Wolfgang Jira
email: wolfgang.jira@bfel.de

Katja Ziegenhals

Karl Speer

A GC/MS method for the determination of 16 European priority polycyclic aromatic hydrocarbons in smoked meat products and edible oils

Keywords: edible oils, GC/MS, HRMS, PAH, smoked meat products

A GC/MS method for the determination of 16 European priority polycyclic aromatic hydrocarbons in smoked meat products and edible oils

Wolfgang Jira, Katja Ziegenhals, Karl Speer

INTRODUCTION

For the first time Jung and Morand reported the presence of PAH in vegetable oils and fats in 1962. Further investigations followed by Sagredos et al. 1979[START_REF]Polycyclic aromatic-hydrocarbons in native vegetable-oils[END_REF], Speer and Montag 1988[START_REF] Dennis | Factors affecting the polycyclic aromatic hydrocarbon content of cereals, fats and other food-products[END_REF]. Investigations of the contents of individual PAH in smoked and barbecued meat products (Simko 2002[START_REF] Jira | Polycyclic aromatic hydrocarbons (PAH) in smoked meat products and liquid smokes[END_REF] were primarily focused on benzo[a]pyrene [START_REF] Kazerouni | Analysis of 200 food items for benzo[a]pyrene and estimation of its intake in an epidemiologic study[END_REF] or the 16 PAH compounds approved by the U.S. Environmental Protection Agency (EPA-PAH) [START_REF] Mottier | Quantitative determination of polycyclic aromatic hydrocarbons in barbecued meat sausages by gas chromatography coupled to mass spectrometry[END_REF].

Since 1 April 2005 the Commission Regulation (EC) No 208/2005 of 4 February 2005 has been restricting maximum levels for benzo [a]pyrene in different food groups: Oils and fats 2 µg/kg, foods for infants and young children 1 µg/kg; smoked meat and smoked fish 5 µg/kg; unsmoked fish 2 µg/kg; crustaceans and cephalopods 5 µg/kg and bivalve molluscs 10 µg/kg. Furthermore, the European Commission (2005/108/EC) recommended that the member states should investigate not only the contents of benzo [a]pyrene in these food groups but also other polycyclic aromatic hydrocarbons (PAH) seen as carcinogenic by the Scientific Committee on Food (SCF). These are 15 PAH compounds which, due to their toxic properties, are of major concern to human health and should be monitored to make possible long-term exposure assessments (SCF, 2002). These 15 "SCF-PAH" are: benzo with European Union Member States in order to collect data for these 16 EU priority PAH [START_REF] Wenzl | Analytical methods for polycyclic aromatic hydrocarbons (PAHs) in food and the environment needed for new food legislation in the European Union[END_REF].

Lately, the total contents of the 15 SCF-PAH were analysed in different Danish meat products by [START_REF] Duedahl-Olesen | Polycyclic aromatic hydrocarbons (PAH) in Danish smoked fish and meat products[END_REF] This new method is based on the method [START_REF] Jira | A GC/MS method for the determination of carcinogenic polycyclic aromatic hydrocarbons (PAH) in smoked meat products and liquid smokes[END_REF]) developed at the Institute for Chemistry and Physics, Federal Research Centre for Nutrition and Food (BfEL), Kulmbach, Germany, and was adapted to the recommendation of the European Commission (2005/108/EC). The performance of the analytical method for edible oils was demonstrated in a proficiency test (IRMM 2006). Its suitability for meat products was shown by investigating suspicious samples. Smoked salts and smoke flavourings can also be analysed by using this method [START_REF] Kleinhenz | Dioxin and polychlorinated biphenyl analysis: Automation and improvement of clean-up established by example of spices[END_REF].

MATERIALS AND METHODS

Materials

The native PAH standard solutions (BcL,BaA,CHR,BbF,BkF,BjF,BaP,IcP,DhA,BgP,DlP,DeP,DiP,DhP,, 2-methylchrysene (2MC), 3-methylchrysene (3MC), 4-methylchrysene (4MC), 5MC, 6-methylchrysene (6MC), triphenylene (TP), dibenzo[a,c]anthracene (DcA), benzo[a]fluoranthene (BaF)) were obtained from Dr.

Ehrenstorfer GmbH (Augsburg Germany). The isotope labelled PAH standard solutions were purchased from Promochem (Wesel, Germany) and the fluorinated PAH standards from Biochemical Institute for Environmental Carcinogens Prof. Dr. Gernot Grimmer-Foundation (Grosshansdorf, Germany).

All solvents were obtained in picograde quality from Promochem (Wesel, Germany). The drying material (poly(acrylic acid), partial sodium salt-graft-poly(ethylene oxide)) was purchased from Sigma Aldrich (Munich, Germany), Bio Beads S-X3 (200 -400 mesh) from Bio-Rad Laboratories (Munich, Germany) and silica gel from Merck (Darmstadt, Germany).

Glass microfibre filters (18 mm i.d.) were obtained from Dionex (Idstein, Germany). The PTFE-Filters (1 µm pore size, 25 mm i.d.) and the SPE-Cartridges (12 mm i.d.) were purchased from Alltech (Unterhaching, Germany).

Sample preparation for the analysis of meat products

Accelerated solvent extraction (ASE): About 5 g homogenised meat product were levigated with the same amount of the drying material poly(acrylic acid), partial sodium salt-graftpoly(ethylene oxide). The resulting material was poured into 33-mL cells, which were locked with glass microfiber filters at the outlet end of the extraction cells. Afterwards, 50 µL of a PAH standard mixture containing isotope labelled ( 13 C and 2 H) and fluorinated PAH Gel permeation chromatography (GPC): The evaporated ASE-extract was dissolved in 4.5 mL cyclohexane/ethylacetate (50:50 v/v) and filtered through a PTFE filter with a pore size of 1 µm. The GPC column (25 mm i.d.) was filled with Bio-Beads S-X3 (weight of filling 60 g).

Samples were eluted at a flow rate of 5 mL/min applying cyclohexane/ethylacetate (50:50 v/v) (Dump time 0 -36 min, Collect time 36 -65 min). The solvent was removed with a rotary evaporator, and the eluate was dried in a nitrogen stream.

Solid phase extraction (SPE): This clean-up step to remove more polar substances was performed automatically with a modified ASPEC Xli (Automatic. Sample Preparation with Extraction Columns) [START_REF] Kleinhenz | Dioxin and polychlorinated biphenyl analysis: Automation and improvement of clean-up established by example of spices[END_REF] from Gilson (Bad Camberg, Germany). This system was modified with a fitting rack, teflon funnels and teflon tubes. Silica, dried for 12 h at 550°C, was deactivated with 15% water. 1 g dried deactivated silica was filled into commercial 8-ml SPE columns (12 mm i.d.). After conditioning the columns with 3 mL cyclohexane the samples were applied and eluted with 10 mL cyclohexane.

Preparation for GC/MS analysis: The dried eluate of SPE was dissolved in 1 mL isooctane and 50 µL of the PAH-recovery standard mixture (benzo[a]anthracene-d 12 and benzo[a]pyrene-d 12 in isooctane) and transferred to a 1 mL tapered vial. The remaining sample was carefully concentrated in a nitrogen stream to a volume of about 50 µL.

GC/MS analysis

The GC/HRMS analysis of PAH was performed on a HP 5890 II gas chromatograph with a split/splitless injection port. The GC was equipped with a VF-17ms capillary column (60 m x 0.25 mm i.d., 0.25 µm film thickness) purchased from Varian (Darmstadt, Germany). Helium was used as carrier gas at a constant pressure of 27 psi. The injection temperature was 300°C and the injection volume was 1 µL (splitless). The following temperature program was used: isothermal at 50°C for 1 min, at 25°C/min to 280°C, at 1°C/min to 330°C, isothermal at 330°C for 30 min.

The quantification of PAH by GC/HRMS was performed by using a VG Autospec (Waters, Manchester, UK) working in the EI positive ion mode using an electron energy of 35 eV. The transfer line temperature and the ion source temperature were maintained at 280°C and 250°C, respectively. The resolution of the MS was tuned to 8000 (10% valley definition). The PAH were analysed in a 4-function Selected Ion Registration (SIR) experiment.

The GC/MSD analysis was performed on an Agilent 7890 GC coupled with an Agilent 5975 inert mass spectrometric detector. The GC was also equipped with a VF-17ms (Varian, Darmstadt, Germany) capillary column (60 m x 0.25 mm i.d., 0.25 µm film thickness).

Helium was used as carrier gas at a constant flow rate of 1 mL/min. 1 µL was injected while the injector port was held at 325°C and operated in pulsed splitless mode. Initially, the oven temperature was set to 50°C (1 min hold), then to 280°C at 30°C/min, to 340°C at 1°C/min and to 350°C at 1.5°C/min kept constant for 2 min. The mass spectrometer was operated in a selected ion monitoring (SIM) mode with an electron impact ionization of 70 eV. The For the determination of the ratio of TP and CHR with GC/MSD a VF-Xms (Varian, Darmstadt, Germany) capillary column (60 m x 0.25 mm i.d., 0.25 µm film thickness) was used. The oven temperature was initially set at 50°C (1 minute hold) and heated to 320°C at 3.5°C/min. Helium was used as carrier gas at a constant flow rate of 1 mL/min. 1 µL was injected while the injector port was held at 325°C and operated in the pulsed splitless mode.

Sample preparation for the analysis of edible oils

Approximately 0.9 g of edible oil was dissolved in 3.5 mL cyclohexane/ethylacetate (50:50 v/v), 50 µL of the internal PAH standard were added and the mixture was chromatographed by GPC. The following clean up steps were carried out as described for meat products.

RESULTS AND DISCUSSION

Applying the GC/MS method (HRMS and MSD) developed here it is possible to achieve gas chromatographic separations of isomers which are otherwise difficult to separate. First, a sufficient chromatographic separation of CPP and CHR was achieved (see Figure 1A).

