Klaas M Jonker

Jeroen J H C Tilburg

Geke Hagele

Enne De Boer

G H Hägele

Species identification in meat products using real-time PCR

Keywords: Real-time polymerase chain reaction (PCR), DNA-hybridization, species identification, TaqMan

come

Introduction

Animal species identification is an important means to check food labelling that is required by EU-regulation. Moreover the species of origin can be relevant to consumers for economic, medical and religious reasons. Earlier methods were based on identification of proteins by means of electrophoretic and/or immunological methods [START_REF] Hsieh | Development of a monoclonal antibody specific to cooked mammalian meats[END_REF], Skarpeid et al. 1998). However, these techniques are not reliable for identifying species in complex heated products as a result of deterioration of the proteins. DNA is more stable than proteins during heat processing and although it can be fragmented by heat, modern DNA-techniques still allow us to identify DNA from the different species present in a sample [START_REF] Ebbehøj | The use of a molecular technique for the detection of porcine ingredients in the Malaysian food market[END_REF][START_REF] Lockley | DNA-based methods for food authentication[END_REF]. Also DNA unlike specific proteins is present in all organs and tissues. Hybridization and PCR-techniques are now available for this purpose. In many publications the PCR-RFLP technique has been described for the identification of species. However this technique is inadequate for mixtures [START_REF] Partis | Evaluation of a DNA fingerprinting method for determining the species origin of meats[END_REF][START_REF] Girish | Meat species identification by polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) of mitochondrial 12S rRNA gene[END_REF]. Real-time PCR-techniques are especially suitable for heated products because small fragments of DNA can still be amplified and identified [START_REF] Hird | Effect of heat and pressure processing on DNA fragmentation and implications for the detection of meat using real-time polymerase chain reaction[END_REF]. PCR has proved to be a highly specific method for among others, the detection of minor amounts of pork-DNA in Halal-products (Farouk et al. 2006). It is possible to determine the amount of DNA of the different species in the sample although inhibitors can influence the results. Furthermore the amount of DNA from e.g. muscle, liver or connective tissue is different [START_REF] Marx | Die Frischmasse sowie die Konzentration an DNA, RNA und Protein in verschiedenen Geweben von Schweinen unterschiedlichen Alters[END_REF]. Consequently it is possible that for a mixture of equal amounts of lean beef and fat pork the proportion of beef DNA to pork DNA will be 80 to 20 instead of the expected 50 to 50. So in most cases only a semi-quantitative estimate of the meat content of the different species present is possible. In spite of these drawbacks real-time PCR to our opinion is a very sensitive technique for the identification of species in heatprocessed meat products.

Materials and methods

Method development

Mixtures of 10 to 0.1 % chicken, turkey, pork (lean meat and fat separately), horse and sheep in beef and 10 to 0.1 % beef in pork were prepared and packaged in plastic bags. The mixtures were heated at 100 °C in a water bath for 5, 15 and 45 min or heated in an autoclave at 115 °C for 10 min (table 1). These samples were used for the development and evaluation of a real-time PCR-method. Next crossreactivity of kangaroo, deer, roe, goat, ostrich, pheasant, goose, domestic duck, mallard, guinea fowl, quail and pigeon for the determination of pork, beef, chicken, turkey, mutton and horse was investigated. Finally the method was tested by analysing more than 150 samples of various processed meat products for pork, beef, chicken, turkey, mutton and horse, using both DNA-hybridization and real-time PCR.

These samples were obtained from retail-shops.

[insert table 1 about here]

Design of TaqMan primer and probe sets

Gene sequences for the six species of interest were obtained from GenBank. It concerned five sequences from cytochrome b (cytb) gene for pork, horse, mutton, chicken and turkey and one from satellite IV DNA for beef. Accession numbers for the gene sequences are X56295 (pork), X56284 (mutton), AY235571 (chicken), AY15157979 (turkey), HRSMTCB20 (horse) and AF446392 (beef).

Primers and probes for these species were designed using Primer Express software (Applied Biosystems, Warrington, UK). Primers and probes were obtained from Eurogentec, Seraing, Belgium. Probes were labelled with reporter dye 6carboxyfluorescein (FAM) and Black Hole Quencher (BHQ) (table 2).

[insert table 2 about here] Sample DNA extraction and purification DNA was extracted by mixing 5 g of the homogenized sample in 20 ml buffer (0.4 M NaCl, 0.01 M Tris-HCl, 0.002 M EDTA, pH 8.0) in a Waring Blender. From this homogenate 750 µl was added to 50 µl of 10% SDS and 100 µl of 0.2% proteinase K and incubated at 65°C overnight. For severely heated samples, the Wizard DNA Clean-up System (Promega, Southampton, UK) was used according to the manufacturer's instructions. Small fragments from highly damaged DNA were better recovered in this way [START_REF] Janssen | Species identification in meat by using PCR-generated satellite probes[END_REF]. In all other cases DNA was obtained by adding 250 µl of saturated NaCl solution to the incubated mixture, centrifuging for 15 min at 10 000 x g. To 500 µl of the supernatant 1 ml of cold (-20°C) ethanol was added and after centrifuging for 15 min at 10 000 x g the pellet was dried and dissolved in Tris-EDTA buffer, pH 7.5 and kept at -20°C until used. This solution and the solution obtained with the Wizard Clean-up System were diluted to a level of 0.1 to 0.15 µg/µl with Milli Q water. In the real-time PCR assay 2.5 µl of this solution (DNA template) was used in the reaction mixture.

Real time PCR assays

The Lightcycler 2.0 (Roche Diagnostics, Mannheim, Germany) was used for the species identification of pork, chicken, turkey, mutton and horse. Amplification reactions were performed with the LightcyclerTaqMan Master Mix (Roche Diagnostics, Mannheim, Germany), containing 2.5 µl template, 500 nM sense and antisense primer and 250 nM probe in 20 µl. The following cycling protocols were used: 95°C for 10 min, followed by 28 cycles for pork, 30 cycles for chicken and turkey, 32 cycles for mutton and 35 cycles for horse, consisting of 95°C for 10 sec and 60°C for 15 sec per cycle and finally cooling to 40°C for 30 sec.

For the species identification of beef the ABI 7000 SDS (Applied Biosystems, Warrington, UK) was used as the reagents for the Lightcycler contained Bovine Serum Albumin (BSA) to prevent adhesion of DNA to the glass-capillaries. Amplification reactions were performed with the qPCR Master Mix (Eurogentec, Seraing, Belgium), containing 2.5 µl template, 500 nM sense and antisense primer and 250 nM probe in 25 µl. For the detection of beef the following cycling protocol was used: 50°C for 5 min, 95°C for 10 min, followed by 25 cycles of 95°C for 15 sec and 60°C for 1 min.

Hybridization assays

Samples were also examined with DNA hybridization using a previous described method [START_REF] Janssen | Species identification in meat by using PCR-generated satellite probes[END_REF]. Shortly 2 µl (2µg) of sample DNA and 2 µl (2 µg -2 ng) of a serial dilution of the DNA to be determined were spotted on a positively charged nylon membrane and hybridization with species-specific probes labelled with digoxigenin took place overnight. After stringency washings (2xSSC 300mM NaCl, 30 mM Na 3 citrate, 0.1% SDS at 42°C), the hybrids (bound probes) were detected by an immunochemical procedure, using an anti-digoxigenin antibody conjugated with alkaline phosphatase and a chromogenic alkaline phosphatase substrate. After comparison of the spots the sample was classified as positive (+), negative (-) or inconclusive (+/-).

Results and discussion

We have described a convenient, sensitive and specific real-time PCR system for the species identification in meat products using the Lightcycler 2.0 (Roche) for pork, mutton, horse, chicken and turkey and using the ABI Prism 7000 SDS (Applied Biosystems) for beef by using TaqMan probes. The primers and probes were designed to work at an annealing temperature of 60 °C, the reagents used allow the polymerase to perform at an extension temperature of 60 °C. The method can be adjusted to the users interest. When high sensitivity is needed the number of cycles can be increased. On the other hand, all species can be analysed using the same number of cycles. In that case the sensitivity for each species will be different. The species identification was set up on the Lightcycler 2.0 for most of the species because this type of apparatus is more sensitive and the software is user-friendlier than the ABI Prism. Species identification of the species mentioned can be performed on the ABI Prism although detection limits are higher (results not shown). It was not possible to use the Lightcycler 2.0 for beef due to the fact that Bovine Serum Albumin In this study we prepared mixtures (table 1) of 10 to 0.1 % chicken, turkey, pork (meat and fat separately), horse and mutton in beef (table 1: A-E and L-P) and 10 to 0.1 % beef in pork (table 1: F-K). The mixtures were heated as described earlier.

These samples were used for the evaluation of the real-time PCR determinations (table 3a-d). In these tables and in table 4 the crossing points (CP-values) for the LightCycler and the crossing threshold (Ct-values) for the ABI Prism are given. The identification of species is performed in complex products; therefore cross-reactivity and sensitivity were studied for a large panel of species. In addition to beef, pork, mutton, horse, chicken and turkey, the following species were also tested: kangaroo, deer, roe, goat, ostrich, pheasant, goose, domestic duck, mallard, guinea fowl, quail and pigeon (table 4). By adjusting the number of cycles it was possible to detect levels as low as 0.1 ng for pork, mutton, horse, chicken and turkey and 0.2 ng for beef. No cross-reaction was observed with pork, mutton and horse. Cross-reaction was only observed with chicken (dear ≥ 125 ng), turkey (horse 250ng) and beef (pigeon ≥ 50 ng). In practice this is no problem as these high amounts of DNA are not likely to be present in meat samples.

[insert table 3a-d and table 4 about here ]

It was possible to detect all species in the test sample down to 0.1 % irrespective the heat treatment. Taking into account some cross-reactivity of high amounts of DNA (≥ 125 ng) from other species, sensitivity could be increased by increasing the number of cycles. In this way it was possible to determine small amounts of DNA of beef in protein hydrolysates, used as additives in chicken and fish. Also small amounts of pork could be determined in Halal products. At the cost of specificity amounts as small as 0.001% of the species evaluated could be detected.

Quantification of the meat content was not possible due to the fact that the amount of DNA and inhibitors in muscle meat and fat is not the same and the composition and processing conditions of the commercial samples were not known. The practicability of the method was tested by analysing more than 150 samples of various processed meat products. The real-time PCR results were compared with the DNA-hybridization results. There was overall similarity (figure 5). Compared to DNA-hybridization the real-time PCR proved to score better for sensitivity, specificity, time and interpretation.

[insert table 5 about here]

Conclusions

A highly sensitive and specific real-time PCR system for the species identification in products of animal origin has been presented. The use of primers resulting in small (< 100bp) amplicons makes the method very suitable for the determination in severely processed meats, where DNA is highly fragmented. Using the speciesspecific primers and probes it is possible to detect amounts of 0.25 ng DNA of beef, pork, mutton, horse, chicken and turkey. Only amounts of more than 50 ng DNA of pigeon, more than 125ng DNA of deer and of 250ng DNA of horse show crossreactivity in the determination of respectively beef, chicken and turkey. For routine examination species of pork, lard, horse, mutton, chicken and turkey in mixtures can be detected down to a level of about 0.05% and beef to a level of about 0.1 %. By fine-tuning the number of cycles sensitivity can be adjusted, so using this real-time PCR system minimal amounts down to 0.001% of the species evaluated can be detected in all kinds of processed food products. By comparison of DNA-hybridization it was found that the real-time PCR gives a better sensitivity and specificity. Furthermore real-time PCR is saving time and the interpretation of the results is easier. 95ºC for 10 min., X cycles (95ºC 10 sec.; 60ºC 15 sec.) and cooling at 40ºC for 30 sec., program on the ABI Prism 7000 SDS for beef exists of the following steps: 50ºC for 5 min., 95ºC for 10 min and 25 cycles (95ºC 15 sec.; 60ºC 1 min.).

