edible tissue constituted only 5% of the Acceptable Daily Intake (ADI).

Despite the fact that no MRL has been established for residues of sodium salicylate, the residue control plans of several EU-Member States include the control of sodium salicylate in edible animal products as well as in feed and animal drinking water. Most commonly, sodium salicylate is determined by HPLC with fluorescence detection after aqueous extraction of the sample [START_REF] Venema | Determination of acetylsalicylic acid and salicylic acid in foods, using HPLC with fluorescence detection[END_REF]Flagerty et al, 2002). For tissue samples, often a sample clean-up step by means of solid phase extraction is applied [START_REF] Pirker | Simultaneous determination of gentisic, salicyluric and salicylic acid in human plasma using solid-phase extraction, liquid chromatography and electrospray ionization mass spectrometry[END_REF][START_REF] Rikilt | Poulty and porcine muscle tissue -Determination of sodium salicylate -HPLC. RIKILT Standard Operation Procedure RSV A[END_REF]. [START_REF] Ec | Commission Decision 2002/657/EC of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results[END_REF]. Results of analysis obtained during routine analysis can therefore be regarded as confirmed results in view of CD 2002/657/EC and hence formally those results need no further confirmatory analysis by e.g. mass spectrometric techniques. However, in every-day practice interferences can be observed with LC-Flu despite its alleged specificity.

In this paper we report the observations we recently made with respect to the conclusiveness of LC-Flu results and the possibility of obtaining false non-compliant findings.

Experimental

Chemicals

Reagents and solvents were of analytical grade unless stated otherwise. Acetonitrile, formic acid and sodium hydroxide were purchased from Merck (Darmstadt, Germany); sodium salicylate was purchased from VWR International (Amsterdam, The Netherlands). Calibrant solutions were prepared by diluting aqueous stock

Instrumentation

The LC-Flu system comprised of a Gilson 234 auto injector (Gilson, Middleton, WI, USA), a Gilson 307 HPLC pump (Gilson, Middleton, WI, USA), a 10*3 mm PRP-1 precolumn (Hamilton, Bonaduz, Switzerland), a 150*4.1 mm, 5 µm PRP-1 analytical column (Hamilton, Bonaduz, Switzerland), a thermostatic oven (Jones Chromatography, Glamorgan, UK) maintained at a temperature of 25°C and a Jasco 1520 fluorescence detector (Jasco, Easton, MD, USA), set at λ ex 300 nm, λ em 400 nm. The system was run isocratic at a flow rate of 1.0 ml min -1 using water/acetonitrile/formic acid (60/40/0.1 v/v/v) as mobile phase. Sodium hydroxide (1M) was added at 0.15 ml min -1 to the effluent of the analytical column using a Gilson minipulse 3 peristaltic pump (Gilson, Middleton, WI, USA) and a teflon mixing coil (2 m * 0.8 mm).

For LC-MS/MS confirmatory analysis, a Waters Quattro Ultima tandem mass spectrometer (Waters Corporation, Milford, MA) was used equipped with an ESI ion source operated in negative ionisation mode. A Waters Alliance 2690 LC system was used equipped with a 3*150 mm, 5 µm Water Symmetry ® C 18 analytical column and a thermostatic oven (Spark, Emmen, The Netherlands) maintained at a temperature of 35°C. Chromatography was performed at 0.4 ml min -1 using an isocratic system of water/acetonitrile/formic acid (50/50/0.1; v/v/v). The column effluent was split 2:1 before it entered the ESI ion source. The interface was operated at optimised settings Being a small molecule, salicylate (m/z 137) undergoes only few characteristic fragmentations upon collision induced dissociation in negative ESI mode. Only two product ions were observed at a collision energy of 30 eV: a major fragment identified as phenolate anion at m/z 93 (loss of 44, corresponding to the loss of CO 2 ) and a minor fragment identified as dicyclopentenyl anion at m/z 65 (loss of 72, corresponding to the combined loss of CO 2 and CO) (Figure 1). Hence the precursor ion together with these two product ions were selected for multiple reaction mode (MRM) data acquisition.

(Insert Figure 1 about here)

Samples

Fifty (50) samples of animal drinking water were collected at farms in the Netherlands where broilers were raised and transported to the laboratory for analysis. To mimic the HPLC-mobile phase composition, 6 ml aliquots were mixed with 4 ml acetonitrile and 10 µl formic acid. The samples were subsequently filtered and analysed with LCfluorescence without further sample preparation. For the LC-Flu analysis every tenth sample was analysed with addition of sodium salicylate at a level of 50 µg l -1 . Samples for which LC-Flu indicated the presence of sodium salicylate at 1 µg l -1 or higher were subjected to LC-MS/MS confirmatory analysis. To this end each suspect