Although CPP (MW = 226) has another molecular weight than BaA and CHR (MW = 228, each), a sufficient chromatographic separation of CPP and CHR is required for a reliable quantification of CPP because BaA and CHR also show fragment ions with the molecular weight MW = 226 [START_REF] Nyman | Comparison of two clean-up methodologies for the gas chromatographic/mass spectrometric determination of low nanogram/gram levels of polynuclear aromatic-hydrocarbons in seafood[END_REF]. Therefore, an unsatisfactory chromatographic separation would adulterate the quantification of CPP. In contrast, a chromatographic separation of CHR and TP on such a column is not possible. Second, with the help of this method a satisfactory chromatographic separation of the six methylchrysene-isomers is achievable (see Figure 1B). Therefore, the exact quantification of 5-methylchrysene (5MC) is possible. Third, an important chromatographic challenge is the separation of the benzofluoranthenes. The base line separation of benzo[b]fluoranthene, benzo[j]fluoranthene and benzo[k]fluoranthene is possible by means of a 60 m VF-17ms GC column (see Figure 1C). Also, benzo[a]fluoranthene does not coelute with the benzofluoranthenes. Fourth, DhA can be detected independent of dibenzo[a,c]anthracene (DcA) (see Figure 1D). Fifth, also the quantification of dibenzopyrenes (MW = 302) is easier to carry out with a GC column with 50% polyphenylsiloxane than with 5% polyphenylsiloxane because in this way a better chromatographic separation of the isomers is achievable. It is well known that there are a number of isomeric PAH compounds with MW = 302 amu (Schmidt et al. 1987, Schubert et al. 2003). In Figure 2 the chromatographic separation of PAH isomers with m/z = 302 in strongly smoked raw sausage on a VF-17ms is presented. For the qualitative detection of the different PAH compounds using GC/MSD, in addition to the retention time and the molecular ion (M+), a third criterion is needed to identify a substance with a high selectivity because of the low resolution of the MSD. A second ion in addition to the quantifier ion is called qualifier ion and was used for the purpose of verification [START_REF] Wenzl | Analytical methods for polycyclic aromatic hydrocarbons (PAHs) in food and the environment needed for new food legislation in the European Union[END_REF]). The ratio between the quantifier ion and the qualifier ion had to match a specific ratio (see Table I Since a gas-chromatographic separation of the two isomers TP and CHR could not be obtained with a VF-17ms capillary column, a VF-Xms capillary column had to be used. With a low heating rate of 3.5°C/min not only a separation of TP and CHR but also a sufficient separation of BaA, CPP, TP and CHR was achieved (see Figure 3).

PAH in edible oils

The reliability of the described analytical method was demonstrated by participation in a proficiency test (IRMM 2006). Three edible oils spiked with PAH, sunflower oil, olive oil, maize oil and one native sunflower oil, were analysed in duplicate. The results of GC/HRMS were submitted to IRMM. Subsequently, the same extracts were measured by GC/MSD. These results were not submitted to the IRMM. Both results determined were compared with the real spiked concentrations (Table III). In the case of the native sunflower oil the results were compared to the median of the PAH concentration of the different participants of the collaborative trial.

The results determined by both GC/MS-systems were in a good agreement. The recoveries for different PAH compounds ranged from 75% to 95%. The repeatability was better in the case of MSD. The averaged relative standard deviations (RSD) of all PAH compounds were 17% for MSD and 22% for HRMS. In order to test the suitability of the described GC/HRMS method developed at the BfEL Kulmbach, 22 smoked raw meat products (mainly raw smoked hams) which looked like very strongly smoked products were collected from different producers at the end of 2005.

PAH in smoked meat products

The resulting benzo[a]pyrene contents of these products were in the median below 0.15 µg/kg and therefore classified as being relatively low. With the exception of one sample, which showed a benzo[a]pyrene content of 18 µg/kg (Table IV), all of the samples were below the new maximum level of 5 µg/kg. Including the sample with a BaP content of 18 µg/kg four samples were above the old maximum level in Germany of 1 µg/kg. The sample with a benzo[a]pyrene content of 18 µg/kg was a smoked belly of pork with a strongly fading, black surface.

For the clarification of the question, in which quantitative ratio the present leading substance benzo [a]pyrene is compared to the other investigated PAH compounds, the contents determined for the 15 priority PAH compounds and the JECFA-PAH benzo[c]fluorene were added to calculate the percentage contributions of the single PAH compounds to the total content of the 16 PAH compounds. The result of these calculations is presented in Figure 4.

Hereby, the results were differentiated between PAH with a higher (marked dark) and a lower (marked bright) carcinogenic potential. The differentiation between these two groups resulted from the toxic equivalent factors (TEF) in the literature (Müller et al. 1995). In this way a higher carcinogenic potential was assumed for PAH with a TEF ≥ 0.89 and a lower carcinogenic potential for TEF ≤ 0.11. The results clearly show that within the group of PAH compounds with a higher carcinogenic potential benzo[a]pyrene (BaP) represents the largest contribution to the total content of the 16 investigated PAH compounds. The contribution of BaP in these samples was 8% of the total PAH content (median) and varied from approximately 6% to 12%. The contributions of the other PAH compounds with a higher carcinogenic potential (dibenzo[a,h] 13 dibenzo[a,h]pyrene (DhP) and dibenzo[a,l]pyrene (DlP)) were significantly lower and were in the median range of about 1% and lower. Consequently, benzo[a]pyrene was the dominating compound within the group of PAH with a higher carcinogenic potential and therefore, seems to be justified to be used as the leading substance in smoked meat products. The percentage contributions of the other PAH with a low carcinogenic potential show relatively high variations.

After developing the method for the separation of TP and CHR the ratios of these isomers were analysed in 25 samples from different meat products. Some of these samples were the same samples that looked like very strongly smoked products because of their black surface.

But some of the samples that looked very strongly smoked had very low concentrations, which could not be analysed by using the GC/MSD. For these measurements the MSD was not sensitive enough. Therefore, samples of bacon were analysed additionally.

The results showed that the ratio of TP/CHR varied also within the ham and bacon samples (Table V). The maximum value was reached for wild boar ham. Except for chrysene the 16 EFSA-PAH can be detected separately from each other and from interfering isomers not classified as priority. This method also allows an analysis with low limits of quantification (LOQs). Such a sensitive analytical method is necessary for the determination of PAH in smoked meat products with low PAH contents. Otherwise, a quantification of PAH in meat samples with lower concentration is very difficult. (Reinik et al. 2007).

The contents of the 16 European priority PAH compounds in the analysed suspicious samples of smoked meat products point out that BaP can be used as the leading substance in smoked meat products. However, the suitability of BaP as a marker for carcinogenic PAH compounds in smoked meat products has to be confirmed by the investigation of representative samples.

In particular, next to a representative study it is important to get more information about the content of DlP in meat products. DlP has been in the spotlight of scientific interest recently because toxicological investigations indicated that DlP probably shows a hundredfold higher toxicity in comparison to BaP [START_REF] Mcclure | Fifteenth International Symposium on Polycyclic Aromatic Compounds[END_REF]Scheny 1995, Müller et al. 1995). In consideration of this potentially extremely high toxicity DlP should not to be underestimated -although the contents in the investigated smoked meat products were very low. The GC chromatograms show that a 50% phenyl capillary column is superior to a 5% phenyl capillary column [START_REF] Jira | A GC/MS method for the determination of carcinogenic polycyclic aromatic hydrocarbons (PAH) in smoked meat products and liquid smokes[END_REF], Rose et al. 2007), especially because of a better chromatographic separation of the high number of PAH compounds with MW = 302 (Schubert et al. 2003).

Toxicological investigations indicated that CHR has a much stronger carcinogenic potential than TP (Müller et al. 1995). CHR und TP elute on polar columns at the same retention time.

Because of their identical molecular mass they could only be quantified in sum. Most studies consider only the content of CHR. For the determination of the TP/CHR-ratio a separate method was developed. Applying this method, differing ratios of TP/CHR within different samples were analysed and as a result the content of TP is not to be neglected. Because of these results, the previously description "CHR" should be replaced by "TP and CHR", to avoid an over-evaluation of the carcinogenic potential of the sample.

  [a]anthracene (BaA), benzo[b]fluoranthene (BbF), benzo[j]fluoranthene (BjF), benzo[k]fluoranthene (BkF), benzo[ghi]perylene (BgP), benzo[a]pyrene (BaP), cyclopenta[cd]pyrene (CPP), dibenzo[a,h]anthracene (DhA), dibenzo[a,e]pyrene (DeP), dibenzo[a,h]pyrene (DhP), dibenzo[a,i]pyrene (DiP), dibenzo[a,l]pyrene (DlP), indeno[1,2,3-cd]pyrene (IcP), chrysene (CHR) and 5-methylchrysene (5MC). In addition, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) identified benzo[c]fluorene (BcL) as a PAH compound that should be monitored as well (JECFA 2005). These 16 EU priority PAH need to be analysed in different food groups. EFSA has established an on-line analytical database in collaboration

  . After saponification, liquid liquid extraction and solid phase extraction, the chromatographic separation was carried out via a 60 m J&W DB-5 capillary column. Then in 2007, the results of 27 PAH including the 15 SCH-PAH in oils were presented by Rose et al. Again, the sample preparation included saponification, liquid liquid extraction and solid phase extraction. Also here a 60 m 5% phenyl capillary column and a low resolution mass spectrometer for separation and quantification were used. This paper describes an analytical method without saponification for the determination of the 16 EU priority PAH compounds in the fat-containing food groups, edible oils and smoked meat products. The method includes accelerated solvent extraction (ASE) and the highly automated clean up steps gel permeation chromatography (GPC) and solid phase extraction (SPE). The determination of the PAH was carried out by applying HRGC in combination with a high resolution mass spectrometer. For separation of the individual components a 60 m 50 % phenyl capillary column was found suitable.

  fluorobenzo[c]fluorene, benzo[a]anthracene-13 C 6 , chrysene-13 C 6 , 5methylchrysene-d 3 , benzo[b]fluoranthene-13 C 6 , benzo[k]fluoranthene-13 C 6 , benzo[a]pyrene-13 C 4 , benzo[g,h,i]perylene-13 C 12 , dibenzo[a,h]anthracene-d 14 , indeno(1,2,3-cd)pyrene-d 12 , dibenzo[a,e]pyrene-13 C 6 , dibenzo[a,i]pyrene-13 C 12 and 13-fluorodibenzo[a,l]pyrene in isooctane) were added. The extraction was performed with an ASE 200 from Dionex (Sunnyvale, USA) and carried out with n-hexane at 100°C and 100 bar at a static time of 10 min. The flush volume was 60% and the purge time 120 s. Two static cycles were accomplished. The solvent of the extract was evaporated in a water bath (40°C) using a nitrogen stream.

  blanks were simultaneously analysed for to detect present PAH in parallel to each series of samples passing the extraction and cleanup procedures using drying material instead of real samples.

  ion source and the quadrupole mass analyser were kept at 300°C and 150°C, respectively.