F

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Food Additives and Contaminants

  is added to the reaction mixture to prevent adhesion of DNA to the glasscapillaries.

  a = forward primer; b = reverse primer; c = Probe; d = Nucleotide positions for pork are in reference to the genomes of S. sucrofa mitochondrial cytb gene (X56295), for beef to the genomes of Bos Taurus satellite IV DNA (AF446392), for mutton to the genomes of Ovis aries mitochondrion cytb gene for cytochrome b (X56284), for chicken to the genomes of Gallus gallus mitochondrion complete genome (AY235571), for turkey to the genomes of Meleagris gallopavo cytochrome b gene (AY157979), for horse to the genomes of Equus caballus mitochondrial gene for cytochrome b (HRSMTCB20); e = program on the Lightcycler 2.0 for pork, mutton, chicken, turkey and horse exists of the following steps:

Table 3a .

 3a Skarpeid, H.J., Kvaal, K., and Hildrum, K.I., 1998, Identification of animal species in ground meat mixtures by multivariate analysis of isoelectricfocusing protein profiles. Sensitivity of the real-time PCR for species identification.

		Species	Pork	Beef	Mutton	Horse Chicken Turkey
	Number of cycles Electrophoresis, 19, 3103-3109. threshold 0.1 28	25 0.5	32 0.1	35 0.1	30 0.1	30 0.1
		primers/probe:	Var	bossatIV	ovis	Equus	gallus Mgal cytb
	Species	DNA-conc ng/ul *) CP-value Ct-value CP-value CP-value CP-value CP-value
		100	17.09	12.35	20.12	21.02	17.94	17.90
		10	20.77	15.69	23.59	24.75	21.13	20.80
		1	24.75	19.90	28.08	29,35	25.20	25.32
		0,1	27.76	23.11	31.82	32.18	29.45	27,78
	o r F o r F o r 0,05 *) Vial containes 2.5 µl F o r	28.80	neg	32.10	35.60	30.25	28.63
		P P P P
		e e e e
		e r e r e r e r
			R R R R
				e e e e
				v i e v i e v i e v i e
					w w w w
					O n l O n l O n l O n l
							y y y y

Table 3b .

 3b Sensitivity of heated mixtures in beef.

	Mixture of beef with		pork	mutton	horse chicken	turkey
	chicken, turkey, pork	primers/probe:	Var P/F/R ovis P/F/R Equus P/F/R gallus P/F/R Mgal cytb P/F/R
	horse, mutton (%)	Heat-treatment CP-value CP-value CP-value CP-value	CP-value
		10% 5 min 100 °C	20,75	20,66	22,84	20,94	21,45
	A		15 min 100 °C	20,52	19,84	21,16	n.d.	21,34
			45 min 100 °C	20,69	20,30	21,62	n.d.	21,90
			10 min 115 °C	21,49	21,34	22,78	n.d.	21,20
	B	5% 5 min 100 °C 15 min 100 °C 45 min 100 °C 10 min 115 °C 1% 5 min 100 °C F o r	22,80 22,69 21,68 22,86 26,84	22,84 22,81 21,92 23,09 29,55	23,68 24,04 23,03 23,27 32,36	22,52 20,49 22,18 21,88 27,92	22,91 22,43 22,57 22,70 26,97
	C		15 min 100 °C	26,48	27,46	29,91	26,36	25,98
			45 min 100 °C 10 min 115 °C P	26,45 27,06	25,20 25,22	28,76 29,95	24,24 25,04	24,71 25,30
	D	0.5 % 5 min 100 °C 15 min 100 °C e 45 min 100 °C 10 min 115 °C 0.1 % 5 min 100 °C e 24,80 25,42 25,52 26,01 26,15 r	25,01 25,52 25,69 26,01 26,78	26,81 27,64 27,76 27,99 29,22	24,69 25,46 25,82 25,30 26,98	25,10 25,75 25,96 26,19 27,30
	E		15 min 100 °C 45 min 100 °C	27,58 27,24 R	28,92 28,12	30,07 29,99	27,54 27,77	27,67 27,85
			10 min 115 °C	28,49 e 30,33	30,13	28,05	28,64
	n.d. : not determined		v i e
					w
						O n l
							y

Table 3c .

 3c Sensitivity of heated lard in beef.

	Mixture of beef	primers/probe:	Var P/F/R
	with pork-lard (%)	Heat-treatment CP-value
		10% 5 min 100 °C	20,75
	L		15 min 100 °C	20,52
			45 min 100 °C	20,69
			10 min 115 °C	21,49
		5% 5 min 100 °C	22,80
	M N	15 min 100 °C 45 min 100 °C 10 min 115 °C 1% 5 min 100 °C 15 min 100 °C F o r	22,69 21,68 22,86 26,84 26,48
			45 min 100 °C	26,45
		10 min 115 °C 0.5 % 5 min 100 °C P	27,06 24,80
	O P	15 min 100 °C 45 min 100 °C e 25,42 25,52 10 min 115 °C 26,01 0.1 % 5 min 100 °C 26,15 15 min 100 °C 27,58 e r
			45 min 100 °C 10 min 115 °C	27,24 28,49 R
				e
				v i e
				w
				O n l
				y

Table 3d .

 3d Sensitivity of heated beef in pork on the ABI 7000.

			number of cycles	25
			Treshold:	0.5
	Mixture of pork	primers/probe:	bossatIV P/F/R
	with beef (%)		Behandeling	Ct-value
		10%	5 min 100	15.54
	F		15 min 100	15.65
			45 min 100	15.91
			10 min 115	15.85
		5%	5 min 100	17.37
	G		15 min 100	17.76
			45 min 100	17.89
	H I	10 min 115 5 min 100 15 min 100 45 min 100 10 min 115 5 min 100 15 min 100 o 1% 0.5 % F r	18.33 19.03 19.27 19.42 19.86 20.61 20.30
	K	0.1 %	45 min 100 10 min 115 5 min 100 P 15 min 100 45 min 100 e 10 min 115 e r 20.39 20.53 21.85 21.91 22.15 22.33
				R
				e
				v i e
				w
				O n l
				y

Table 4 .

 4 Cross-reaction of the real-time PCR for species identification.

	cross-reactions Species	Pork	Beef	Mutton	Horse Chicken Turkey
		Number of cycles	28	25	32	35	30	30
		threshold	0.1	0.5	0.1	0.1	0.1	0.1
		primers/probe:	Var	bossatIV	ovis	Equus	gallus Mgal cytb
	Species	DNA-conc ng/ul *) CP-value Ct-value CP-value CP-value CP-value CP-value

Table 5 .

 5 Comparison of the results for hybridization and real-time PCR.

	Primers + probe	Number of samples tested	Hybridisation results		Real-time results
				pos	neg	+/-	pos	neg
	Chicken		20 (chicken indicated)	19	1	-	20	-
			32 (chicken not indicated)	-	32	-	5	27
	Turkey		13 (turkey indicated)	11	1	1	12	1
			115 (turkey not indicated)	3	111	1	18	97
	Pork		27 (pork indicated)	25	-	2	27	-
			72 (pork not indicated)	2	64	6	6	66
	Mutton		65 (mutton not indicated)	1	63	1	3	62
	Horse		65 (horse not indicated)	-	64	1	4	61
	Beef		70 (beef indicated)	38	28	4	68	2
			41 (beef not indicated)	4	33	4	13	28
	pos :	species identification positive
	neg :	species identification negative
	+/-: species identification inconclusive

Table 1 .

 1 Composition of test samples made by mixing lean (muscle) meat.

			Food Additives and Contaminants
	Beef with five species added:
		A	B	C	D	E
	Beef %	33.7	58.7	78.7	81.2	83.2
	Chicken %	10	5	1	0.5	0.1
	Turkey %	10	5	1	0.5	0.1
	Pork %	10	5	1	0.5	0.1
	Horse %	10	5	1	0.5	0.1
	Mutton %	10	5	1	0.5	0.1
	Additives a %	16.3	16.3	16.3	16.3	16.3
	Pork with admixture of beef:
		F	G	H	I	K
	Pork %	73.3	78.7	82.7	83.2	83.6
	Beef %	10	5	1	0.5	0.1
	Additives a %	16.3	16.3	16.3	16.3	16.3
	Beef with admixture of lard:
		L	M	N	O	P
	Beef %	73.3	78.7	82.7	83.2	83.6
	Lard %	10	5	1	0.5	0.1
	Additives a %	16.3	16.3	16.3	16.3	16.3
	a Additives 16.3% = 9.25% water + 2% NaCl/NaNO 2 + 5% starch + 0.05% ascorbic acid.
	Portions of all mixtures were heated for 5, 15 or 45 min at 100°C or for 10 min at 115 °C.

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Table 2 .

 2 Primers and probes for species identification in products.

	Species	Target	Pr.&Probe DNA sequence 5'→ → → → 3'

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


Zigmas Ežerskis
email: zigmas.ezerskis@jrc.it

S Pastorelli

C Contini

C Simoneau

Survey of 2-Ethylhexanoic acid in baby food

Keywords: 2-ethylhexanoic acid (2-EHA), solid phase microextraction (SPME), polyacrylate fiber, gas chromatography/mass spectrometry (GC/MS), PVC gasket seals, baby food, tolerable daily intake (TDI), exposure assessment

A solid phase microextraction method was developed and used to perform a survey of 2-ethylhexanoic acid (2-EHA) in 63 samples of baby food from 13 EU countries, Norway and Switzerland. The levels of 2-EHA did not exceed 3.4 mg kg -1 . The concentrations of 2-EHA in 53 samples (84%) were in the range between less than 0.1 and 0.5 mg kg -1 . Ten samples showed the presence of the compound in the range between 0.5 and 3.4 mg kg -1 . An exposure assessment showed that intake of 2-EHA does not exceed the TDI for infants of 6-12 months old except one occurrence when taking the worst-case food consumption data available and the highest level of contamination of the survey. In the other cases the levels of 2-EHA are at 13-fold below the TDI.

Introduction

Salts of 2-ethylhexanoic acid are used as thermal plasticisers/stabilisers during the production of PVC sealing of twist-off caps in order to render the seals thermo-stable (HSDB). The PVC gaskets form an airtight seal preventing microbiological and other contamination. Up to now, very limited information is available on the occurrence of 2-EHA in foods. The presence of 2-EHA in baby food and fruit juice packed in glass jars and closed with metal lids, which where sealed by a plastic gaskets was recently detected in a number of the samples from German suppliers and are suspected to be the result of its migration from the gasket seals into the food under the thermal sterilisation (Elss et al. 2004). Amounts of 2-EHA in baby food and fruit juices ranged from 0.25 to 3.2 mg kg -1 and from 0.01 to 0.59 mg l -1 , respectively (Elss et al. 2004). The levels of 2-EHA determined in baby foods on the Norwegian market were from 0.08 to 0.6 mg kg -1 (NSCFS 2005).

2-EHA is not listed on the positive list for plastics in the contact with food (EC 2006) and there is currently no specific migration limit level for 2-EHA in food contact materials at the EU level. But the substance is included in the Synoptic Document (EC 2005). The latter implies that in the future the compound might be included in the positive list (EC 2006) and the specific or overall migration limit (60 mg kg -1 ) might be applied. Meanwhile it has not been clarified whether the gaskets of metal closures are within the scope of the EU Directive on plastics (EC 2003a). According to the procedure for complying with the provisions of Directive 2002/72/EC on plastic materials and articles in contact with foodstuffs, which is described in Practical Guide 2-EHA is considered negative for mutagenicity but positive for clastogenicity in vitro (NSCFS 2005).

The toxicity of 2-EHA on reproduction was studied on rabbits and rats and it was found out that the substance is foetotoxic at high doses [START_REF] Hendrickx | Assesment of the developmental toxicity of 2-ethylhexanoic acid in rats and rabbits[END_REF]. The presence of 2-EHA is linked to peroxisome proliferation of liver cells in rats and mice (Lampen et al. 2003, Sundberg et al. 1994).

According to Directive 67/548/EEC, Annex VI (EC 1992) the German Federal Institute for Occupational Safety and Health classified 2-EHA as suspected to cause harmful effects on embryos and labelled it as R63, category 3. US Environmental Protection Agency (EPA) announced that 2-ethylhexanoic acid may present an unreasonable risk of oncogenicity, developmental toxicity and subchronic toxicity [START_REF] Epa | Chemical Information Collection and Data Development (Testing)[END_REF].