Results and discussion

Although the therapeutic dose for oral administration of sodium salicylate is around 300 mg l -1 , in this survey a low (1.0 µg l -1 ) cut-off concentration was used. It can be reasoned that in case of illicit use of sodium salicylate via the drinking water, low concentration of residues may persist in the drinking water system for a considerably longer period of time than the actual medication period. Detection of residues of salicylate in the drinking water, even at very low concentrations, may indicate illegal use of this drug in the past. From the water samples taken at the broiler farms analysis by LC-Flu indicated that a substantial number (18 out of 50) contained sodium salicylate in a concentration exceeding 1 µg l -1 (Table I). In another 12 samples amounts ranging between 0.3 (the estimated LoD of the method) and 1.0 µg l -1 were detected. One sample (entry 15) contained a very high concentration sodium salicylate, estimated (after 1000-fold dilution) at 375 mg l -1 . Figure 2 presents typical LC-Flu chromatograms obtained for a standard solution, a blank and two positive samples (entries 15 and 16). No signal was detected in the blank samples while for the samples fortified at 50 µg l -1 a mean recovery of 88% was obtained (range 80-105%). For the suspect samples in the low concentration range (1-30 µg l -1 ), analytical results were very consistent: both the retention time and peak width were very stable and matched with the standard solutions and no other peaks were present in the chromatograms. Furthermore, standard addition of sodium salicylate to the samples did not result in any change of the retention time, nor to detectable peak (Insert Table I andFigure 2 about here) Listed as a B-group substance, strictly speaking using HPLC-fluorescence as detection technique and applying the criteria of CD 2002/657/EC is sufficient to acquire data suitable for confirmation of the analyte's identity. However, the percentage of positive samples (above 1 µg l -1 ) was surprisingly high (36%) but at the same time the majority of the concentrations found was low; 1000-fold or more below the regular therapeutic dose. Sodium salicylate has been detected in the effluent of waste water treatment plants and in surface water, but always in concentrations well below 1 µg l -1 (La Farré et al 2001, [START_REF] Marchese | Determination of nonsteroidal anti-inflammatory drugs in surface water and wastewater by liquid chromatography-tandem mass spectrometry[END_REF]. As these findings could be of considerable significance for further enforcement actions, it was decided to use LC-MS/MS for additional confirmation.

Each suspect sample was re-analysed by LC-MS/MS in duplicate and with addition of sodium salicylate at approximately the same concentration that was determined by LC-Flu. The results are given in Table I. Figure 3 presents the extracted ion chromatograms obtained for the same samples as shown in figure 2. It is clear that with LC-MS/MS no sodium salicylate could be detected in sample 16. In fact not a trace of the analyte could be detected while the limit of detection of the LC-MS/MS method was below 1 µg l -1 in drinking water. Sodium salicylate was detected and confirmed in the standard addition sample. The result for sample 16 is representative for all samples in which low concentrations of sodium salicylate were detected by LC- Recently we encountered another case in our laboratory in which a HPLCfluorescence method produced aberrant results. In the analysis of avermectines in raw milk, derivatisation is used followed by fluorescence detection (λ ex 365 nm, λ em 470 nm) [START_REF] Schenck | Multiresidue determination of abamectin, doramectin, ivermectin and moxidectin in milk using liquid chromatography and fluorescence detection[END_REF][START_REF] Danaher | Development and optimization of an improved derivatisation procedure for the determination of avermectins and milbemycins in bovine liver[END_REF][START_REF] Rikilt | Poulty and porcine muscle tissue -Determination of sodium salicylate -HPLC. RIKILT Standard Operation Procedure RSV A[END_REF]. Again this is generally considered a highly specific method and suitable for confirmatory analysis. Recently we participated in a proficiency study on avermectins in reconstituted milk. The study comprised 7 samples with unknown concentrations of a number of avermectins, including eprinomectin (EPM), moxidectin (MOX), emamectin (EMA), abamectin (ABA), doramectin (DOR) and ivermectin (IVM). The proficiency test samples were received in a lyophilised state and were reconstituted with water before analysis.

However, for the control blank and fortified samples raw milk was used, as no lyophilised control milk was available (nor was it supplied by the proficiency test organiser). As can be seen from Figure 4 the blank raw milk sample produced a very clean chromatogram with only small background interferences and all 6 avermectines could easily be detected and quantified, down to sub-ng/ml concentrations. In contrast, the reconstituted milk samples produced chromatograms containing significantly more and higher background interferences. In Figure 4 the chromatogram obtained for one of the proficiency test samples is given as an example.

(Insert Figure 4 about here)