  ) to show a positive result. The ratios need to be determined by measuring an external PAH standard solution. The quantification of the different PAH compounds is based on the comparison of the peak area of the most abundant ion (M + ) of the native PAH compound with the peak area of the corresponding deuterated, 13 C-labelled or fluorinated internal standards. As not all of the 16 PAH compounds are commercially available as isotope labelled standard compounds, it was necessary to use response factors for the quantification of PAH with no corresponding internal standards (cyclopenta[c,d]pyrene, benzo[j]fluoranthene and dibenzo[a,h]pyrene). These response factors were determined by the analysis of a PAH standard mixture containing the 16 PAHcompounds. The range of linearity of both GC/MS-systems was determined by an automated statistical test and ranged from 1 or 5 to 1000 pg/µl. GC/HRMS and GC/MSD show different limits of detection (LOD) and limits of quantification (LOQ). The LOD, which is defined as the lowest concentration of an analyte detectable in a sample (signal-to-noise ratio = 3:1) and the LOQ, which is defined as the lowest content of the analyte that can be measured with reasonable statistical certainty (2007/333/EC) (signal-to-noise ratio = 9:1) for the different PAH compounds in smoked meat products, are shown in TableII. Two groups are defined: 1) PAH with a lower molecular weight which can be detected more sensitively than 2) PAH with a high molecular weight. The two different groups are:1. BcL, BaA, CHR, CPP, 5MC,BbF, BkF, BjF, BaP, BgP , DhA, DlP, DeP, DiP, DhP

  anthracene (DhA), dibenzo[a,e]pyrene (DeP),

  of both GC/MS methods (HRMS and MSD) a reliable determination of the 16 European priority PAH compounds in smoked meat products and edible oils is possible.

Figure 1 .

 1 Figure 1. Structures, abbreviations and molecular weights of the 16 priority PAH

Figure 5 .

 5 Figure 5. Linear range HRMS (1 -1000 pg/µL)

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. Chromatograms of the priority PAH and some isomers on a VF-17ms

  Chromatograms of the priority PAH and some isomers on a VF-17ms 190x94mm (150 x 150 DPI) Chromatographic separation of PAH isomers with m/z=302 in a smoked raw sausage on a VF-17ms 252x120mm (150 x 150 DPI) Chromatogram of the separation of triphenylene (TP) and chrysene (CHR) 267x131mm (150 x 150 DPI) Percentage contribution of single PAH to the total content of the 16 EFSA-PAH in smoked meat products

Table II .

 II LOD und LOQ in meat products

Table I .

 I Ratio qualifier to quantifier ion of different PAH compounds

	Table II. LOD und LOQ in meat products
	Table III. Contents of PAH determined with both GC/MS-systems in three spiked and one
		native oil
	Table IV. Contents of PAH in smoked meat products
	Table V.	Ratios of triphenylene to chrysene in smoked meat products

Table I .

 I Ratio qualifier to quantifier ion of different PAH compounds

	Table II. LOD und LOQ in meat products
			meat products
		Qualifier/Quantifier Ion 5 g sample weight
		MSD		BcL 108/216 HRMS	10.0
				BaA 114/228	11.0
	µg/kg	LOD	LOQ	CHR 113/228 LOD LOQ	11.5
				CPP 113/226	16.7
	group 1	0.01	0.03	5MC 215/242 BbF 126/252 0.003 0.009	11.9 12.5
	group 2	F o r 0.02 0.06 F o r	BkF 125/252 BjF 125/252 BaP 126/252 IcP 138/276 DhA 139/278 BgP 138/276 0.01 0.03	9.5 12.6 13.2 21.7 13.8 18.7
				DlP 150/302	28.8
			DeP 150/302 DiP 150/302 DhP 150/302 e P P e	15.0 9.7 10.3
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Table III :

 III Contents of PAH determined with both GC/MS-systems in three spiked and one native oil

		spiked sunflower oil		spiked olive oil		spiked maize oil	native sunflower oil
				median			median			median			median
		HRMS MSD	conc.*	HRMS MSD	conc.*	HRMS MSD	conc.*	HRMS MSD	conc.*
	benzo[a]anthracene	0.9	0.8	0.9	2.7	2.7	2.9	7.9	8.1	7.8	5.6	5.5	5.7
	benzo[a]pyrene	1.0	0.8	0.9	2.5	2.2	2.3	7.6	7.1	7.5	4.8	4.3	4.7
	benzo[b]fluoranthene	6.3	6.3	6.6	1.2	1.1	1.1	2.1	1.9	1.9	5.0	4.6	4.6
	benzo[c]fluorene	1.6	1.4	1.5	6.7	5.9	6.9	0.7	0.6	0.7	1.0	0.8	1.5
	benzo[ghi]perylene	8.6	9.2	9.5	1.6	1.3	1.5	2.9	2.8	2.8	3.7	3.2	3.6
	benzo[j]fluoranthene	2.8	2.7	2.7	10.2	10.6	10.0	1.4	1.2	1.1	2.7	2.6	2.6
	benzo[k]fluoranthene chrysene cyclopenta[cd]pyrene dibenzo[a,e]pyrene dibenzo[a,h]anthracene dibenzo[a,h]pyrene F 1.0 1.1 7.1 2.0 1.3 2.1 o 0.7 1.0 8.2 1.6 1.0 1.7 r	0.8 1.0 6.3 1.7 1.0 1.8	2.3 10.1 1.1 1.1 3.1 1.1	2.2 10.0 1.0 0.7 2.8 0.7	2.3 9.9 1.1 0.8 2.9 0.8	7.3 2.3 2.1 6.3 8.9 7.0	7.3 2.5 2.1 6.2 9.3 7.1	7.4 2.3 1.8 6.8 11.0 6.8	2.6 8.1 2.5 1.0 1.1 0.6	2.4 7.6 2.4 0.7 0.8 0.1	2.4 7.5 2.6 0.8 0.8 -
	dibenzo[a,i]pyrene dibenzo[a,l]pyrene Indeno[1,2,3-cd]pyrene 5-methylchrysene	1.0 7.3 1.9 1.0	0.6 7.0 1.6 P 0.6 7.5 1.8 0.7 0.9 e	7.2 1.9 6.2 0.4	7.2 2.0 6.1 0.3	6.8 2.1 6.9 0.8	2.1 1.1 2.8 3.6	1.8 0.7 2.3 2.5	1.9 1.0 0.8 3.5	0.9 0.5 3.0 -	1.0 0.2 2.8 -	0.3 0.6 3.2 -
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Table IV .