Several analytical methods for determination of 2-EHA are described in the literature. °C for 2 h. Further quantification was performed by GC/MS (EI) in SIM mode using internal standardtrans-cinnamic acid. [START_REF] Horn | Plasticizer metabolites in the environment[END_REF] used chloroform for the extraction of 2-EHA from water and sediments. Subsequent analysis was done by GC/MS using RTX-5 capillary column. Few authors reported quantitative headspace SPME analysis of volatiles in foods and beverages (Roberts et al. 2000[START_REF] Ai | Solid Phase Microextraction for Quantitative Analysis in Nonequilibrium Situations[END_REF][START_REF] Górecki | Effect of Sample Volume on Quantitative Analysis by Solid-phase Microextraction, Part 1[END_REF][START_REF] Hawthorne | Solventless determination of caffeine in beverages using solid-phase microextraction with fused-silica fibers[END_REF]. Also Pinho et al. (2002) used a headspace solid phase microextraction (SPME) method in combination with GC/MS for the extraction, identification and quantification of butanoic, hexanoic, octanoic and decanoic acids in ewe cheeses. The method of standard additions and isotope dilution assays (IDA) were used in the latter work for the accurate quantification of the free fatty acids.

In our work we applied external calibration method for headspace SPME analysis using synthetic matrices, which were prepared by choosing carefully the components and quantities in order to match the real samples as much as possible. Also the sampling conditions were optimised in order to reach maximum reproducibility for the standards and the samples. Usually external calibration is not so accurate as standard additions method or IDA due to the fact that a synthetic matrix could hardly match that of authentic sample (Pinho et al. 2002). But for our purpose we assumed the compromise between accuracy on one side and moderate amount of the samples on the other side.

Materials and methods

Chemicals

2-Ethylhexanoic acid (≥99.5%, Sigma-Aldrich, Milan, Italy) was used as standard material. Ultrapure water prepared using a Milli-Q filter system (Direct Q5 Millipore, Milan, Italy). Metaphosphoric acid (Sigma-Aldrich, Milan, Italy) was used for the preparation of solutions.

Samples

For the calibration 3 types of home-made baby food was prepared by mixing water, rice, vegetables (courgettes, carrots, tomatoes, onions), chicken breast, olive oil and salts at different quantities in order to reproduce commercial types of the food. Real food samples were of several different types: a) meat or fish, rice or pasta and vegetables; b) meat or fish and vegetables; c) rice or pasta and vegetables; d) vegetables. Fat content in each type of the food was from 2.5 to 3.5 %. Sixty three real samples taken for the 2-EHA analysis were part of those which were used for the ESBO chlorohydrins survey (Piccinini et al. 2004).

Sample preparation

Each real baby food sample was prepared by weighing 5 g of the food in a 20 ml headspace vial. 5 ml of 5 % metaphosphoric acid solution, 2 ml of distilled water and magnetic stirring bar were added to each vial before sealing hermetically. Real samples were conditioned in the water bath heated at 45 °C for 10 min under magnetic stirring (IKA, Germany) in order to achieve the partition equilibrium between the liquid and vapour phases. 85 µm polyacrylate SPME fiber (Supelco, USA) was used for the extraction procedure. SPME fiber was exposed to the headspace for 5 min. The analyte was desorbed in the GC injector port at 260 °C. Blank runs were performed between each extraction to ascertain that the fiber is free from 2-EHA. The samples used for calibration were prepared by spiking of home-made baby food at six different concentrations (0.1; 0.2; 0.5; 1; 2 and 4 mg kg -1 ) with 2-EHA by adding 0.05, 0.1, 0.25, 0.5, 1 and 2 ml of 2-EHA solution in water, respectively to 5 g of the food in 20 ml headspace vial. The concentration of 2-EHA was 10.3 mg l -1 (at this concentration 2-EHA is completely soluble in water). Afterwards, 5 ml of 5 % metaphosphoric acid and different amount of water (1.95, 1.9, 1.75, 1.5 and 1 ml) was added in the spiked samples with the concentrations of 2-EHA at 0.1, 0.2, 0.5, 1 and 2 mg kg -1 , respectively.

The other conditions for the preparation of the standards were the same as for the real samples. The adjusting of the standard sample volumes with water was performed in order to have the equal (as much as possible) headspace volumes for standard and real samples.

Capillary gas chromatography/mass spectrometry (GC/MS)

The calibration and real samples were analysed using gas chromatograph CP-3800

Calibration

The calibration curves for 3 different types of food: a) meat, rice, and vegetables; b) meat and vegetables; c) rice and vegetables were constructed for quantification of the analyte in real samples. 2-EHA was quantified by means of the different calibration curves, which were constructed by analysing spiked home-made food at six different concentrations of 2-EHA (from 0.1 to 4 mg kg -1 ) as described above. Quantification was based on peak area integration. External calibration approach was used for quantitative analysis. Correlation coefficients for the all three curves were higher than 0.99. For each type of the real food sample corresponding calibration curve (in the terms of matrices similarity) was used in order to decrease as much as possible the influence of matrices differences on the quantification results when using external calibration method. The samples analysed were from 13 EU countries: Austria, Belgium, Denmark, France, Germany, Greece, Ireland, Italy, Netherlands, Portugal, Spain, Sweden, United Kingdom, also from Switzerland and Norway. The typical GC/MS total and extracted ion chromatograms of baby food sample contaminated with 2-EHA are shown (see Figure 1).

Results and Discussion

The results on concentrations of 2-EHA in different type of baby food are summarized (see Table I). It shows that concentration of 2-EHA ranges from non-detectable (less than 0.1 mg kg -1 ) up to 3.4 mg kg -1 . The concentration of 2-EHA was below 0.1 mg kg -1 in 27 of the samples (42.9 %). In 26 samples (41.3 %) the amount of 2-EHA ranged between 0.1 and 0.5 mg kg -1 . The total percentage of the samples with the concentration of 2-EHA from non-detectable (less than 0.1 mg kg -1 ) to 0.5 mg kg -1 was 84 % (53 out of 63). The later values were consistent with the levels of 2-EHA determined in baby foods on the Norwegian market, which were from 0.08 to 0.6 mg kg -1 (NSCFS 2005).

Three baby food samples from Norway were analysed so far in this survey. The amounts found indicated non-detectable (less than 0.1 mg kg -1 ), 0.3 and 0.4 mg kg -1 (see Table I), which were within the range of former study (NSCFS 2005). The average concentration of 2-EHA was 0.6 mg kg -1 , not taking into account the samples in which the amount of 2-EHA was below 0.1 mg kg -1 . It's in good agreement with the results of twenty baby food samples from 11 German suppliers in which the average concentration of 2-EHA was 0.55 mg kg -1 (Elss et al. 2004). The concentration of 2-EHA in two samples from Germany showed the values: 0.28 and 1.2 mg kg -1 (see Table I) and were within the range of former study (Elss et al. 2004). Distribution of concentrations of 2-EHA in the baby food samples is shown (see Figure 2). Comparison of the amounts of 2-EHA found in baby food samples with different content (meat or fish, rice or pasta and vegetables; meat or fish and vegetables; rice or pasta and vegetables; vegetables) shows no distinct correlation. It seems that amount of fat or water and the other constituents in the final product not influences so much for the migration of 2-EHA into the food. Almost exclusively the highest values of 2-EHA were found in the samples from Spain (see Table I). More careful examination showed that 4 samples (out of 7 from Spain, with the concentration of 2-EHA from 1 to 3.4 mg kg -1 ) were delivered from one supplier. This supplier was not found in the information list of food samples, which were delivered from the other countries studied. Also it's worth to note that the concentration of 2-EHA in the two similar samples: "ES-V-05" and "ES-V-07" (see Table I), delivered from the same supplier, differs almost by factor of 10.

Taking into account the findings from this study it is possible to state that the concentration of 2-EHA in the food depends more on technological processes of thermal sterilization and has not so much to do with the content of the food. Also it might be possible that the amount of 2-EHA salts in PVC gasket seals correlates with the concentrations of the substance in the food (from this study it was not evident because the PVC gasket seals were not analysed). The presumption that the concentration of 2-EHA in the food depends on technological process is even more evident from the fact that 43 % of the samples from this study were non-contaminated with 2-EHA. No tolerable daily intake (TDI) or acceptable daily intake (ADI) for 2-EHA is established by either the EC Scientific Committee for Food (SCF), European Safety Authority (EFSA), Joint FAO/WHO Expert Committee on Food Additives (JECFA) or the U.S. Food and Drug Administration (FDA). The exposure assessment was based on the estimation of TDI -0.25 mg kg -1 /day for 2-EHA, which was taken from the opinion published by the Norwegian Committee for Food Safety (NSCFS 2005). The exposure assessment showed that intakes of 2-EHA do not exceed the TDI for infants of 6-12 months old except one occurrence when taking the worst-case food consumption study available and the highest level of contamination of the survey. In the other cases the levels of 2-EHA are at 13-fold below the TDI.

F

  ), metal lids covered with PVC gasket seals are not considered as plastic materials or articles, because they are not made exclusively from plastic. In this case only the general article 3 of the FrameworkRegulation 1935[START_REF] Ec | Occurrence of 2-ethylhexanoic acid in foods packed in glass jars[END_REF][START_REF] Ec | Occurrence of 2-ethylhexanoic acid in foods packed in glass jars[END_REF]) should be applicable as harmonized European legislation.2-EHA has been included in List "6B" by Scientific Committee of Food (SCF) as the substance suspected of having toxic properties (other than carcinogenic) (EC 2005).

Elss

  et al. (2004) used Likens-Nickerson simultaneous distillation-extraction (SDE) over 2 h with a pentane-diethyl ether mixture (1:1, v/v) for concentration of 2-EHA and fruit juices. Afterwards, the quantification was done by GC/MS (EI) in full scan mode using internal standard -2-methyl-1-pentanol. For determination of 2-EHA in urine samples Wahl et al. (2001) used cleaning procedure with Chromosorb P without prior extraction and subsequent silylation with MTBSTFA at 60

(

  Varian, USA) equipped with 1079 universal capillary injector and a triple quadruple mass spectrometer 1200L MS/MS (Varian, USA). The narrow bore (0.8 mm ID) deactivated SPME injection liner (Varian, USA) and VF-1ms fused silica capillary column (60 m; 0.25 mm ID; 0.25 µm) (Varian, USA) were used with the GC/MS. The instrument was operated under the following conditions: injector temperature -260 °C, for 2 min, then split mode until 3 min with split flow rate -50 ml/min and finally with split vent opened at the flow rate of 10 ml/min for carrier gas saving. Column oven temperature program: 50 °C (3 min), then 4 °C /min to 220 °C. Transfer line temperature -250 °C. The masspectromer was operated in EI ionisation (70 eV) mode by scanning the ions from 35 to 150 m/z at the first quadruple. Scan time -0.5 s; ion source temperature used -250 °C. Confirmation of the presence of 2-ethylhexanoic acid in the real samples was based on the retention time and on the mass spectrum of 2-EHA. For the quantification the sum of the most intense ions of 2-EHA fragmentation (m/z 73 and m/z 88) was used.

Figure 1 .

 1 Figure legends:

Figure 2 .

 2 Figure 2. Distribution of concentrations of 2-ethylhexanoic acid (2-EHA) in the baby food samples: baby food type (X axis); proportion of the samples, % (Y axis); concentration range of 2-EHA, mg kg -1 (Z axis).

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

Acknowledgements

We thanks all the countries, who provided baby food samples for this survey.