We reported this sample (by duplicate analysis) to contain 28, 1.6 and 1.1 µg l -1 MOX, EMA and IVM, respectively. We correctly labelled a peak eluting at 5.15 min as not being EPM (retention time 5.25 min, relative difference 2%). However, we learned from the organiser of the proficiency study that we had reported for this sample a deviant (false non-compliant) result for emamectin. It was stated by the organiser that EMA had not been added to any of the samples. Re-examination of the raw analytical data did indeed reveal that the retention time of the peak (8.48 min) was slightly different from that of the EMA standard in the control sample (8.39 min). The retention time difference is only 1% and well below the criterion of 2.5% as specified in CD 2002/657/EC [START_REF] Ec | Commission Decision 2002/657/EC of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results[END_REF]. This minor difference was found consistently in the duplicate sample as well as in two other samples for which we had reported the presence of EMA (producing two more false positive results). This small retention time difference had escaped attention during the analysis of the raw data and even if it had been noted it would probably not have triggered further action since it complied with the criteria. Furthermore, it should be noted that this peak was prominently present in three samples but virtually absent in the remaining 4 samples. All samples were run in duplicate and produced consistent results. On the basis of the combined information the reported findings of EMA formally remained justifiable. At this moment it is not yet clear if EMA was detected by any of the other 30 participants (the final report is still pending). However, the conclusion seems justified that the usefulness and conclusiveness of HPLC-Fluorescence strongly depends on the quality of the produced sample extracts and the absence or presence of matrix interferences or other artefacts. When these interferences occur infrequently, it becomes difficult to recognise them as such and to avoid false positives.

Conclusions

From an analytical perspective, animal drinking water could be considered as a simple matrix with little or no risk of interferences. For this reason we used HPLCfluorescence to analyse sodium salicylate in drinking water samples collected at poultry farms. LC-Flu indicated low levels of sodium salicylate to be present in a substantial number of samples. The identity of sodium salicylate could not be confirmed by LC-MS/MS, however. It was concluded that LC-Flu analysis had generated false-positive results for these samples, probably due to an artefact or an unknown compound that was present in variable amounts. This case and the one described on the analysis of avermectin residues in milk illustrates that despite the fact that LC with fluorescence detection is generally regarded as an acceptable confirmatory method for B-group substances, false-non-compliant results can occur, especially when the analysis is performed at trace levels. The specificity of fluorescence detection can be questioned and consequently so can its conclusiveness.

Considering the possible impact that non-compliant analytical results also for B-group substances may have for the individual farmer as well as on trade, product image EPM but an interference on the basis of a slightly different retention time. The peak labelled as EMA (quantified as 1.6 µg l -1 ) was not recognized being an artifact and was reported as a false positive. ). The peak labelled with an asterisk was identified correctly not to be EPM but an interference on the basis of a slightly different retention time. The peak labelled as EMA (quantified as 1.6 µg l-1) was not recognized being an artifact and was reported as a false positive.

  . Nonetheless it is suspected that sodium salicylate is sometimes used during the last period of broiler fattening to relieve the animals from pain. Therefore, sodium salicylate was recently targeted in a routine survey in drinking water supplied to broilers. Analysis of the samples was carried out using HPLC with fluorescence detection. According to Commission Decision 2002/657/EC, HPLCfluorescence (LC-Flu) is a suitable confirmatory method for B-group substances that exhibit native fluorescence or exhibit fluorescence after either transformation or derivatisation and when the performance criteria for chromatographic conditions are fulfilled

  40/0.1 v/v/v) to obtain solutions containing 1, 2, 5, 10, 20, 50, 100, 200 and 500 µg l -1 .

  desolvation gas flow 550 l h -1 , cone gas flow 150 l h -1 ).

  concentration as determined by LC-Flu.

  non-compliant findings.

  analysis, hence the non-compliant findings by LC-Flu could not have been caused by low-level contamination of the samples, but must have been caused by a fluorescent artefact present in part of the water samples. LC-MS/MS analysis yielded one positive case: in sample 15 a high concentration (400 mg l -1 ) of sodium salicylate was detected and its identity was confirmed according to the criteria of CD 2002/657/EC. (Insert Figure 3 about here)

  HPLC-Fluorescence and LC-MS/MS analysis of sodium salicylate in drinking water samples obtained at broiler farms. Concentrations are given in µg l -1 . Only those samples are listed that contained more than 1 µg l -1 according to LC-Flu analysis.

Figure 1 .

 1 Figure 1. MS/MS fragmentation pathway of sodium salicylate, negative ESI, collision

Figure 2 .

 2 Figure 2. LC-Flu chromatograms of animal drinking water samples analysed for sodium salicylate. A: standard solution (1 µg l -1 ) B: blank control sample, C: sample 16 (28 µg l -1 ) and D: sample 15 (1000x diluted, 375 mg l -1 )

Figure 4 :

 4 Figure 4: Analysis of avermectines in milk by HPLC-Fluorescence. A: Blank raw milk sample. B. Raw milk sample spiked with 2.0 µg l -1 eprinomectin (EPM), moxidectin

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

 1234 Figure 1. MS/MS fragmentation pathway of sodium salicylate, negative ESI, collision energy 30 eV.

Table I .

 I

	Sample code	LC-Flu	LC-MS/MS
	1	1.2	<1
	2	14	<1
	3	2.5	<1
	4	6.2	<1
	5	2.2	<1
	6	2.0	<1
	7	1.0	<1
	8	2.9	<1
	9	5.1	<1
	10	2.4	<1
	11	1.4	<1
	12	1.5	<1
	13	5.9	<1
	14	1.7	<1
	15	375 000	400 000
	16	28	<1
	17	4.3	<1
	18	1.5	<1

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