 IV Contents of PAH in smoked meat products

	µg/kg	BcL BaA CPP	CHR + TP	5MC BbF BjF BkF BaP BgP DhA	IcP	DeP	DhP	DiP	DlP
	smoked ham	1.59 2.02 1.42 2.22 0.38 0.87 0.94 0.66 1.03 0.61	0.20 0.62	0.10 <0.03	0.05	0.07
	smoked ham	0.19 0.32 0.41 0.36 0.05 0.20 0.22 0.15 0.23 0.21	0.06 0.20	0.04 <0.01 <0.03 <0.03
	smoked ham	0.05 0.09 0.05 0.14 0.03 0.09 0.07 0.05 0.08 0.10 <0.03 0.08 <0.03 <0.01 <0.01 <0.03
	smoked ham	0.79 0.84 0.91 0.78 0.06 0.38 0.42 0.29 0.50 0.39	0.08 0.35	0.05 <0.01	0.04	0.03
	smoked ham	0.14 0.14 0.11 0.20 0.02 0.12 0.10 0.07 0.12 0.15	0.03 0.11 <0.03 <0.01	0.03 <0.03
	smoked ham smoked ham smoked ham smoked ham	0.36 0.93 0.36 3.55 0.39 0.80 0.64 0.29 0.54 0.79 0.12 0.49 0.48 0.65 0.03 0.41 0.38 0.25 0.40 0.37 <0.03 0.27 0.17 0.47 <0.01 <0.01 <0.01 <0.01 0.03 <0.01 <0.03 <0.01 0.05 0.08 0.05 0.12 0.03 0.09 0.07 0.05 0.08 0.11 <0.03 0.08 <0.01 <0.01 <0.01 <0.01 0.18 0.15 0.16 0.20 0.01 0.10 0.10 0.06 0.09 0.16 <0.01 0.10 <0.01 <0.01 <0.01 <0.01 F o r F o r
	smoked ham smoked ham smoked ham smoked ham smoked ham	0.95 0.79 0.56 4.29 0.2 P P 0.12 0.12 0.03 0.15 0.04 0.07 0.05 0.03 0.06 0.06 <0.03 0.05 <0.01 <0.01 <0.01 <0.01 0.59 1.29 0.43 0.76 0.26 0.03 0.16 <0.01 <0.01 <0.01 <0.01 4.60 4.70 2.93 4.99 0.96 5.00 1.14 2.49 2.31 1.34 0.17 1.25 0.20 <0.03 e e 0.12 <0.03 2.25 4.47 4.04 4.27 0.34 2.66 3.14 2.25 3.89 2.60 0.27 2.57 0.48 0.06 0.32 0.05 0.08 0.12 0.08 0.15 0.03 0.13 0.13 0.10 0.18 0.20 <0.03 0.18 0.04 <0.03 <0.03 <0.01 e r e r
	smoked ham smoked ham smoked ham	0.06 0.06 0.03 0.09 0.02 0.05 0.04 0.03 0.04 0.08 <0.03 0.04 <0.03 <0.01 R R 0.09 0.10 0.04 0.19 0.03 0.06 0.04 0.03 0.04 0.05 <0.03 0.03 <0.03 <0.01 <0.01 <0.01 <0.03 <0.01 0.01 0.03 0.02 0.05 0.02 0.03 0.02 0.01 0.02 0.04 <0.01 <0.03 0.005 <0.01 <0.01 <0.01 e e
	smoked ham smoked ham smoked ham	0.09 0.07 0.02 0.11 0.03 0.04 0.02 0.02 0.03 0.04 0.004 0.02 <0.03 <0.03 <0.03 <0.03 0.13 0.15 0.04 0.18 0.04 0.08 0.06 0.04 0.08 0.07 <0.03 0.05 <0.03 <0.03 <0.03 <0.03 0.06 0.06 0.03 0.09 0.02 0.04 0.03 0.02 0.03 0.05 <0.03 0.03 <0.03 <0.03 <0.03 <0.03 v i e v i e
	belly pork ham smoked wild boar	13.88 13.48 13.04 13.69 2.54 16.04 19.38 16.46 17.63 19.29 w w	6.21 21.03	3.09	0.27	1.93	2.88
	ham	0.86 0.82 0.23 3.22 0.21 0.61 0.51 0.24 0.49 0.52 O n 0.13 0.41 l O n l	0.09 <0.03	0.07	0.06
					y y

Table V .

 V Ratios of triphenylene (TP) to chrysene (CHR) in smoked meat products

	meat product	ratio TP/CHR	meat product	ratio TP/CHR
	raw ham_1	0.39	bacon_8	0.37
	raw ham_2	0.11	bacon_9	0.49
	raw ham_3	0.51	bacon_10	0.28
	raw ham_4	0.24	bacon_11	0.25
	raw ham_5	0.32	bacon_12	0.46
	belly of pork	0.12	bacon_13	0.36
	bacon_1	0.37	wild boar ham	2.70
	bacon_2	0.34	smoked ham_1	0.46
	bacon_3	0.25	smoked ham_2	0.20
	bacon_4	0.53	smoked ham_3	0.18
	bacon_5	0.54	smoked ham_4	0.17
	bacon_6	0.47	smoked ham_5	0.35
	bacon_7	0.28

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Centre for Nutrition and Food (BfEL),


Keywords: Exposure assessment, Immunoassays Additives/Contaminants: Aflatoxins, Mycotoxins aflatoxin, aflatoxin-albumin, biomarkers Egypt, serum 3

Aflatoxins are potent liver carcinogens that frequently contaminate dietary cereals 2 within developing regions. Aflatoxin exposure has been predicted within Egypt but to 3 date no studies have measured the level of aflatoxin-albumin (AF-alb) adducts as a 4

an hepatocellular carcinoma (HCC) case control study within Egypt. In this pilot survey 6 AF-alb was detected in 24/24 samples from individuals without HCC diagnosis 7 (geometric mean 9.0pg/mg range 3.5-25.8pg/mg), whilst 7/22 samples from HCC cases 8 had detectable AF-alb (geometric mean 2.6pg/mg (range nd -32.8pg/mg). These AF-9 alb data do not represent a case control comparison due to inherent difficulties in 10 comparing markers of dietary intake between controls and patients with disease. 11 Although these data are limited, the potential health consequences of aflatoxin exposure 12 in this region merit further investigation. The most common and toxic of the naturally occurring aflatoxins is aflatoxin B 1 4 (AFB 1 ), and has been classified as a class 1A human liver carcinogen by the 5 International Agency of Research on Cancer (IARC 2002). The highest aflatoxin 6 exposures have been observed in parts of Africa, China and Southeast Asia, areas which 7 are also characterized by a high incidence of hepatocellular carcinoma (HCC) (IARC 8 2003). HCC is the fifth most common cancer in the world with around 500,000 new 9 cases annually, and HCC represents the third most common cause of cancer mortality in 10 the world (IARC 2003). The main risk factors for HCC in addition to aflatoxin are 11 hepatitis B virus (HBV) and hepatitis C viruses (HCV) (IARC 1994(IARC , 2002)). Given the 12 burden of HCC in many regions of the world with mortality rates only slightly lagging 13 incidence rates, it is important to understand the contribution of all putative etiological 14 agents within a given region. In Egypt the prevalence of HCC is increasing (El-Zayadi 15 for the mutation (Bressec et al., 1991;Hsu et al., 1991;[START_REF] Wild | Environmental and genetic determinants of aflatoxin-albumin adducts in the 19 Gambia[END_REF]. In response, 1 we have undertaken a pilot biomarker survey to further assess the prevalence of 2 aflatoxin exposure within Egypt. The level of aflatoxin-albumin (AF-alb) adduct in 3 serum reflects the biologically effective aflatoxin dose, and serves as a useful aflatoxin 4 exposure biomarker in humans (Sabbioni et al., 1987 , 2006, 2007a, 2007b). However, to date there has not been an assessment of 9 aflatoxin exposure in Egypt using the more robust measure of the level of AF-alb 10 adducts in serum. The AF-alb adduct is particularly valuable in this respect as it 11 provides a good indicator of exposure over the previous two the three months 12 (Montesano et al., 1999;[START_REF] Wild | The toxicology of aflatoxins as a basis for public health 10 decisions[END_REF]. This manuscript describes an 13 exploratory study in which the levels of AF-alb were measured for a subset of sera from 14 individuals within an Egyptian HCC case-control study, previously reported by Ezzat et 15 al., (2005). The prevalence and levels of exposure in this pilot study were compared to 16 exposure patterns reported elsewhere. 17 individuals, including 22 cases (aged 36-74; 18 males, 4 females) and 24 controls (aged 1 37-73; 20 males, 4 females), for whom blood samples were available, were tested for 2 the level of serum AF-alb adduct. All of these individuals were from the Cairo 3 metropolitan area or governorates within the Nile Delta, and all were HBsAg negative. 4

The subset of cases for this AF-albumin study were selected because they had TP53 5 gene mutation results available from a previous analysis (El-Kafrawy et al. 2005); 6 controls were randomly selected from the total pool of controls to match these cases on 7 age, gender and region distributions. However, due to difficulties in comparing 8 biomarkers related to dietary intake where one group are patients with disease, this 9 manuscript is not attempting to present case control data in relation to HCC causation. 10 11 AF-albumin ELISA 12 The levels of AFB 1 -albumin adduct were determined by albumin extraction, digestion 13 and ELISA as previously described (Chapot and Wild 1991). The detection limit was 14 3pg AFB 1 -lysine equivalents per mg of albumin (pg/mg). Three positive and one 15 negative control samples were analyzed with each batch of samples. Samples were 16 measured in quadruplicate on two separate days; CVs were less than 25%. AF-alb data 17 were natural log transformed to better approximate a normal distribution. Table 1, 18 reports the percentage of Egyptian samples with detectable AF-alb, and for clarity 19 adduct levels are back transformed and presented as geometric means. 20 21

Results and Discussion 22

Aflatoxin is a potent hepatocarcinogen, and given that exposure can occur throughout 23 life it is important to understand the pattern of exposure in regions potentially at risk 24 from this toxin. AF-alb has been used as a specific biomarker of exposure in West 25 [START_REF] Turner | Hepatitis B 14 infection and aflatoxin biomarker levels in Gambian children[END_REF][START_REF] Turner | Modification of immune 18 function through exposure to dietary aflatoxin in Gambian children[END_REF](Turner et al., , 2005b(Turner et al., , 2007;;Gong et al., 2002Gong et al., , 2004)), and exposure based 2 on this biomarker is frequent and at high levels. By comparison, there is less 3 information from Egypt on aflatoxin exposure using biomarkers but levels are predicted 4 to be lower than in West Africa (El- [START_REF] El-Nezami | Ability of a mixture of lactobacillus and propionibacterium to influence the 7 faecal aflatoxin content in healthy Egyptian volunteers: a pilot clinical study[END_REF]Hatem et al., 2005). In this 5 pilot study of Egyptian samples, AF-alb adducts were detected in 31 of 46 (67.4%) of 6 the samples (geometric mean 5.0pg/mg (range non detectable to 32.8pg/mg), see Table 7 1. Of the 24 individuals without HCC, all were positive for AF-alb (n=24, geometric 8 mean 9.0pg/mg; range 3.5 -25.8pg/mg. By contrast fewer individuals, 7 of 22 (31.8%), 9

with HCC had detectable adducts, and overall adduct levels were lower (p<0.001) in 10 cases (geometric mean 2.6pg/mg; range non detectable to 32.8pg/mg). HCC is 11 frequently diagnosed late, and due to the advanced state of the disease it is known that 12 individuals diagnosed with HCC in Africa have modified or restricted diets, leading to 13 lower aflatoxin exposures (Kirk et al., 2005). Dietary surveys were not conducted 14 within this study to specifically address this issue. AF-alb adducts were not observed for 15 the eight HCC cases carrying the TP53 Ser249 mutation, which may again simply 16 reflect such changes in dietary intake for HCC cases. In addition, if aflatoxin exposure 17 were causative for the reported TP53 Ser249 mutations in these eight HCC cases, this 18 would be as a consequence of chronic exposure over years or decades, thus a measure of 19 recent exposure would not neccesarily correlate with the mutation (Kirk et al., 2005). 20