(available at http://ecb.jrc.it/DOCUMENTS/Classification-Labelling/Annex_VI.pdf). EC, 2003a, Commission Directive 2002/72/EC relating to plastic materials and articles intended to come into contact with foodstuffs. Official Journal, L 039, 1-42 (available at http//:europa.eu.int/comm/food/food/chemicalsafety/foodcontact/2002-72_en.pdf). EC, 2003b, Food contact materials, Practical Guide (available at http://ec.europa.eu/food/food/chemicalsafety/foodcontact/practical_guide_en.pdf). EC, 2004, Regulation (EC) No. 1935/2004 European Parlament and the Council of 27 October 2004 on materials and articles intended to come into contact with food repealing Directives 80/590/EEC and 89/109/EEC (available at http://europa.eu.int/eurlex/lex/LexUriServ/site/en/oj/2004/1_338/1_33820041113en00040017.pdf). EC, 2005, Synoptic document -provisional list of monomers and additives notified to European Commission as substances which may be used in the manufacture of plastics or coatings intended to come into contact with foodstuffs, updated 25 July 2005 (available at http://ec.europa.eu/food/fs/sfp/food_contact/synoptic_doc_en.pdf). EC, 2006, Food contact materials, substances listed in EU directives on plastics in contact with food (updated at 16 August 2006) (available at http://ec.europa.eu/food/food/chemicalsafety/foodcontact/eu_substances_aug2006_en. pdf).


Arvid ; Fromberg

Arne Højgård

Lene Duedahl-Olesen

Analysis of polycyclic aromatic hydrocarbons in vegetable oils combining gel permeation chromatography with solid phase extraction cleanup

Keywords: PAH, Process contaminants -PAH's Analysis, PAHs, benzo[a]pyrene, method validation, vegetable oil, sunflower oil, rapeseed oil, grape seed oil, sesame oil, GPC, SPE

Analysis of polycyclic aromatic hydrocarbons in vegetable oils combining gel permeation chromatography with solid phase extraction cleanup

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are a group of hundreds of organic compounds, which contain two or more fused aromatic rings made up of carbon and hydrogen. PAHs are formed by the incomplete combustion of organic material like wood, coal or oil. Studies of various matrices, such as coal combustion effluents, motor vehicle exhaust, used motor lubricating oil and tobacco smoke, have shown that PAHs are found in the outdoor air and the indoor environment. However, for non-smokers, human exposure to PAHs occurs mainly via food consumption (IPCS, 1998;European Commission, 2002). The occurrence of PAHs in foods is due to the formation of PAHs during food processing, such as drying, smoking, and domestic food preparation such as barbecuing, roasting and frying.

Although studies on animals exposed to individual PAHs, most notably benzo[a]pyrene, have shown various toxicological effects, such as haematological effects, reproductive and developmental toxicity and immunotoxicity, the critical effects are genotoxicity and cancer. Only very few single PAHs and a few complex mixtures have been toxicologically risk assessed. Recently, the European Scientific Committee on Food (SCF) has risk assessed 33 PAHs. The Committee concluded that 15 PAHs showed clear evidence of genotoxicity, and 14 of these were carcinogenic to animals (European Commission, 2002). However, SCF recommended to evaluate the usefulness of In 2001, a processing problem caused a general contamination of European olive pomace oils. The awareness of processing problems and the publication in 2002 of SCF's health risk assessment of PAHs exposure from foods (European Commission, 2002) has given rise to novel interests in the field of PAHs in foods. In April 2005, EC maximum levels for benzo [a]pyrene (European Commission, 2005) were adapted for e.g. oils and fats intended for human consumption or use as an ingredient in foods (2 µg kg -1 wet weight). At the same time, minimum performance criteria for sampling methods and for methods of analysis for the official control of benzo [a]pyrene levels in foodstuffs were adapted in a Commission Directive (European Commission, 2005a).

Commission recommendations on further investigation into the levels of the 15 EU priority PAHs in certain foods were also given (European Commission, 2005b).

Vegetable oils, naturally free of PAHs, are contaminated by technological processes like smoke drying of oil seeds or indirectly by environmental sources, such as exhaust gases from traffic or other combustion derived atmospheric particles deposited on the crops during growing. However, the PAHs content in vegetable oils can be radically reduced during refining, the final level depending on the refining conditions adopted [START_REF] Hopia | Margarines, butter and vegetable oils as sources of poilycyclic aromatic hydrocarbons[END_REF][START_REF] Larsson | Polycyclic aromatic hydrocarbons in crude and deodorized vegetable oils[END_REF]IPCS, 1998;Moret et al., 2000). Previously, benzo[a]pyrene concentrations below the European Commission maximum limit of 2 µg kg -1 have been reported for virgin olive oils [START_REF] Hopia | Margarines, butter and vegetable oils as sources of poilycyclic aromatic hydrocarbons[END_REF][START_REF] Moret | HPLC determination of polycyclic aromatic hydrocarbons in olive oils[END_REF][START_REF] Menichini | Polycyclic aromatic hydrocarbons in olive oils on the Italian market[END_REF][START_REF] Speer | Determination and distribution of polycyclic aromatic hydrocarbons in native vegetable oils, smoked fish products, mussels and oysters, and bream from the river Elbe[END_REF][START_REF] Lodovici | Polycyclic aromatic hydrocarbon contamination in the Italian diet[END_REF]European Commission, 2004). In comparison, surveillance data reflecting targeted sampling of vegetable oils showed that benzo[a]pyrene concentrations in olive pomace oils exceeded the European maximum limit for 83% of 268 samples (European Commission, 2004). [START_REF] Pupin | Benzo(a)pyrene in olive oils on the Brazilian market[END_REF] found benzo[a]pyrene concentrations for olive oils (n=7) of Argentine origin up to 164 µg kg -1 , with no further explanation for the high concentrations. Van der Wielen and co-workers (2006) reported benzo[a]pyrene concentrations up to 85 µg kg -1 in olive oils (n=170), while other vegetable oils (n=170) for human consumption only showed benzo[a]pyrene concentrations up to 9 µg kg -1 . Crude rapeseed oils have been reported to contain benzo[a]pyrene concentrations ranging from 1.4 to 64 µg kg -1 (Dennis et al., 1991), while grape seed oils have been reported to contain average benzo[a]pyrene concentrations of 4.2 µg kg -1 (n=92) (European Commission, 2004) and from 8.6 to 44 µg kg -1 (n=20) (Moret et al., 2000).

Sunflower oils (n=201) collected in seven European member states revealed an average value of 3.2 µg kg -1 benzo[a]pyrene (European Commission, 2004) with 29% of all samples exceeding the EU maximum limit of 2 µg kg -1 .

Literature including results of PAHs for types of vegetable oils other than olive oils have been rare and if other types of vegetable oils were included, only a few samples were reported e.g. one linseed and one sesame oil [START_REF] Speer | Determination and distribution of polycyclic aromatic hydrocarbons in native vegetable oils, smoked fish products, mussels and oysters, and bream from the river Elbe[END_REF], two refined [START_REF] Larsson | Polycyclic aromatic hydrocarbons in crude and deodorized vegetable oils[END_REF] three refined maize oils (Dennis et al., 1991). Furthermore no results have been presented for samples taken after the introduction of the EU maximum limit.

Previously, the German Society for Fat Science fixed a limit of 25 µg kg -1 for the total sum of PAHs and a limit of 5 µg kg -1 for the sum of PAHs of five or more aromatic rings [START_REF] Speer | Determination and distribution of polycyclic aromatic hydrocarbons in native vegetable oils, smoked fish products, mussels and oysters, and bream from the river Elbe[END_REF][START_REF] Swetman | Contamination of coconut oil by PAH[END_REF]. In addition a calculation of the sum of genotoxic PAHs analysed are included in the present study.

Traditionally, determination of PAHs has relied on procedures generally consisting of an extraction step (e.g. liquid-liquid partition or saponification) followed by one or more purification procedures (e.g. column chromatography or solid phase extraction (SPE)).

Analytical determination has usually been carried out by HPLC coupled to fluorometric or UV detection, or through high resolution capillary GC coupled to flame ionization detection (FID) or mass spectrometry (MS) [START_REF] Hopia | Margarines, butter and vegetable oils as sources of poilycyclic aromatic hydrocarbons[END_REF][START_REF] Larsson | Polycyclic aromatic hydrocarbons in crude and deodorized vegetable oils[END_REF]Dennis et al., 1991;[START_REF] Menichini | Polycyclic aromatic hydrocarbons in olive oils on the Italian market[END_REF]Moret et al., 2000;Moret and Conte 2000).

Among different purification procedures silica gel columns, used alone or in combination with other materials, have been widely used (Moret and Conte 2000). In 1996 Moret and co-workers evaluated the use of SPE silica cartridges for olive oils, and these cartridges were also employed by [START_REF] Swetman | Contamination of coconut oil by PAH[END_REF] for coconut oil.

The present study presents a semi-automatic method for the determination of PAHs in edible oils using a combined Gel Permeation Chromatography (GPC) and Solid Phase Extraction (SPE) clean-up. The clean-up techniques are based on those described by (1990). The method takes advantage of automatic injections and sample handling using a Gilson apparatus and is validated for the determination of a large number of important PAHs in vegetable oils. The analytical method is able to meet the criteria for the official control of benzo[a]pyrene levels in foods laid down by the Commission of the European Communities. The study also includes new data on olive oils and report values for oils commonly used for food preparation in Denmark, but rarely reported in the literature. These oils include rapeseed oils, sunflower oils, grape seed oils and sesame oil.

Methods and materials

Chemicals

All solvents used were HPLC-grade or pro analysi. Bio-Beads S-X3, 200-400 mesh was obtained from Bio-Rad Laboratories and Silica Isolute SPE columns 500 mg/3 ml from International Sorbent Technology Limited. Gel permeation chromatography (GPC) About 1.5 g of edible oil was dissolved in cyclohexane-ethyl acetate 1:1 (v/v), and the internal standard containing 13 C 6 or D 12 labelled PAHs was added. Half of the sample solution was then added, via a Gilson ASPEC XL apparatus, onto a glass column (15 x 500 mm) packed with pre-swollen and washed Bio-Beads S-X3 corresponding to 24 g dry material. The column was eluted using cyclohexane-ethyl acetate 1:1 (v/v) at a flow rate of 1.5 ml min -1 . The first fraction (0-64 ml) eluting from the column containing lipids was diverted to waste, after which the PAHs fraction (65-115 ml) was collected.

Subsequently the other half of the sample was cleaned up on the column and the combined PAHs fraction was collected, including in total 100 ml of eluate. The solvent was evaporated on a rotary evaporator and subsequently concentrated to 1 ml by blowing with nitrogen.

Silica column clean-up

A pre-packed silica SPE column was used to remove residue lipids and other disruptive compounds from the GPC cleaned sample using the Gilson ASPEC XL apparatus. After conditioning of the column with cyclohexane, the concentrated sample was added to the SPE column followed by elution using cyclohexane. The cleaned up sample was transferred into a GC-vial equipped with a pointed bottom and the solvent was evaporated to around 50 -100 µl and afterwards 100 µl of the internal injection standard (Benzo[e]pyrene D 12 ) was added to the GC-vial. Finally the sample was concentrated to 100 µl and analysed using GC-MS (gas chromatograph equipped with a mass spectrometer). The entire sample preparation procedure for sample preparation of edible oils is outlined in Figure 1.

GC-MS analysis

The GC-MS analyses were preformed using a 50m DB-5MS (J&W) column, 0.25 mm inner diameter, 0.25 µm film thickness. The ionisation mode was electron impact at 70eV. The carrier gas used was helium at 1 ml min -1 . 2 µl of the cleaned sample was injected splitless with a splitless time of 1 min. The injector was held at 280°C and the transfer-line at 280°C. The GC oven was held at 90°C for 1 min. then raised to 270°C at a rate of 7°C min -1 ; further raised to 280°C at a rate of 1°C min -1 and finally to 320°C at a rate of 5°C min -1 ; and held at 320°C for 10 min. The compounds were identified and quantified by comparing retention times and responses with those of standard mixtures.

The criteria for identification were same retention times as the standard within ± 2%, correct relative abundance of the two characteristic ions within ± 15%. Compounds formula, mol weight, retention times, Limit of detection (LOD) and limit of quantification (LOQ) are listed in Table 1.