For the non HCC cases, a non-significant trend (p>0.05) towards higher adduct levels 21 was observed for those from rural areas (geometric mean 11.2pg/mg; 95%CI: 7.7, 22 16.2pg/mg, n= 9) compared to those in urban areas (geometric mean 7.9pg/mg; 95%CI: 23 5.8, 10.7, n=15), possibly reflecting less varied diets and reliance on basic dietary 24 staples in rural settings, as observed in The Gambia [START_REF] Wild | Environmental and genetic determinants of aflatoxin-albumin adducts in the 19 Gambia[END_REF]. A similar but 25 again non-significant trend was also observed within the cases (data not shown). 1 Numbers limit both the interpretation of this observation, and further analysis of the 2 data by age, sex, or seasonality. 3 A comparison of AF-alb adducts in this study and a number of other countries, 4 measured using the ELISA methodology, are made in Table 1. The frequency and level 5 of exposure in West African countries is clearly higher, and for many individuals in 6

West Africa the levels exceed 100pg/mg [START_REF] Wild | Environmental and genetic determinants of aflatoxin-albumin adducts in the 19 Gambia[END_REF]Turner et al., 2007). A 7 similar high prevalence of AF-alb was reported for adults in Ghana [START_REF] Dash | Determinants of the variability of aflatoxin-albumin adduct levels in Ghanaians. 24[END_REF], 8 however, differences in assay methodology restrict further comparison with their data. 9

Limited surveys in Western Europe revealed no positive samples for this adduct 10 (reviewed by [START_REF] Wild | Molecular dosimetry of aflatoxin 13 exposure: contribution to understanding the multifactorial etiopathogenesis of primary 14 hepatocellular carcinoma with particular reference to hepatitis B virus[END_REF]. The frequency and levels of exposure in these Egyptian 11 samples are therefore considered to be intermediate in comparison with the low risk and 12 high risk areas for exposure, and to be more in line with exposures recently observed by 13 Scussel et al., (2006) in Brazil, or previously reported from Thailand (reviewed by Wild 14 et al., 1993). 15 16 Urine samples and breast milk provide additional bio-fluids with which to monitor 17 aflatoxin exposure. AFM 1 is a major metabolite of AFB 1 and is the main aflatoxin that 18 is sequestered into breast milk following maternal exposure to AFB 1 [START_REF] Zarba | Aflatoxin 22 M1 in human breast milk from The Gambia, west Africa, quantified by combined 23 monoclonal antibody immunoaffinity chromatography and HPLC[END_REF]19 IARC 2002). The frequency of AF-alb in our present study are in line with recent 20 observations of aflatoxin M 1 in breast milk samples from Egyptian mothers 21 (Polychronaki et al., 2006) Depending on sex and geographic location within Egypt the HBV surface antigen 1 (indicative of current infection) was detected in 3-11% of the total case-control study 2 population [START_REF] Ezzat | Associations of 20 pesticides, HCV, HBV, and hepatocellular carcinoma in Egypt[END_REF], and HBV was estimated to contribute 5-10% to the 3 burden of HCC in Egypt. In regions where co-exposure to aflatoxin and chronic carriage 4 of HBV infection occur, a synergistic interaction in the development of HCC has been 5

reported [START_REF] Ross | Urinary aflatoxin biomarkers and risk of hepatocellular carcinoma[END_REF][START_REF] Qian | A follow-up study of urinary markers of aflatoxin exposure and liver cancer 14 risk in Shanghai, People's Republic of China[END_REF][START_REF] Wang | Hepatocellular carcinoma and aflatoxin 6 exposure in Zhuqing Village, Fusui County, People's Republic of China[END_REF], with the relative risk 6 of HCC increasing up to 60-fold with co-exposure. Given the level of chronic HBV 7 carriage in Egypt, the significance of potential co-exposure to aflatoxin is of concern. 8 HCV accounts for nearly 90% of the HCC burden in Egypt [START_REF] Ezzat | Associations of 20 pesticides, HCV, HBV, and hepatocellular carcinoma in Egypt[END_REF], 9 however, to date no interaction between HCV infection and aflatoxin exposure are 10 reported in the literature. 11 12The purpose of this survey was a pilot investigation of the prevalence of AF-alb 13 adducts, as a biomarker of aflatoxin exposure, within Egypt. To our knowledge the 14 measurement of this validated biomarker to assess exposure in this region has not 15 previously been reported. Our data indicate moderate levels of aflatoxin exposure is 16 occurring within this limited survey, though larger studies are needed to investigate both 17 the sources and potential consequences of exposure to this potent toxin in Egypt. 18

  assess exposure. A limited number of sera were available from 5

13

 13

  during both cultivation and storage post-harvest (IARC 2002). 3

  et al., 2005), and deaths due to HCC more than doubled in the previous two decades 16 increasing from 2.4 per 100,000 in 1987 to 5.2 per 100,000 in 2000 (El-Sawry et al., 17 2001). Major etiological factors in this region are clearly HCV (Angelico et al., 1997; 18 Saeed et al., 1991; El-Zayadi et al. 2005, El-Kafrawy et al. 2005) and HBV (Attia, 19 1998), with a possible independent role for occupational pesticide exposures (Ezzat et 20 al. 2005). To date the potential role of aflatoxins in HCC development in Egypt has not 21 been fully explored. We recently reported that 8 of 41 liver samples from Egyptian 22 HCC cases contained a specific transversion mutation (AGG to AGT (arginine-to-23 serine) at codon 249) within the TP53 tumour suppressor gene (TP53 Ser249) (El-24 Kafrawy et al., 2005). This specific mutation is suggestive of aflatoxin being causative 25

  control study was conducted from 1999 -2004 to study viral and environmental 21 risk factors associated with hepatocellular carcinoma (HCC) in Egypt. Detailed methods 22 of recruitment have been previously documented (Cowgill et al. 2004; Ezzat et al. 23 2005). In brief the study consisted of 343 confirmed cases of HCC and 524 non-cancer 24 controls matched by age, sex, and region of current residence. A subset of 46 25

  African adults[START_REF] Wild | Environmental and genetic determinants of aflatoxin-albumin adducts in the 19 Gambia[END_REF] Turner et al., 2005a;[START_REF] Dash | Determinants of the variability of aflatoxin-albumin adduct levels in Ghanaians. 24[END_REF] and children 1

  in which 138/388 (35.6%) were positive for this metabolite. 22The difference in prevalence may reflect the longer half-life of the albumin adduct, 23 approximately 3 weeks (Chapot and Wild, 1991), compared to AFM 1

Food

  Renganathan E,Gandin C, Fathy M, Profili MC, Refai W, De Santis A, 3 Nagi A, Amin G, Capcaccia L, Callea F, Rapicetta M, Badr G, Rocchi G, 1997. Chronic 4 liver disease in the Alexandria governorate, Egypt: contribution of schistosomiasis and 5 hepatitis virus infections. Journal of Hepatology. 26: 236-243. 6 7 Attia MA. 1998. Prevalence of hepatitis B and C in Egypt and Africa. Antiviral 8 Therapeutics 3:1-9. 9 10 Bressac, B., Kew, M., Wands, J., and Ozturk, M. Selective G to T mutations of p53 11 gene in hepatocellular carcinoma from southern Africa. Nature 350:429-431, 1991. 12 13 Chapot B, and Wild CP. 1991. ELISA for quantification of aflatoxin-albumin 14 adducts and their application to human exposure assessment., p. 135-155. In M. Warhol, 15 D. van Velzen, and G. R. Bullock (ed.), Techniques in Diagnostic Pathology. Gong Y, Cardwell K, Hounsa A, Egal S, Turner PC, Hall AJ, Wild CP. 2002. Dietary 1 aflatoxin exposure and impaired growth in young children from Benin and Togo: cross 2 sectional study. British Medical Journal 325: 20-21. 3 4 Gong Y.Y., Hounsa A., Egal S., Turner P.C., Sutcliffe A.E., Hall A.J., Cardwell K. 5 Wild C.P. (2004) Post-weaning exposure to aflatoxin results in impaired child growth: a 6 longitudinal study in Benin, West Africa. Environmental Health Perspectives. 112(13Sheashaa HA, Abdel Fatah MF, Ibrahim AZ, Gaber OA. 2006 Does 10 aflatoxin as an environmental mycotoxin adversely affect the renal and hepatic 11 functions of Egyptian lactating mothers and their infants? A preliminary report. 12 International urology and nephrology. 38(2): 339-42. 13 14 Hatem NL, Hassab HM, Abd Al-Rahman EM, El-Deeb SA, El-Sayed Ahmed RL. 2005. 15 Prevalence of aflatoxins in blood and urine of Egyptian infants with protein-energy 16 malnutrition. Food and Nutrition Bulletin 26: 49-56. 17 18 Hsu, I.C., Metcalf, R.A., Sun, T., Welsh, J.A., Wang, N.J., and Harris, C.C. Mutational 19 hotspot in the p53 gene in human hepatocellular carcinomas. Nature 350:427-428, 1991. 20 21 International Agency for Research on cancer (IARC) 1994, Hepatitis viruses, 22 monographs on the evaluation of carcinogenic risks to humans. IARC Sci. Publ. 59 23 Lyon, France. 24 Page 12 of 16 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Research on cancer (IARC) (2003). Cancer in Africa: 5 epidemiology and prevention. Lyon (France): IARC Scientific Publication 153 6 7 Kirk GD, Camus-Randon AM, Mendy M, Goedert JJ, Merle P, Trepo C, Brechot C, 8 Hainaut P, Montesano R. 2000. Ser-249 p53 mutations in plasma DNA of patients with 9 hepatocellular carcinoma from The Gambia. Journal of the National Cancer Institute 92: 10 148-153. 11 12 Kirk GD, Lesi OA, Mendy M., Szymanska K., Whittle H., Goedert J.J., Hainaut P., 13 Montesano R. (2005) 249(ser) TP53 mutation in plasma DNA, hepatitis B viral 14 infection, and risk of hepatocellular carcinoma. Oncogene24(38):5858-67. 15 16 Kirk GD, Camus-Randon AM, Mendy M, Goedert JJ, Merle P, Trepo C, Brechot C, 17 Hainaut P, Montesano R. 2000.. 249(ser) TP53 mutation in plasma DNA, hepatitis B 18 viral infection, and risk of hepatocellular carcinoma. Oncogene. ;24(38):5858-67. 19 20 Montesano R, Hainaut P, Wild CP. 1997 Hepatocellular Carcinoma: From gene to 21 public health Journal of the National Cancer Institute 89 1844

Table 1 :

 1 Aflatoxin-albumin adducts in adults from selected countries.

	1
	2
		COUNTRY	AF-Alb (pg/mg)	Prevalence of	Reference
		(n)	Geometric Mean	positive samples
			(range)	(%)
		Egypt
		Total	5.0 (nd -32.8)	31/46 (76)
		HCC cases	2.6 (nd -32.8)	7/22 (31.8)	This report
		Controls	9.0 (nd -25.8)	24/24 (100)
		The Gambia
		Rural	61.8 (57.3 -66.7)#	272/272 (100)	Wild et al., 2000
		Peri-urban	39.1 (31.1 -49.1)#	83/84 (98.8)
		Guinea##	18.4 (nd-66)	245/250 (98)	Turner et al., 2005b
		The Gambia	40.4 (4.8-260.8)	119/119 (100)	Turner et al., 2007
		Brazil*	10.7 (nd -57.3)	31/59 (62)	Scussel et al., 2006
		France/ Poland	0 (nd)	0/74 (0)	Wild et al., 1993
	3	nd. non-detected
	4	* Geometric means of positives only
	5	# Geometric means and 95%CI
	6	## Data obtained for blood samples in Feb/Mar
	7	$ -Infants aged 4 months old.

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Acknowledgements

This study was supported by grant number R01CA85888 from the U.S. National 21 Institutes of Health, and grant number TSA R6/81/1 (ID RCP 02/58) from the Regional 22 Office for the Eastern Mediterranean, World Health Organization. Authors thank Mrs 23 Anne Sutcliffe for technical assistance. AS and PCT were supported by NIEHS Grant 24 No. ES06052.


Frank Welle
email: welle@ivv.fraunhofer.de

Roland Franz

SiO x layer as functional barrier for PET bottles towards potential contaminants from post-consumer recycled poly(ethylene terephthalate)

Keywords: Food contact materials, Migration -diffusion, Packaging -PET, Packaging -recycling Functional barrier, PET recycling, SiO x coating, challenge test, food packaging, migration

The barrier effect of a silicon oxide (SiO x ) coating on the inner surface of PET bottles, in terms of the ability to reduce the migration of post-consumer compounds from the PET bottle wall into the food simulants 3% acetic acid and 10% ethanol, was investigated. The barrier effect was examined by artificially introducing model substances (surrogates) into the PET bottle wall to represent a worst case scenario.

Test bottles with three different spiking levels up to about 1000 mg kg -1 per surrogate were blown and coated on the inner surface. The SiO x coated bottles and the noncoated reference bottles were filled with food simulants. From the specific migration of the surrogates with different bottles wall concentrations the maximum concentrations of the surrogates in the bottle wall corresponding to the migration of 10 µg l -1 were determined. It was shown that the SiO x coating layer is an efficient barrier towards post-consumer compounds. The maximum bottle wall concentrations of the surrogates corresponding to the migration of 10 µg l -1 were in the range of 200 mg kg -1 for toluene and about 900 mg kg -1 for benzophenone. Consequently, the SiO x coating allows use of conventionally recycled post-consumer PET flakes (without a super-clean recycling process) for packaging aqueous and low alcoholic foodstuffs (under cold-fill conditions) and prevents the food from migration of unwanted contaminants from post-consumer PET.

into the PET material may migrate from the recyclate containing bottle into the foodstuff. In case of a too high concentration of the post-consumer compounds in the bottle wall, the migration might pose a health risk for the consumer. Since measurement of bottle wall concentrations for each and every bottle is impossible, so-called challenge tests have been developed in which model chemicals (also named surrogates) are artificially introduced into a recycling process. As a safety parameter or as cleaning efficiency criterion, for these surrogates a migration limit of 10 µg l -1 in food from a bottle produced in a challenged recycling process has been generally accepted [START_REF] Fda | Points to Consider for the Use of Recycled Plastics in Food Packaging: Chemistry Considerations[END_REF], ILSI 1998[START_REF] Bgvv | Use of mechanical recycled plastic made from polyethylene terephthalate (PET) for the manufacture of articles coming in contact with food[END_REF], Franz 2004a[START_REF] Begley | Recycled polymers in food packaging: migration considerations[END_REF], Franz et al 1998[START_REF] Franz | Post-consumer poly(ethylene terephthalate) for direct food contact applications -final proof of food law compliance[END_REF][START_REF] Franz | Post-consumer Poly(ethylene terephthalate) for Direct Food Contact Application -Challenge-Test of an Inline Recycling Process[END_REF]. More recently, the French Food Safety Authority AFFSA has defined 1.5 µg l -1 as a target migration limit which should not be exceeded (AFFSA 2006). It is assumed, when taking exposure scenarios also into account, that with these performances a recycling process would remove any unknown contaminant which might be able to migrate and would be capable to produce a recycled PET food contact material which can safely be reused for direct food contact.

In the last decade, several super-clean recycling technologies have been established

in industrial or pilot plant scale in several countries [START_REF] Mueller | PET in beverage packaging[END_REF]. All of these technologies have been shown to be able to reduce the concentration levels of super-clean recycling process has therefore a migration potential which is similar to virgin PET [START_REF] Franz | Recycling packaging materials[END_REF]). An overview of the different recycling technologies and food contact application conditions is given on the internet homepage of the US FDA [START_REF] Fda | Points to Consider for the Use of Recycled Plastics in Food Packaging: Chemistry Considerations[END_REF].

Another possibility to prevent the migration of unwanted post-consumer compounds into PET bottled foodstuffs is the use of appropriate functional barriers. A functional barrier is defined as a package construction or barrier layer which limits the extent of migration of a component from the package to the food or food simulant in amounts below an accepted value threshold or migration limit of no concern. However, it should be noted that a functional barrier is not necessarily an absolute barrier but a function of the given food packaging application. The migration kinetics of a functional barrier system is in general characterized by an initial time lag phase followed by a much less steeper migration curve line compared to the functional barrier free package [START_REF] Feigenbaum | Functional barriers: Properties and evaluation[END_REF][START_REF] Dole | Modelling of migration from multi-layers and functional barriers: Estimation of parameters[END_REF]. The lag time is defined as the time which a substance needs to cross a functional barrier package construction and to reach the food contact surface. Typically the lag time is responsible, that during the shelf life of the product the migration limit is not exceeded. In the special case of PET bottles or sheets, an outside and inner layer of virgin PET acts as a functional barrier. In the core layer of such a multi-layer structure conventionally recycled PET is located in a certain amount. The virgin layer on the inner side with contact to the foodstuff can reduce the migration into the foodstuff to levels under the general accepted migration limit of 10 µg l -1 (Franz et al. 1996, Welle and[START_REF] Welle | Recycled Plastics and Chemical Migration into Food[END_REF]. In comparison to conventional monolayer PET bottles, the manufacturing of multilayer PET bottles is more expensive. Another problem is that the virgin layer might be contaminated with post-consumer compounds by in-situ diffusion during bottle manufacturing with the consequence that the partially contaminated virgin layer might have then a lower barrier effect [START_REF] Franz | Presentation and experimental verification of a physico-mathematical model describing the migration across functional barrier layers into foodstuffs[END_REF][START_REF] Piringer | Migration from food packaging containing a functional barrier: Mathematical and experimental evaluation[END_REF] In the literature some studies can be found dealing with the barrier properties of SiO x coatings inside of PET bottles. For instance, a diamond-like carbon (DLC) layer on PET bottles enhances the gas permeation barrier [START_REF] Boutroy | Hydrogenated amorphous carbon film coating of PET bottles for gas diffusion barriers[END_REF][START_REF] Yamamoto | Oxygen transmission of transparent diamond-like carbon films[END_REF][START_REF] Finch | Diamond-like Carbon, a barrier coating for polymers used in packaging applications[END_REF]. In another study the barrier properties of carbon coated PET against post-consumer contaminants was investigated [START_REF] Cruz | Study of barrier properties and chemical resistance of recycled PET coated with amorphous carbon through a plasma enhanced chemical vapour deposition (PECVD) process[END_REF]. The results of this study, however, do not reflect the barrier properties potential of SiO x layers which, in our opinion, might be due to the laboratory coating scale character of the investigated samples. In a study with a SiO x coated PET film [START_REF] Dimitroulas | Permeation of methylethylketone, oxygen and water vapor through PET films coated with SiOx: Effect of temperature and coating speed[END_REF] found that the barrier against the chemical substance methylethyl ketone increases by a factor of 7-8 at room temperature.