[Insert Table 1 here]

Method validation

The analytical method has been validated according to the Danish guidelines for validation of methods in chemical analytical laboratories (Danak 2003) [START_REF] Iso | General requirements for the competence of testing and calibration laboratories[END_REF]. The validation protocol for validation of the analytical method included linearity, limit of detection, limit of quantification, repeatability and intermediate reproducibility. As a supplement to the method validation the measurement uncertainty was calculated according to the guidelines for expression uncertainty (GUM) [START_REF] Iso | IUPAC, IUPAP, OIML: Guide to the expression of uncertainty in measurement[END_REF]. Included in the calculations were the uncertainty of the calibration standards used in the method including the contributions from purity and gravimetric determination of standards and solvents used for preparation of the standards. In addition the relative standard deviation of the results were included.

Sampling of vegetable oils

The Regional Veterinary and Food Control Authorities in Denmark sampled vegetable oils from merchants. The samples included 52 samples of olive oil, 8 samples of rapeseed oil, 4 samples of sunflower oil, 4 samples of grape seed oil and one sample of sesame oil. All samples were for human consumption, to be used either in private homes or in restaurants or catering firms.

Results and discussion

Method development

A part of the method development included the selection of suitable internal standards. [START_REF] Bolt | The preparation of 13 C-labled standards of polycyclic aromatic hydrocarbons and other micropollutants: Synthetic and QA/QC considerations[END_REF] External standards of all the compounds in solvent were used for the determination of the calibration curves, using the approximate concentrations of 0 µg L -1 , 1 µg L -1 , 2 µg L -1 , 10 µg L -1 , 30 µg L -1 , 100 µg L -1 , and 250 µg L -1 in toluene to ensure a well fitted calibration curve. For the curve fit function, a linear regression line going through point of origin was chosen, i.e. a function (Y= αX) with Y as the area ratio between the compound and the internal standard, α as the slope and X as the concentration. All standard curves had correlation coefficients above 0.99 for the linear regression through the data points and therefore a satisfactory linear range. In Figure 2 the residual plot for the calibration curve of benzo[a]pyrene is displayed. All residuals were between ± 0.07 µg L -1 and the studentized residuals were between ± 2.

[Insert Figure 2 here]

Limit of detection and limit of quantification

In order to determine the limit of detection (LOD) and limit of quantification (LOQ) 14 olive oil samples were spiked with 1 µg kg -1 of the compounds. The individual PAH level of each of the spiked olive samples was determined using the analytical method, and concentrations were calculated. LOD and LOQ were calculated using three and six times the standard deviation on the calculated amount in the spiked samples, respectively. The study of sensitivity indicated that the limit of detection ranged from 0.2 to 1.5 µg kg -1 oil for the individual compounds, and the estimated LOQs ranged from 0.3 to 3.0 µg kg -1 oil (Table 1).

The combination of GPC and further SPE clean-up resulted in relatively clean extracts, avoiding problems with chromatographic interferences for most compounds. Absolute The recovery of each compound was calculated after the concentration in the unspiked sample (olive oil) was subtracted from that found in the spiked sample and divided by the theoretical spiked concentration. Generally the average recoveries for all three spiking levels were 60 to 120 percent, except for acenaphthene (59% for the recovery at 1 µg kg -1 ) and benzo[b]fluoranthene (142% for the recovery at 2 µg kg -1 ) which were influenced by coeluting peaks, and phenanthrene (up to 178%), fluoranthene (up to 188%) and pyrene (up to 153%) at the low spiking levels as the incurred level of the individual PAH in the unspiked sample was high compared to the spiking levels.

Considerable uncertainty was therefore applied to these results. This fact can also be where PRSD is calculated using the Horwitz equation [START_REF] Horwitz | Evaluation of analytical methods used for regulation of foods and drugs[END_REF] The recovery for the internal standards has been calculated using the internal injection standard (benzo[e]pyrene D 12 ) expressing the performance of the analytical method.

The average recoveries of the internal standards for the three spiking levels are between 79 and 109 percent (Table 3).

[insert Tabel 3 about here]

In order to ensure quality during method validation, all analytical GC-MS series included analyses of a NIST CRM 1647d reference material, consisting of a standard solution in acetonitrile containing all the PAH compounds, except benzo[j]fluoranthene, benzo[e]pyrene and perylene. Furthermore, all series included the clean-up and analysis of at least one unspiked oil sample. The method has also been tested in a interlaboratory study provided by the UK Food Analysis Performance Assessment Scheme (FAPAS®)

with excellent result e.g. Benzo[a]pyrene: FAPAS® assigned value 18.66 µg/kg, result 18.5 µg/kg, z-score 0.0 [START_REF] Fapas | Enviromental contaminants FAPAS ® Series 6 Round 18[END_REF].

Benzo[a]pyrene levels in vegetable oils

The average concentration of benzo[a]pyrene (Table 4), the total sum of PAHs, the sum of genotoxic PAHs, and the sum of light and heavy PAHs (Table 5) were calculated using half of the LOD for results below LOD. Of the 69 vegetable oils analysed only one sample of sunflower oil at 11 µg kg -1 benzo[a]pyrene exceeded the EU maximum limit of 2 µg kg -1 (subtracted into a separate line in Table 4). Two of four grape seed oils [Insert Table 4 here]

In the present study the data reported on rapeseed oils, the average concentration of benzo[a]pyrene and the sum of PAHs (Table 5), correspond to the low levels previously reported for two deodorized rapeseed oils [START_REF] Larsson | Polycyclic aromatic hydrocarbons in crude and deodorized vegetable oils[END_REF] and rapeseed oils collected in three member states (European Commission, 2004). Refined rapeseed oils have been reported with benzo[a]pyrene concentrations well above 2 µg kg -1 and with increased levels of benzo[a]pyrene even for crude rapeseed oils (1.4 -64 µg kg -1 ) (Dennis et al., 1991).

Levels of benzo[a]pyrene in sunflower oils were found to range from <0.2 to 0.8 µg kg - 1 , similar to the levels found by Speer and co-workers (1990).

In sesame oil, both the benzo[a]pyrene concentration (0.2 µg kg -1 ) and total sum of PAHs (11 µg kg -1 ) were similar to the levels found for rapeseed and sunflower oils.

However, only one sample was analysed and more data is needed for this type of oil. The total sum of PAHs for vegetable oils (Table 5) includes 17 PAHs and was calculated by omitting phenanthrene, which was not detected in this survey. For sunflower oils 50% or more of the total concentration of PAHs analysed were carcinogenic (3.7 µg kg -1 out of 7.4 µg kg -1 and 93 µg kg -1 out of 172 µg kg -1 ), while for the vegetable oils in general less than 25% of the total sum of PAHs were found to belong to the genotoxic compounds.

Total sum of PAHs in vegetable oils

[Insert Table 5 here] Three grape seed oils, three virgin olive oils and one single sunflower oil had a total sum of PAHs above the suggested limit of 25 µg kg -1 for the total sum of PAHs [START_REF] Speer | Determination and distribution of polycyclic aromatic hydrocarbons in native vegetable oils, smoked fish products, mussels and oysters, and bream from the river Elbe[END_REF][START_REF] Swetman | Contamination of coconut oil by PAH[END_REF]. While more and more PAHs are included in the analysis, the limit of 25 µg kg -1 for the total sum of PAHs has been remarked as unrealistic, especially if dealing with unrefined products such as extra virgin olive oil containing up to 110 µg kg -1 PAHs [START_REF] Speer | Determination and distribution of polycyclic aromatic hydrocarbons in native vegetable oils, smoked fish products, mussels and oysters, and bream from the river Elbe[END_REF][START_REF] Moret | HPLC determination of polycyclic aromatic hydrocarbons in olive oils[END_REF]. For data obtained in this study it is important to notice that when omitting phenanthrene, the PAH compound most abundant in e.g. grape seed oil, lower total sums of PAHs are obtained which might explain the lower results.

The average sum of PAHs in the virgin olive oil samples was in agreement with previous studies [START_REF] Menichini | Polycyclic aromatic hydrocarbons in olive oils on the Italian market[END_REF][START_REF] Moret | HPLC determination of polycyclic aromatic hydrocarbons in olive oils[END_REF]Moret and Conte, 2000) found to be higher than the average sum of PAHs for the olive oils (a blend of refined and virgin oils) (Table 5). These results has previously been explained by the olive oil blend being exposed to a PAHs reduction in the oil refining processes [START_REF] Larsson | Polycyclic aromatic hydrocarbons in crude and deodorized vegetable oils[END_REF].

Two grape seed oils and two sunflower oils had a sum of heavy PAHs (5 or more aromatic rings) exceeding 5 µg kg -1 . Using the limit of the sum of heavy PAHs corresponds well to the fact that 12 of 15 genotoxic compounds assessed by SCF (European Commission, 2002) contain five or more aromatic rings.

Comparing the level of light PAHs for all oil samples, only sunflower (app. 50%) and rapeseed oils (70%) have levels of light PAHs less than 90% of the total sum of PAHs, indicating the use of a refining process. For vegetable oils the refining process includes a bleaching step, a treatment with activated carbon removing a considerable amount of PAHs, while the deodorizing step removes some of the light PAHs and has little effect on the heavy PAHs. Therefore a total reduction in the amount of light PAHs indicates the inclusion of a refining step in the vegetable oil production.

Conclusions

The presented analytical method makes it possible to meet the performance criteria for the limit of detection (LOD) and the recovery, as described in the EC document laying down methods of analysis for the official control of the levels of benzo[a]pyrene in foods. The criteria for LOD is set to no more than 0.3 µg kg -1 and the recovery between 50% and 120%. Horrat values for the spiking level 1 µg kg -1 , 2 µg kg -1 and 3 µg kg -1 .

F

Recovery [%] RSD r [%] RSD R [%]

Horrat value 1 µg kg -1 2 µg kg -1 3 µg kg -1 1 µg kg -1 2 µg kg -1 3 µg kg -1 1 µg kg -1 2 µg kg -1 3 a) µg kg -1 1 µg kg -1 2 µg kg -1 3 µg kg

  pyrene as a marker for the carcinogenic PAHs in food and to evaluate the risk assessment of the carcinogenicity of PAHs in food based on the level of benzo[a]pyrene.

  for spiked olive oil samples relative to the internal standard (ISTD) added to the sample before clean-up. An example of the chromatogram for benzo[a]pyrene spiked at low level (1 µg kg -1 ) and the correlating internal standard benzo[a]pyrene D12 are illustrated in Figure3left and right, respectively. ]fluoranthene, being identified using the same mass (m/z: 252), were only partly resolved. The peaks were split into two at the valley and quantified separately during the first part of the method development and method validation. However, it was recognised that it was not possible to quantify the two compounds individually, as high standards derivations were observed during validation mainly expected to be due to the problem of splitting the peaks.Recovery, repeatability, intermediate reproducibility and measurement uncertaintyIn order to validate the analytical method, recovery results as well as the repeatability and intermediate reproducibility for olive oil spiked with the compounds were determined (Table2). Three concentrations were used for spiking the oil during the method validation. For spiking level 1 approximately 1 µg kg -1 of PAHs standard was added to the olive oil, for spiking level 2: approximately 2 µg kg -1 was added and for spiking level 3: approximately 3 µg kg -1 was added. The repeatability includes analyses of a number of individual samples cleaned up and analysed in the same batch. The intermediate reproducibility includes clean-up and analysis of samples by different analysed in separate series using individual calibration curves and spiked samples made with two different olive oils.

  seen from the repeatability and intermediate reproducibility results. The HORRAT values were calculated (McClure and Lee, 2003): HORRAT= RSD [%] / PRSD [%]

  a]pyrene concentrations of 1.7 and 1.8 µg kg -1 . All the other samples have contents below the EU maximum limit set in April 2005(European Commission, 2005), and most samples have contents even below the LOD.

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. Procedure for sample preparation of edible oils.

  observed instability of deuterium labelled PAHs due to the loss of deuterium from the labelled compounds. Carbon-13 labelled compounds are therefore

	preferred. However, benzo[a]pyrene D12 was preferred as the internal standard for
	benzo[a]pyrene, as carbon labelled benzo[a]pyrene only contains four carbon-13 atoms
	per molecule and a test showed a peak at the retention time and m/z of benzo[a]pyrene
	probably due to loss of H from the molecule or due to benzo[a]pyrene as impurity in the
	labelled standard. Difficulties with the loss of deuterium from benzo[a]pyrene D12 have
	F o r not been observed. Bolt and co-workers did not report any problems with the loss of deuterium from benzo[a]anthracene, and benzo[a]anthracene D12 was therefore used as the internal standard. F o r
		P P
	Calibration curves	e e r e e r
		R R
		e e
		v i e v i e
		w w
		O n l O n l
		y y
		10 11

Table 1 .