The objective of this study was to investigate and to evaluate the barrier effect of a market scale commercially available SiO x coating process. The barrier effect was examined by artificially introducing model substances (surrogates) into the bottle wall in a worst case scenario. The surrogates were chosen according to the principles recommended by American and European Guidelines [START_REF] Fda | Points to Consider for the Use of Recycled Plastics in Food Packaging: Chemistry Considerations[END_REF][START_REF] Fda | Points to Consider for the Use of Recycled Plastics in Food Packaging: Chemistry Considerations[END_REF], ILSI 1998[START_REF] Bgvv | Use of mechanical recycled plastic made from polyethylene terephthalate (PET) for the manufacture of articles coming in contact with food[END_REF], Franz et al. 2004a). After the contamination, the spiked PET bottles were industrially coated with a SiO x layer. The specific migration of the surrogates into food simulants from the bottle wall through the SiO x barrier layer was

Materials and Methods

Selection of Surrogates

The barrier properties of the SiO x coating was determined by introducing chemicals (surrogates) into the bottle wall of PET test bottles. The applied surrogates are in compliance with the four categories of organic compounds: high volatile and polar, high volatile and non-polar, low volatile and polar, low volatile and non-polar. The following model compounds were used for the migration tests: toluene, chlorobenzene, phenyl cyclohexane, benzophenone and methyl stearate.

Spiking of the Test bottles

Batches of 1.1 kg of dried PET resin were weighed into glass jars. For the desired amount of preforms several batches were prepared in parallel. Subsequently the target weight of each of the chemicals were added to each of the jars. The jars were tightly sealed and shaken to disperse the chemical mixture onto the surface of the dried PET pellets. The sealed jars were stored at 38°C for 7 days. Each day, the jars were shaken to thoroughly mix the resin and chemicals. At the end of the seven days storage time, the material was removed from the storage environment and be kept sealed until it is injection molded into 24.5 g preforms. From the preforms 0.3 l PET bottles were blown. Subsequently one part of the bottles was coated with the investigated SiO x coating process whereas the other part of the (non-coated) bottles was used as reference. The concentrations of each surrogate and contamination level were determined in the coated as well as in the non-coated reference bottles.

Coating of the test bottles

The coating film is deposited on the surface of PET bottles using a plasma impulse chemical vapour deposition (PICVD) process. Starting substances for the SiO x coating are hexamethyl disiloxane (HMDSO) and hexamethyl disilazane (HMDSN).

The barrier coating is a quartz-like surface with almost the stochiometric composition of SiO 2 . The maximum average thickness of the barrier layer is about 100 nm. The SiO x coating is approved by the American health authority FDA under food contact substance notification FCN 329.

Migration testing

The specific migration of the surrogates was measured into the food simulants 3%

Determination of the bottle wall concentrations of the surrogates

The PET test bottles were cut in small pieces of approximately 0.5 x 0.5 cm. 1.0 g of each PET sample was placed in a 5 ml glass vial. 1.0 ml 1,1,1,3,3,3-hexafluoro-isopropanol (HFIP) was given to the PET material and stored for 1 d at 60 °C in order to swell the PET matrix. Then 2.0 ml iso-propanol was added for 1 d at 60 °C to extract the swollen matrix. The extract was decanted from the polymer and stored for 4 h at .

Determination of the surrogate concentration in migration solutions by purge and trap gas chromatography

The concentration of the volatile and medium volatile surrogates toluene, chlorobenzene, phenyl cyclohexane in the food simulants was determined using purge and trap gas chromatography (p&t GC) with flame ionisation detection (FID).

Sample preparation: a) Acetic acid: 5 ml of the 3% acetic acid migration solution was neutralised with 1 ml of 20% caustic soda (NaOH) solution in water. Subsequently

Determination of the surrogate concentrations in migration solutions by GC/MS (SIM)

The 3% acetic acid and 10% ethanol migration solutions were extracted three times with n-hexane (20 ml of food simulant with 8 ml, 5 ml and 5 ml of n-hexane).

Subsequently the concentrations of methyl stearate was determined using GC/MS in the selective ion mode (SIM). The mass fragment m/z = 74 was used for quantification. m/z = 87 was used as qualification ion. GC/MS System: Shimadzu QP Quantification was achieved by external calibration using standard solutions of the surrogates.

Determination of the benzophenone concentrations in migration solutions by HPLC (UV)

The migration solutions were analysed by HPLC (UV). HPLC equipment: Dionex, column: Phenomenex, Synergi Fusion RP 150×2.0 mm, 4 µm particle size, guard column, mobile phase: 60% acetonitrile and 40% water, isocratic elution, flow rate: 0.3 ml min -1 , oven temperature: 25 °C, detection at 254 nm, injection volume: 10 µl.

Detection Limits

The detection limits were determined according to DIN 32645. The detection limits of the applied methods for the determination of the surrogates in the different food simulants are given in Table 1.

Place here Table 1 Results and Discussion

Spiking Levels of Test Bottles

The concentration of the surrogates in the bottle wall was analysed after manufacturing of the spiked bottles as well as after coating. The concentrations are measured by extraction of the PET bottle wall with HFIP/iso-propanol and gas chromatographic analysis. The results are summarised in

Specific migration testing

The specific migration testing was performed using three different detection methods. Volatile surrogates were quantitatively determined using a purge and trap gas chromatographic method. The method shows detection limits and a good linearity of the calibrations curves. However, the method is limited to the volatile toluene, chlorobenzene and phenyl cyclohexane only. For benzophenone an HPLC/UV method was applied. In the case of methyl stearate, the analytical detection in the migration solutions at detection limits below 10 µg l -1 failed due to interferences and analytical artefacts. The applied gas chromatographic separation with mass spectrometric determination in the single ion mode (GC MS (SIM)) was not sensitive enough for the determination of methyl stearate in the 10 µg l -1 range.

Therefore the results of methyl stearate were not used for the evaluation of the maximum bottle wall concentrations. However, the concentrations of methyl stearate in the migration solutions after storage for 10 d at 40 °C was below the analytical detection limit of 13.0 µg l -1 (3% acetic acid) and 25.4 µg l -1 (10% ethanol). The results of the specific migration testing for toluene and chlorobenzene with the contact conditions 10 d at 40 °C into 3% acetic acid and 10% ethanol are given in Table 3. For toluene and chlorobenzene the experimental migration kinetics follows the prediction by changing the bottle wall concentration (c P,0 ). The concentration of phenyl cyclohexane in migration simulants after storage for 10 d at 40 °C are for all investigated bottle wall concentrations (coated and non-coated) below the analytical detection limits of 0.9 µg l -1 and 1.1 µg l -1

, respectively. For benzophenone only the bottles with the highest concentrations were measured. The migration was below the analytical detection limit of 8.8 µg l -1

.

Place here

Migration kinetics into 10% ethanol

For the high level spiked test bottles (non-coated and coated) a migration kinetics was established up to a storage time of 30 d at 40 °C. Table 4 summarizes the results of the migration kinetics for toluene, chlorobenzene, phenyl cyclohexane and benzophenone in 10% ethanol. As a result the surrogates toluene and chlorobenzene follow this linear correlation (Figure 1) between the square root of time and the migration for the non-coated as well as for the SiO x coated test bottle which indicates that the migration follows Fick's law. For phenyl cyclohexane and for benzophenone, the concentrations in the migration solutions are too low for a correlation.

Place here Table 4 Place here Figure 1 Maximum concentrations of surrogates in the bottle wall

For the evaluation of food law compliance of the PCR PET containing and SiO x coated PET bottles a maximum migration of 10 µg l -1 into food simulants at 40 °C was applied. It should be noted that this 10 µg l -1 migration limit is not an officially by EU Directives recognised threshold of no concern for potential contaminants from PCR plastics (see introduction). The maximum acceptable concentrations of surrogates in the bottle wall, which corresponds to a 10 µg l -1 migration value in the investigated 300 ml bottle, can be calculated from the correlation between the bottle wall concentration and the resulting specific migration into the investigated food simulants due to the fact that according to migration theory the bottle wall concentration correlates linear with the concentration in food simulant for a specific migrant. Such a correlation after storage for 10 d at 40 °C was established for the surrogates toluene and chlorobenzene for the non-coated test bottles (Table 3).

From the slope of the linear correlation the maximum concentration could be estimated directly. The intercept was not taken into account. For phenyl cyclohexane and benzophenone as well as for toluene and chlorobenzene in the SiO x coated 12 bottles, the concentrations in the migration solutions were below 10 µg l -1 even at the highest bottle wall concentrations. The estimated maximum acceptable concentrations in PET material which correlate with a 10 µg l -1 migration limit in food simulants are summarised in Table 5. In the cases of a migration below the analytical detection limits, the highest concentration level in the spiked test bottles was used as maximum acceptable bottle wall concentration. For the SiO x coated bottles the maximum concentrations in the bottle wall are much higher due to the additional barrier effect in comparison to the non-coated reference bottle.

Place here Table 5 Conclusions

From analytical screening tests on PET recyclate samples taken from the market, it has been shown, that the concentrations of foreign compounds in production batches of conventionally recycled PCR PET do not exceed a concentration level of about 50 mg kg -1 for any individual post-consumer compounds [START_REF] Franz | Recycling packaging materials[END_REF], Franz et al 2004b). Chemical compounds, which are introduced into the bottle wall by misuse of PET bottles for the storage of e.g. household chemicals have high levels in the individual bottle. However, due to the rarity of this event and due to the dilution of the contaminated PET flakes during washing with non-contaminated flakes, the average residual concentration of chemical compounds from misuse are more likely to be <10 mg kg -1 (Franz et al 2004b). Therefore, the artificially established initial concentrations used in this study and especially at the high spiking level would be far above any relevant concentrations of post-consumer compounds occurring in practice and would represent a worst-case scenario for the inputmaterial.

From the ratios of the slopes of the migration kinetics (concentration in food simulant versus square root of time) the barrier effect for the SiO x coating layer for reducing the migration of toluene and chlorobenzene in 10% ethanol was determined to 37 and 29, respectively. Therefore, the data shows the SiOx layer significantly reduces migration. It can be assumed, that the barrier effects for other surrogates or post- . Due to the more favourable surface/volume ratios of larger bottles, the estimated maximum levels given in Table 8 can be considered as worse case. Therefore the maximum levels of possible contaminants in the bottle wall will increase with increasing bottle size (increasing surface/volume ratio).