 1 Using the method for the determination of the levels of polycyclic aromatic hydrocarbons in vegetable oils revealed low levels of the compounds in many of the olive oil samples and other vegetable oils with higher levels. Olive oils of virgin origin were confirmed as having higher concentrations of PAHs compared to olive oils (a blend of refined and virgin oils) with a slightly lower fraction of light PAHs in olive oils (80% compared to 90% for virgin oils). Generally, the benzo[a]pyrene levels and the sum of PAHs for olive oils, including virgin olive oils, was lower in this study than the levels previously reported. A low fraction of light PAHs was remarked for the sunflower oils, while grape seed oils contained the highest concentrations of benzo[a]pyrene, heavy PAHs, genotoxic PAHs as well as the total sum of PAHs. Compounds included in the method and method performance. LOD and LOQ expressed as µg kg -1 oil.

	o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	19

,2,3-c,d]pyrene D 12 C

	F o r P e e r R e v i e w	O n l y
		22 D 12	288.4	43.9	-	-
	Dibenzo[a,h]anthracene	C 22 H 14	278.4	44.2	0.9	1.8
	Benzo[g,h,i]perylene	C 22 H 12	276.3	45.2	0.2	0.3
							1

Table 2 .

 2 Recovery, repeatability relative standard deviation (RSD r ), intermediate reproducibility relative standard deviation (RSD R ) and

Table 3 .

 3 Recovery of labelled compounds for the spiking level 1 µg kg -1 , 2 µg kg -1 and 3 µg kg -1 [percent].

	-1

Table 4 .

 4 Average, ranges and median of benzo[a]pyrene concentration in µg kg -1 for vegetable oils analysed with country of origin including values for references.Deod. = deodorized oil. Origin codes: AT = Austria, BE = Belgium, DE = Germany, DK = Denmark, ES = Spain, FR = France, GR = Greece, IT = Italy, NL = Holland.

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	1

Table 5 .

 5 Average and ranges of total sum of PAHs, sum of genotoxic PAHs and sums of light and heavy PAHs in µg kg -1 for vegetable oils analysed. ) Genotoxic PAHs include the sum of benzo[a]anthracene, chrysene, benzo[b+j]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, indeno[1,2,3-c,d]pyrene, dibenzo[a,h]anthracene, and benzo[g,h,i]perylene. b) Light PAHs (4 or less aromatic rings) include the sum of acenaphthylene, acenaphthene, fluorene, anthracene, fluoranthene, pyrene, benzo[a]anthracene and chrysene c) Heavy PAHs (5 or more aromatic rings) include the sum of benzo[b+j]fluoranthene, benzo[k]fluoranthene, benzo[e]pyrene, benzo[a]pyrene, perylene, indeno[1,2,3-c,d]pyrene, dibenzo[a,h]anthracene, benzo[g,h,i]perylene.

	Vegetable oil	N Total PAHs	Genotoxic PAHs a)	Light PAHs b)	Heavy PAHs c)
			Average	Average	Average	Average
	Olive oil, extra
	virgin	46	16	2.7	15	1.6
	Olive oil	6	8.1	1.9	6.8	1.3
	Rapeseed oil	8	5.5	1.3	4.0	1.5
	Sunflower oil	3	7.4	3.7	3.8	3.7
	Sunflower oil	1	172	93	97	75
	Grape seed oil	4	42	11	35	7.2
	Sesame oil	1	11	1.6	9.8	1.2

a

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

Acknowledgements

We wish to thank Vera Klausen and Vibeke Balswel from the Danish Institute for Food and Veterinary Research, as well as Anni Mårbjerg Thomsen and Thomas Dyekjaer from the Regional Veterinary and Food Administration Center, Region North for their skilful technical assistance with the chemical analyses.


Natthasit Tansakul

Frank Niedorf

Manfred Kietzmann

Sulfadimidine as a model to evaluate pharmacokinetics and residues at various concentrations in laying hen

Keywords: Veterinary drug residues -sulphonamides Food Types: Animal feedingstuffs, Eggs sulfadimidine, laying hen, pharmacokinetic, residue

A low level intake of drugs from the ingestion of contaminated feed may lead to residue problems in food animals. Sulfadimidine (SDD) was used as a model to determine the residue risk at various doses in laying hens. The drug was administered as a single intravenous injection (100 mg kg -1 body weight, BW), as a single oral dose (100, 30, 10, 3, 1 mg kg -1 BW) and via medicated feed for seven consecutive days (30, 10, 3 mg kg -1 BW). Drug levels were determined with HPLC-UV for plasma, yolk and albumen. Pharmacokinetic values, which were calculated using a first-order one-compartment model, residue levels and transfer rates into the eggs, were found to be dose-dependent. Even low doses at 3 and 1 mg kg -1 BW resulted in measurable residues in yolk and albumen one day after a single oral administration. After ingestion of medicated feed at 3 mg kg -1 BW mean drug levels at 0.14 ± 0.01 µg g -1 were found in albumen and at 0.09 ± 0.01 µg ml -1 in plasma. Generally, the residue levels in albumen and plasma were higher than in yolk. These findings demonstrate a residue risk for the consumer even after low level intake of drugs.

Introduction

Antibiotics and coccidiostats are of widespread use in food animals. Improper use or crosscontamination during manufacture of animal feed with these compounds may cause residue problems. Cross-contamination of feed is a well-known problem and may be due to an improper construction of the equipment, poor dust control or inadequate clean-out procedures between sequential batches of feed [START_REF] Fda | Unsafe contamination of animal feed from drug carryover[END_REF]. Even very low levels of antibiotic contamination may result in relevant residues in food, as confirmed by studies on coccidiostats in poultry feed [START_REF] Rokka | The residue levels of narasin in eggs of laying hens fed with unmedicated and medicated feed[END_REF][START_REF] Kennedy | Monensin carry-over into unmedicated broiler feeds[END_REF]) and sulfadimidine in swine [START_REF] Biehl | Sulfamethazine residues in swine[END_REF][START_REF] Mccaughey | Tissue residues in pigs fed on meal contaminated with sulphadimidine during mixing[END_REF]). Sulfonamides are veterinary drugs routinely used in farm animals, and sulfonamide residues in animal products such as egg and milk have recently been investigated [START_REF] Furusawa | Transfer and distribution profiles of dietary sulphonamides in the tissues of the laying hen[END_REF][START_REF] Cavaliere | A simple and sensitve liquid chromatography-mass spectrometry confirmatory method for analyzing sulfonamide antibacterials in milk and egg[END_REF][START_REF] Juhel-Gaugain | European proficiency testing of national reference laboratories for the confirmation of sulfonamide residues in muscle and milk[END_REF]. We therefore used sulfadimidine in laying hens as a model to study the impact of different dosages of sulfadimidine on its pharmacokinetics and risk of residue build-up in eggs.

Briefly, sulfadimidine (SDD) which is also known as sulfamethazine is one of the drugs regularly used in poultry husbandry for its broad-spectrum activities against bacterial and coccidial infections in chicken. The European Union (EU) and the Codex Alimentarius Commission have established a maximum residue limit of sulfonamides (MRL = 0.1 µg g - 1 ) for many kinds of animal products; however, no MRL is established for eggs. Sulfonamides are therefore unapproved for administration in laying hens (EEC 2377/90, Codex Alimentarius).

Previous studies, pharmacokinetic behaviors of SDD in chicken have been described [START_REF] Kietzmann | Beitrag zur Pharmakokinetik von Sulfonamiden beim Geflügel[END_REF][START_REF] Geertsma | Residues of sulphadimidine and its metabolites in eggs following oral sulphadimidine medication of hens[END_REF][START_REF] Nouws | Plasma disposition and renal clearance of sulphadimidine and its metabolites in laying hens[END_REF], and Jian & Punia, 2001). Different doses and routes of SDD residues have also been studied in laying hen [START_REF] Blom | Plasma half-life and the excretion into egg-white and -yolk of three sulphonamides and pyrimethamine after medication of laying hens[END_REF][START_REF] Seib | Zum Rückstandsverhalten von Sulfadimidin in Eiern, Follikeln und Geweben von Legehennen[END_REF][START_REF] Roudaut | Sulphonamide residues in eggs following drug administration via the drinking water[END_REF][START_REF] Furusawa | Transference of dietary verterinary drugs into eggs[END_REF][START_REF] Kan | Residues of veterinary drugs in eggs and possible explanation for their distribution between egg white and yolk[END_REF]. These studies indicated a transfer of sulfonamides into the eggs, but mainly provided data focusing on therapeutic doses. Therefore, information about the pharmacokinetic behaviors and residue risk following the administration of SDD at low doses are scarce. As the extrapolation of high to low-level kinetics is generally regarded to be inadequate [START_REF] Braun | Sources of uncertainty in pharmacokinetic prediction[END_REF] this study was conduced to clarify the residue risk after low-level ingestion of sulfadimidine in laying hens.

Materials and methods

Animals: Healthy laying hens (Lohmann Brown), 22-26 weeks of ages (1.95 ± 0.15 kg), were housed at controlled temperature (20 ± 2°C) and light (14 h day -1 ) in accordance with the animal welfare acts (33-42502-04/917, LAVES). Water and a commercial standard laying hen mash (deuka Deutsche Tiernahrung, Düsseldorf, Germany) were supplied ad libitum.

Reagents and chemicals: All reagents and chemicals used were of HPLC or analytical grade. Acetonitrile, ethyl acetate and n-hexane were obtained from Labscan (Dublin, Ireland). Phosphate hydrogen monohydrate, sodium chloride, hydrochloric acid (Merck, Germany) and 85% acetic acid (AppliChem, Germany) were used. A 0.05 mol l -1 phosphate buffer, pH 3, was prepared by dissolving 0.68 g of phosphate monohydrate with 1000 ml of high purity water, and then adjusted to pH 3 with 85% acetic acid.

Standards: Sulfadimidine (SDD) and sulfadoxine (SDX) were purchased from Sigma-Aldrich (Steinheim, Germany). SDX, as internal standard, was dissolved in methanol at concentrations of 50 µg ml -1 . SDD standard solutions were prepared in distilled water at concentration from 1 to 0.001 mg ml -1 .

Drug administration and sample collection: Groups of six hens each were treated with SDD via intravenous injection (i.v., 1 group), via a single bolus oral administration (p.o., 7 groups), or via medicated feed (3 groups). For the i.v. group SDD was injected as a single bolus via the vena axillaris at 100 mg kg -1 body weight (BW). For the oral administration of a single dose, 100, 30, 10, 3, 1 mg kg -1 BW were used. The three groups receiving medicated feed were treated for seven consecutive days with mixed feed resulting in a dose of 30, 10 and 3 mg kg -1 BW per day. For the analysis, blood samples of 1.5 ml were taken from the brachial vein at pre-determined intervals and heparinised to prevent blood clotting. These were centrifuged at 4100 g for 10 min to separate the plasma, which was placed in a 2 ml micro centrifuge vial for drug extraction. Eggs were collected for 7 days, starting 20 h after the treatment. Egg yolk and egg albumen were separated, and placed into 10 ml polypropylene test tubes. All samples were stored at -20°C pending analysis.

Plasma extraction: 10 µl of SDX solution (50µg ml -1 ) were added to 490 µl of plasma along with 100 µl of 0.1 N HCl and 1 ml of ethyl acetate. After mixing in a vortex mixer for 1 min and centrifugation at 23100 g for 5 min, 600 µl of the supernatant were evaporated under a gentle N 2 stream at 40°C. Yolk extraction: 1 g of yolk was weighed into a 10 ml test tube. The sample was mixed with 2.9 ml distilled water and internal standard (10 µl of 100µg ml -1 of SDX). Sulfonamides were extracted after adding 0.5 ml of 0.1 N HCl and 2 ml of ethyl acetate by mixing a vortex mixer for 2 min. An excessive amount of NaCl (approximately 1.2 g), 100 µl of 1 N HCl and 500 µl of n-hexane were added to break the emulsion. After mixing for 2 min and centrifugation at 4100 g for 5 min, 1.5 ml of the supernatant was taken into a second 10 ml test tube. The remainder was extracted a second time. For this, 1.5 ml ethyl acetate were added, then mixed for 2 min and centrifuged at 4100 g for 5 min. 1.5 ml of the upper phase were combined with the previous one and evaporated to dryness under a gentle stream of N 2 .

Albumen extraction: A 1 g portion of albumen was weighed into a test tube, and 0.5 ml distilled water and 10 µl of SDX (100µg ml -1 ) were added along with 0.5 ml of 0.1 N HCl, 0.9 ml of acetonitrile and 0.1 ml of n-hexane. After mixing in a vortex mixer for 2 min, excessive NaCl (approximately 1.2 g) and 2 ml of ethyl acetate were added. After mixing for 2 min and centrifugation at 4100 g for 5 min 2 ml of the upper phase were evaporated under a gentle N2 stream at 40°C. All residues were reconstituted in 700 µl of eluent and centrifuged at 23100 g for 5 min. A 100 µl aliquot of the clear lower phase was injected into the HPLC system.

HPLC apparatus, conditions and validation:

The HPLC apparatus consisted of an UV detector; Varian ® 2050, solvent delivery system; Beckman ® , programmable solvent module 116, an auto sampler; Gilson, model 231, chromatographic software; 32-Karat™ Software, version 3.0. Separation was achieved on an analytical column (LiChrospher 100 RP-18, endcapped, 125×4, 5 µm; Merck, Germany) and guard cartridge. The temperature was set to 40°C and controlled with a column oven. A mobile phase consisting of 0.05 mol l -1 phosphate buffer (pH 3.0) and acetonitrile at 85:15, v/v and 89:11, v/v was used for plasma and egg analysis, respectively. A flow rate of 1.5 ml min -1 and a wavelength of 268 nm were used throughout.

The method was validated according to FDA requirements (FDA 2001) and was found to have a limit of quantification (LOQ) of 0.025 µg ml -1 for plasma and 0.05 µg g -1 for both albumen and yolk. The limits of detection (LOD) were established to be 0.01 µg ml -1 for plasma and 0.02 µg g -1 for eggs as determined from the calibration curve according to DIN 32645 (1994). As shown in figure 1 no interferences were detected at the retention times of SDD and the internal standard SDX (I.S.). The method was linear in the range between LOQ and 100 µg ml -1 (µg g -1 ) (R²= 0.999). Accuracy, intra-and inter-day precision were determined using quality control samples at concentration of 0.025-0.10 µg ml -1 . For both plasma and eggs, accuracy as expressed as the relative deviations from expected values was less than 15%. Intra-and inter-day precision were less than 10 % except at the LOQ of plasma (16.9%). The recoveries were 81-94 %, 88-97 % and 90 % for plasma, yolk and albumen, respectively.

Pharmacokinetic parameters and drug transfer rate calculation: Pharmacokinetic parameters were calculated using WinNonLin® version 4.1, Pharsight® (USA). A firstorder one-compartment model was used for the calculations. The area under the curve (AUC), half life of absorption (K01_HL), elimination half life (K10_HL), total body clearance (Cl), time of maximum plasma concentration (T max ) and maximum plasma concentration (C max ) were calculated.

For the calculation of the drug transfer rate (TR) into the eggs, the sum of residues (R total ) in albumen and yolk were calculated using the measured concentrations in (c albumen , c yolk ) and mass of albumen and yolk (m albumen , m yolk ) at day i as:

[1] i yolk i yolk i albumen i albumen i total m c m c R , , , , , ⋅ + ⋅ =
For the mass of yolk and albumen, an average weight of six representative eggs was used (13.84 g and 36.28 g, respectively). The transfer rate was calculated from the sum of residues of all eggs measured up to 10 days after administration as:

[2] 100 [%] 10 1 , ⋅ = ∑ = = dose R TR t i i total

Results

The semi logarithmic plots of plasma concentrations vs. time curves of SDD (Mean ± SD) following i.v. and p.o. application are given in figure 2. Pharmacokinetic parameters were not calculated for the p.o. group following 1 mg kg -1 BW due to insufficient data quality. SDD was very quickly eliminated from the plasma. Within 6-32 h after p.o. administration at various doses, the concentrations were lower than the LOQ. The pharmacokinetic parameters calculated assuming first order kinetics are given in table I. After single oral doses, the C max, T max and the K10_HL increased while the Cl decreased with increasing doses (see figure 3).

[Insert figure 1 The residue levels in eggs after single oral doses are summarized in tables II and III. In general, SDD was detectable for longer in yolk than in albumen and plasma. However, in the initial phases considerably higher concentrations of SDD were measured in albumen and plasma compared to yolk. In yolk, residues above the LOQ were detectable up to 8 days following the normal therapeutic dose (100 mg kg -1 BW). By using lower doses of 30, 10, and 3 mg kg -1 BW, drug levels over the LOQ were measured up to 7, 4 and 1 day post single oral doses, respectively. No drug levels in yolk above the LOQ were measured after treatment with 1 mg kg -1 BW. In albumen, drug concentrations above the LOQ appeared for 6, 3, 1, 1, and 1 days after treatment with 100, 30, 10, 3 and 1 mg kg -1 BW, respectively. The calculated transfer rates of SDD into the eggs after single oral doses are dose dependent (table IV). Decreasing doses are related with a diminishing transfer rate.

During treatment with medicated feed for 7 days, approximately equal concentrations were measured in plasma and albumen but higher than yolk (table V), for groups ingesting 30 and 10 mg kg -1 BW. In the group of 3 mg kg -1 BW, residue levels in albumen exceeded even in plasma, but concentrations were lower than LOQ in yolk. At all concentrations, residues rapidly accumulated in eggs, as proved by high concentrations of SDD in eggs collected 20 h after treatment.

[Insert

Discussion

We used sulfadimidine as a model to estimate the risk of a residue build-up in eggs after unintended low-level intake. At the therapeutic dose, pharmacokinetic values in this present study consistent with former studies [START_REF] Geertsma | Residues of sulphadimidine and its metabolites in eggs following oral sulphadimidine medication of hens[END_REF][START_REF] Nouws | Plasma disposition and renal clearance of sulphadimidine and its metabolites in laying hens[END_REF], and Jian & Punia 2001). In general, SDD is known to be rapidly absorbed [START_REF] Botsoglou | Drug residues in foods: Pharmacology, Food safety, and Analysis[END_REF] and quickly eliminated [START_REF] Nouws | Plasma disposition and renal clearance of sulphadimidine and its metabolites in laying hens[END_REF]) after oral therapy. At low doses, however, our data demonstrate that SDD is absorbed even more rapidly in a dose dependent manner, resulting in a decrease of the calculated absorption rate half-life (K 01_HL) to 0.2 h at 3 mg kg -1 BW as compared to 1.2 h at 100 mg kg -1 BW. Correspondingly, the T max value decreases from 4 h to 0.5 h. Furthermore, decreasing doses are associated with larger volumes of total body clearance resulting in dose dependent shorter elimination half-life, which decrease from 9.1 h for 100 mg kg -1 BW to 0.9 h for 3 mg kg -1 BW. Rate-limited absorption may explain the deviations from dose independent total clearance, which is expected using first order kinetics. However, the nearly equal clearance after i.v. and p.o application with the same dose (100 mg kg -1 BW) suggests that in fact also the excretion is rate-limited, with both processes resulting in over-proportional decrease of the AUC after decreasing doses of SDD. The underlying physiologic mechanisms involved in this complex pattern of absorption and excretion of SDD in chicken remain to be clarified. The limited data gathered in this study however do not allow for further interpretations or the application of more complex pharmacokinetic models. Nevertheless, as first order kinetics may not be readily assumed for SDD in chicken, pharmacokinetic parameters formerly established on therapeutic levels can not be extrapolated on subtherapeutic levels. The impact of this on residue levels in eggs is clearly reflected by the transfer rate of SDD, which decreases correlating with the dose.

Our data nevertheless demonstrate that measurable residue levels may occur in eggs even after a single low-level intake at only 1% of the recommended dose. Moreover, during ingestion of feed supplemented with 3% of the therapeutic dose, these residue levels are higher than the MRL of 0.1 µg g -1 established for other tissues throughout.

Interestingly, the accumulation of SDD in different compartments changes with time. According to our results, the SDD concentration at initial phases after single oral administrations and throughout medication via feed is 2 to 4 times higher in albumen than in yolk. However, after single oral doses the SDD level in yolk exceeded that in albumen after about 2 days, supporting previous studies (Geertma et al. 1987[START_REF] Shaikh | Distribution of total 14 C residue in egg yolk, albumen, and tissues following oral ( 14 C) sulfamethazine administration to hens[END_REF]. In addition, SDD residues in yolk were detectable longer after a single oral administration of the therapeutic dose (up to the 8 th day as compared to the 6 th day in albumen, whereas it was eliminated from plasma after only 32 h). We therefore confirm the use of yolk as appropriate matrix for monitoring SDD residues as previously suggested for sulfaquinoxaline [START_REF] Shaikh | Determination of 14 C residue in egg of laying hens administered orally with ( 14 C) sulfaquinoxaline[END_REF]). The reason for this complex pattern of time depended selective accumulation in certain compartments remains unclear, as recently concluded by [START_REF] Kan | Residues of veterinary drugs in eggs and possible explanation for their distribution between egg white and yolk[END_REF], who described none of the formerly studied physicochemical substance properties like pKa value, lipid solubility and protein binding to be clearly linked to the pattern of distribution of sulfonamides between plasma, yolk and albumen.

In conclusion, our study demonstrates the existence of a residue risk due to a low level intake of SDD, as the ingestion of the drug at only 1% of the therapeutic dose resulted in measurable concentrations in eggs. The finding of a clear dose dependence of its pharmacokinetics is of special interest for other substances, as it clarifies, that residue risks should be examined at relevant concentrations and may not be estimated from known highlevel kinetics. ) *, †, ‡ 5, 4 and 3 eggs laid and analyzed, respectively

Figure 1 .

 1 Figure 1. Typical chromatogram obtained from control plasma (B), albumen (D) and yolk (F), respectively. Parts A, C and E show typical chromatogram of fortified plasma (25 ng mL -1 ), albumen and yolk (50 ng g -1 ), respectively. Black arrow indicate peak of sulfadimidine (I.S.= internal standard).

  Figure 3. C max , K10_HL, T max and Cl values of SDD in plasma after single oral administration of different doses (Mean ± SD, n=6).

  indicated while *, † and ‡ = 5, 4, 3 eggs laid and analyzed, respectively. ND = not detected (< LOD)

Figure 1 .

 1 Figure 1. Typical chromatogram obtained from control plasma (B), albumen (D) and yolk (F), respectively. Parts A, C and E show typical chromatogram of fortified plasma (25 ng mL -1 ), albumen and yolk (50 ng g -1 ), respectively. Black arrow indicate peak of sulfadimidine (I.S.= internal standard).

  Figure 3. C max , K10_HL, T max and Cl values of SDD in plasma after single oral administration of different doses (Mean ± SD, n=6).

table II

 II

	about here]
	[Insert table III about here]
	[Insert table IV about here]
	[Insert table V about here]

Table I .

 I Pharmacokinetic characteristics of sulfadimidine in plasma after single intravenous (i.v.) and oral (p.o.) administration in laying hen (Mean ± SD, n=6).

						I.S.
		1000.000						A	i.v. 100 mg/kg
								B	p.o. 100 mg/kg
	I.S. -100 (p.o.) 1379.3 ± 428.1 1.2 ± 0.4 9.1 ± 1.6 82.5 ± 40.0 4.0 ± 0.9 76.5 ± 19.9 D C F o r 0.010 0.100 1.000 10.000 100.000 p.o. 1 mg/kg F o r 100 (i.v.) 1122.0 ± 245.8 5.9 ± 1.1 94.1 ± 27.8 -132.2 ± 16.9 30 (p.o.) 151.5 ± 27.5 0.7 ± 0.6 2.8 ± 0.7 204.2 ± 41.6 1.5 ± 0.8 25.3 ± 5.8 10 (p.o.) 18.5 ± 4.1 0.4 ± 0.4 1.4 ± 0.8 563.2 ± 119.2 0.7 ± 0.5 6.2 ± 1.8 3 (p.o.) 4.2 ± 1.2 0.2 ± 0.3 0.9 ± 0.2 797.3 ± 337.1 0.5 ± 0.3 2.4 ± 1.1 Plasma concentration ( p.o. 3 mg/kg mg kg -1 h µg ml -1 µg h ml -1 h h ml h -1 kg -1 µ g ml ) p.o. 30 mg/kg -1 p.o. 10 mg/kg Dose T max C max AUC K01_HL K10_HL Cl
	0 Figure 2. Plasma concentrations of sulfadimidine (Mean ± SD) versus time after a single 2 4 6 8 10 12 14 16 Time (Min) E I.S. P e e r 0.001 0 6 12 18 24 30 36 42 48 Time (h) intravenous (i.v.) and oral (p.o.) administration (n=6). AUC, area under the curve; K01_HL, half life of absorption; K10_HL, elimination P r e half life; Cl, total body clearance; T max , time of maximum plasma concentration; e C max , maximum plasma concentration.
	0	2	4	6	8 Time (Min)	10	12	F R 14 16 e v i e w R e v i e w
								O n O n
								l y l y
									11

Table II .

 II Sulfadimidine residues in yolk (µg g -1 , Mean ±SD) after single oral administration of different doses.

	Dose (mg kg -1 BW)
	Day after treatment
	1	14.32 ± 6.47 * 3.08 ± 1.52 * 0.42 ± 0.08 0.11 ± 0.05
	2	6.62 ± 2.72 * 0.51 ± 0.01 ‡ 0.07 ± 0.02
	3	3.67 ± 1.17 * 0.44 ± 0.12 * 0.06 ± 0.02 *
	4	2.74 ± 0.69 * 0.32 ± 0.05 †
	5	1.98 ± 0.56 * 0.20 ± 0.04 ‡
	6	1.21 ± 0.59 * 0.13 ± 0.02 *
	7	0.43 ± 0.32 * 0.07 ± 0.03 ‡

Table III .

 III Sulfadimidine residues in albumen (µg g -1 , Mean ±SD) after single oral administration of different doses.TableIV. Transfer rate of SDD into eggs after single oral administration. TableV. Residue concentration of Sulfadimidine (Mean± SD) in plasma, albumen and yolk following medicated feed of different doses (30, 10 and 3 mg kg -1 BW) for seven consecutive days.

								Page 16 of 25
	Dose (mg kg -1 BW)	100	30	10	3	1
	Day after treatment Dose Drug	Residue	Transfer-rate c
		1	49.34 ± 7.03 * 6.25 ± 1.85 * 0.98 ± 0.24 0.28 ± 0.12 0.07 ± 0.02 intake a in whole egg b
	2 (mg kg -1 BW)	6.10 ± 0.43 * 0.16 ± 0.04 † (mg) (µg) (%)	ND	ND	ND
	100	3	195	0.83 ± 0.16 * 0.15 ± 0.14 * 2497.16 1.28	ND	ND	ND
	4 5 6 7 8 n=6 unless otherwise indicated while *, †, ‡ = 5, 4, 3 eggs laid and analyzed, 0.38 ± 0.12 * 0.20 ± 0.07 * 0.12 ± 0.03 * ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND < 0.05 ND ND ND respectively. ND = not detected (< LOD) P Day 7 1.48 ± 0.95 0.37 ± 0.12 0.09 ± 0.02 2.97 ± 0.90 † 0.43 ± 0.14 0.13 ± 0.01 r Day 6 2.91 ± 1.08 0.31 ± 0.10 0.07 ± 0.01 2.84 ± 1.25 * 0.40 ± 0.12 0.14 ± 0.02 e Day 5 2.36 ± 2.41 0.43 ± 0.07 0.10 ± 0.03 3.77 ± 1.25 * 0.45 ± 0.06 0.13 ± 0.02 * 1.80 ± 0.52 * 0.16 ± 0.02 <0.05 1.64 ± 0.55 † 0.20 ± 0.02 <0.05 1.55 ± 0.45 * 0.20 ± 0.02 <0.05 e c calculated according to equation [2] Day 4 3.99 ± 1.52 0.44 ± 0.05 0.09 ± 0.03 4.99 ± 1.93 * 0.64 ± 0.05 * 0.14 ± 0.01 2.04 ± 0.60 * 0.15 ± 0.03 <0.05 b calculated according to equation [1] Day 3 4.70 ± 1.88 0.47 ± 0.07 0.09 ± 0.02 4.86 ± 1.44 0.61 ± 0.15 0.15 ± 0.01 1.50 ± 0.70 0.13 ± 0.03 * <0.05 a calculated from dose × average BW, (data not shown) Day 2 3.33 ± 1.97 0.52 ± 0.11 0.09 ± 0.03 5.40 ± 2.58 * 0.62 ± 0.15 0.15 ± 0.02 1.23 ± 0.52 * 0.10 ± 0.03 <0.05 r 1 1.8 2.54 0.14 Day 1 4.87 ± 2.05 0.58 ± 0.17 0.11 ± 0.03 2.50 ± 1.10 0.42 ± 0.07 0.13 ± 0.03 ‡ 0.26 ± 0.14 <0.05 <0.05 o 30 61.5 303.60 0.49 3 5.8 11.68 0.20 Dose (mg kg 3 10 3 30 10 30 10 3 30 -1 BW) F 10 19.1 43.17 0.23 Matrix Albumen (µg g -1 ) Yolk (µg g -1 ) Plasma (µg ml -1 )
	w e i v e R *, †, ‡ 5, 4 and 3 eggs laid and analyzed, respectively
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Table I .

 I Pharmacokinetic characteristics of sulfadimidine in plasma after single intravenous (i.v.) and oral (p.o.) administration in laying hen (Mean ± SD, n=6).

	Dose	AUC	K01_HL	K10_HL	Cl	T max	C max
	mg kg -1	µg h ml -1	h	h	ml h -1 kg -1	h	µg ml -1
	100 (i.v.) 1122.0 ± 245.8	-	5.9 ± 1.1 94.1 ± 27.8	-	132.2 ± 16.9
	100 (p.o.) 1379.3 ± 428.1 1.2 ± 0.4 9.1 ± 1.6 82.5 ± 40.0 4.0 ± 0.9 76.5 ± 19.9
	30 (p.o.) 151.5 ± 27.5 0.7 ± 0.6 2.8 ± 0.7 204.2 ± 41.6 1.5 ± 0.8 25.3 ± 5.8
	10 (p.o.)	18.5 ± 4.1	0.4 ± 0.4 1.4 ± 0.8 563.2 ± 119.2 0.7 ± 0.5	6.2 ± 1.8
	3 (p.o.)	4.2 ± 1.2	0.2 ± 0.3 0.9 ± 0.2 797.3 ± 337.1 0.5 ± 0.3	2.4 ± 1.1
	AUC, area under the curve; K01_HL, half life of absorption; K10_HL, elimination half life; Cl, total body clearance; T max , time of maximum plasma concentration; C max , maximum plasma concentration. F o r
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Table II .

 II Sulfadimidine residues in yolk (µg g -1 , Mean ±SD) after single oral administration of different doses.

	Dose (mg kg -1 BW)	100	30	10	3	1
	Day after treatment
	1		14.32 ± 6.47 * 3.08 ± 1.52 * 0.42 ± 0.08 0.11 ± 0.05	ND
	2		6.62 ± 2.72 * 0.51 ± 0.01 ‡ 0.07 ± 0.02	< 0.05	ND
	3		3.67 ± 1.17 * 0.44 ± 0.12 * 0.06 ± 0.02 *	ND	ND
	4		2.74 ± 0.69 * 0.32 ± 0.05 †	< 0.05	ND	ND
	5 6 7 8 F o r 1.98 ± 0.56 * 0.20 ± 0.04 ‡ 1.21 ± 0.59 * 0.13 ± 0.02 * 0.43 ± 0.32 * 0.07 ± 0.03 ‡ 0.12 ± 0.14 ‡ < 0.05	< 0.05 ND ND ND	ND ND ND ND	ND ND ND ND
	9 10	ND < 0.05 P	ND ND	ND ND	ND ND	ND ND
	e n=6 unless otherwise indicated while *, † and ‡ = 5, 4, 3 eggs laid and analyzed, respectively. ND = not detected (< LOD) e r
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Table III .

 III Sulfadimidine residues in albumen (µg g -1 , Mean ±SD) after single oral administration of different doses.TableIV. Transfer rate of SDD into eggs after single oral administration.

			Food Additives and Contaminants	Page 24 of 25
	Dose	Drug	Residue	Transfer-rate c
	Dose (mg kg -1 BW) Day after treatment 1 2 3 4 5 6 7 8 n=6 unless otherwise indicated while *, †, ‡ = 5, 4, 3 eggs laid and analyzed, 49.34 ± 7.03 * 6.25 ± 1.85 * 0.98 ± 0.24 0.28 ± 0.12 0.07 ± 0.02 6.10 ± 0.43 * 0.16 ± 0.04 † 0.83 ± 0.16 * 0.15 ± 0.14 * 0.38 ± 0.12 * 0.20 ± 0.07 * 0.12 ± 0.03 * 100 30 10 3 1 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND < 0.05 ND ND ND respectively. ND = not detected (< LOD) F o r P e e r R e intake a in whole egg b (mg kg -1 BW) (mg) (µg) (%) 100 195 2497.16 1.28 30 61.5 303.60 0.49 10 19.1 43.17 0.23 3 5.8 11.68 0.20 1 1.8 2.54 0.14 a calculated from dose × average BW, (data not shown) b calculated according to equation [1] c calculated according to equation [2] F o r P e e r R e v i e w O n l y
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Table V .

 V Residue concentration of Sulfadimidine (Mean± SD) in plasma, albumen and yolk following medicated feed of different doses (30, 10 and 3 mg kg -1 BW) for seven consecutive days.

	Matrix		Plasma (µg ml	-1		Albumen (µg g -1 )		Yolk (µg g -1 )
	Dose (mg kg -1 BW)	30	10	3	30	10	3	30	10	3
	Day 1	4.87 ± 2.05 0.58 ± 0.17 0.11 ± 0.03	2.50 ± 1.10 0.42 ± 0.07 0.13 ± 0.03 ‡ 0.26 ± 0.14	<0.05	<0.05
	Day 2	3.33 ± 1.97 0.52 ± 0.11 0.09 ± 0.03	5.40 ± 2.58 * 0.62 ± 0.15 0.15 ± 0.02	1.23 ± 0.52	* 0.10 ± 0.03 <0.05
	Day 3	4.70 ± 1.88 0.47 ± 0.07 0.09 ± 0.02	4.86 ± 1.44 0.61 ± 0.15 0.15 ± 0.01	1.50 ± 0.70 0.13 ± 0.03 * <0.05
	Day 4	3.99 ± 1.52 0.44 ± 0.05 0.09 ± 0.03	4.99 ± 1.93 * 0.64 ± 0.05 * 0.14 ± 0.01	2.04 ± 0.60 * 0.15 ± 0.03 <0.05
	Day 5	2.36 ± 2.41 0.43 ± 0.07 0.10 ± 0.03	3.77 ± 1.25 * 0.45 ± 0.06 0.13 ± 0.02 * 1.80 ± 0.52 * 0.16 ± 0.02 <0.05
	Day 6	2.91 ± 1.08 0.31 ± 0.10 0.07 ± 0.01	2.84 ± 1.25 * 0.40 ± 0.12 0.14 ± 0.02	1.55 ± 0.45	* 0.20 ± 0.02 <0.05
	Day 7	1.48 ± 0.95 0.37 ± 0.12 0.09 ± 0.02	2.97 ± 0.90 † 0.43 ± 0.14 0.13 ± 0.01	1.64 ± 0.55	† 0.20 ± 0.02 <0.05

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