The experimental data from this study demonstrate that SiO x coated PET bottles produced from the contaminated test batch would be in accordance with the 10 µg l -1 migration limit as long as the estimated maximum concentrations given in Table 8 are not exceeded. The maximum bottle wall concentrations of the surrogates are in the range of 200 mg kg -1 (toluene) and about 900 mg kg -1 (benzophenone).

  SiOx layer as functional barrier for PET bottles towards potential contaminants from post-consumer recycled poly(ethylene terephthalate) Introduction The use of post-consumer recycled polyethylene terephthalate (PCR PET) in soft drink bottles has been established for a number of years. Post-consumer PET bottles typically are collected with curbside or deposit collections. Subsequently the PET bottles which have been collected are ground and washed. Labels and closures as well as dirt and surface contamination are removed from the PET recyclates. The resulting conventionally recycled PET flakes can be used for non-food packaging applications (e.g. detergent bottles) or fibres. If the recycled PET flakes are to be reused in food contact applications, further deep-cleansing recycling processes is necessary. Post-consumer substances or compounds from the possible misuse of PET bottles for the storage of e.g. household chemicals which have been absorbed

  in the PET recyclates down to levels which are similar to the concentrations of technical impurities in virgin PET. The output material of a

  difference between the specific migration from the coated and noncoated test bottles represents the barrier effect of the SiO x coating layer towards the applied surrogates. In order to establish a basis for an extrapolation from the worst case scenario of the challenge test to more realistic concentrations of possible contaminants, the PET test bottles were spiked with three different contamination levels (low, medium, high).

  (w/v) acetic acid and 10% (v/v) ethanol. Due to the fact that the SiO x coating might be sensitive towards high pH values, 5 g citric acid per 300 ml was dissolved in the ethanolic food simulant. For migration testing, the bottles were filled with the food simulants (two bottles for each bottle type and food simulant) and stored for 10 d at 40 °C. The bottles were sealed with HDPE closures with an aluminum film on the contact surface in order to prevent sorption of the surrogates into the closures. For the food simulant 10% ethanol a migration kinetic was established. 50 ml aliquots of the food simulant were drawn after 3 d, 6 d, 10 d, 20 d and 30 d. The bottles were refilled after drawing the 50 ml aliquots with the same amount of simulant. The reduction of the concentration of the surrogates by the refilling with food simulants was considered.

  Then it was decanted again from the precipitate and analyzed by GC/FID. Quantification was achieved by external calibration using the standard addition method. Gas chromatograph: HP 5890II, column: SE 10 -30 m -0.32 mm i.d. -0.32 µm film thickness, temperature program: 40 °C (5 min), rate 15 °C min -1 , 240 °C (15 min), pressure: 50 kPa hydrogen, split: 10 ml min -1

  the solution was analysed by purge and trap gas chromatography (FID). b) 10% ethanol: 10 ml of the 10% ethanol migration solution was analysed by purge and trap gas chromatography (FID). Method: Gas Chromatograph: Carlo Erba 5300 Mega, column: ZB 624, length 60 m, inner diameter 0.32 mm, film thickness 1.8 µm, carrier gas: 120 kPa helium, Temperature program: 35 °C (6 min), rate 5 °C min -1 , 90 °C (0 min) rate 10 °C min -1 , 260 °C (15 min). Purge and trap conditions (PTA 3000): sample temperature 40 °C, purge time: 20 min, purge flow 20 ml min -1 , trap temperature: -65 °C, desorption temperature 200 °C, desorption time 7 min, water trap MWT, Tenax ®trap. Quantification was achieved by external calibration using standard solutions of the surrogates.

  : DB VRX -30 m -0.32 mm i.D. -1.8 µm film thickness, temperature program: 40 °C (5 min), rate 15 °C min -1 , 250 °C (15 min), pressure: 64 kPa hydrogen, splitless, interface temperature: 260°C, Injector temperature: 250°C.

  in a similar range. The migration tests were performed on a 0.3 l bottle with a surface of about 280 cm 2

  Therefore the investigated SiO x coating layer is an efficient functional barrier towards recyclate typical compounds. Consequently, this SiO x coating layer would allow the use of conventionally recycled post-consumer PET flakes without super-clean recycling processes for packaging aqueous and low alcoholic foodstuffs under coldfill conditions and prevent the food from migration of unwanted contaminants from post-consumer PET. In addition, it should be noted that the drawn conclusions implicate the worst case assumption that 100% post-consumer recyclate is used for PET bottle manufacturing.AcknowledgementThe author would like to thank SIG Corpoplast for the SiO x coating of the investigated bottles. Special thanks are due to Erik Lüpke (SIG), Hermann Neugebauer (Rexam), Silvia Demiani, Hans Ewender, Alexandra Mauer and Sonja Smolic (Fraunhofer IVV) for experimental contributions and fruitful discussions. Literature AFFSA. 2006. Opinion of the French Food Safety Agency (AFFSA) on the assessment of health risks associated with the use of materials made from recycled

  . The use of conventionally recycled PCR PET in the core layer

	of PET multilayer bottles is therefore today not established on a significant industrial
	scale in Europe.
	Another functional barrier technology is the application of a silicon oxide (SiO x )
	coating on the inner surface of PET bottles. Typically this technology is used to
	reduce the permeation of permanent gases like oxygen or carbon dioxide. Therefore
	SiO x coated PET bottles are on the market for oxygen sensitive foodstuffs like beer and juices. On the other hand, the SiO x layer reduces not only the permeation of F gases. The functional barrier might also reduce the migration of post-consumer o r compounds into the PCR PET bottled foodstuff. If this functional barrier is efficient
	enough, then it would present an alternative option to super-clean technologies and P may allow directly the re-use of conventionally recycled PET without further deep-e r e cleansing of the PCR PET.
	R
	e
	v i e
	w
	O n l
	y

Table 2

 2

	than for volatile substances like the solvents toluene and chlorobenzene. During
	preform and bottle manufacturing of the test bottles, the concentration of the high
	volatile substances are reduced due to vaporisation.
	Place here Table 2
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	10

(mean value from three individual bottles). The contamination levels are slightly higher for the medium and low volatile substances (phenyl cyclohexane, benzophenone, methyl stearate)

Table 3

 3

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

Table 1 :

 1 Detection limits of the applied model compounds in food simulants

	Toluene uncoated
	Chlorobenzene uncoated
	Toluene coated
	Chlorobenzene coated

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Table 2 :

 2 Concentration of the surrogates in the bottle wall of the spiked PET bottles determined after extraction with HFIP/iso-propanol (mean values of the concentrations and standard deviation from three individual bottles)

	Page 19 of 22		Food Additives and Contaminants
	Test bottle type	Concentration [ppm]
		Toluene	Chlorobenzene	Phenyl cyclohexane Benzophenone	Methyl stearate
	low level, non-coated	17.6 ±0.6	26.2 ±0.5	35.3 ±1.0	91.4 ±2.4	107.0 ±2.9
	low level, coated	18.7 ±0.3	26.1 ±0.4	32.2 ±0.4	88.9 ±0.7	104.6 ±1.0
	medium level, non-coated 70.6 ±0.6	101.3 ±0.8	261.7 ±1.7	394.3 ±3.2	400.9 ±3.0
	medium level, coated	57.0 ±0.1	87.6 ±0.3	240.9 ±1.6	396.1 ±2.7	412.5 ±3.4
	high level, non-coated	297.5 ±1.2	357.8 ±1.5	782.3 ±1.4	973.9 ±3.5	1029.1 ±16.3
	high level, coated	203.6 ±1.5	249.5 ±1.8	601.3 ±4.9	882.5 ±7.7	904.1 ±11.5

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Table 3 :

 3 Concentration of toluene and chlorobenzene in migration simulants after storage for 10 d at 40 °C determined by purge and trap gas chromatography

				Food Additives and Contaminants			Page 20 of 22
	Simulant	Migrant	bottle type	concentration in food simulant [µg l -1 ]	slope	Intercept Correlation coefficient R 2
				low level medium	high level
					level
	3% acetic acid Toluene	non-coated	1.4	2.3	14.5	0.0489	-0.2170	0.9862
	10% ethanol	Toluene	non-coated	2.3	8.6	43.4	0.1488	-1.0284	0.9987
	3% acetic acid Chlorobenzene non-coated	3.2	5.7	32.6	0.0928	-1.1818	0.9803
	10% ethanol	Chlorobenzene non-coated	4.5	17.1	65.4	0.1848	-0.9022	0.9996
	3% acetic acid Toluene	coated	<1.0	<1.0	<1.0	/	/	/
	10% ethanol	Toluene	coated	<0.4	<0.4	1.1	/	/	/
	3% acetic acid Chlorobenzene coated	<0.7	<0.7	<0.7	/	/	/
	10% ethanol	Chlorobenzene coated	<0.3	0.3	1.7	0.0086	-0.4875	/

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Table 4 :

 4 Results of the migration kinetics of the model compounds from the test bottles into 10% ethanol at 40 °C

	Page 21 of 22			Food Additives and Contaminants
	storage	concentration [µg l -1 ]
	time [d]	(bottle wall concentration [ppm])
		Toluene		Chlorobenzene	Phenyl cyclohexane	Benzophenone
		non coated	SiO x coated	non coated	SiO x coated	non coated	SiO x coated	non coated	SiO x coated
		(297.5 ±1.2)	(203.6 ±1.5)	(357.8 ±1.5)	(249.5 ±1.8)	(782.3 ±1.4)	(601.3 ±4.9)	(973.9 ±3.5)	(882.5 ±7.7)
	3	24.7	<0.4	32.7	0.3	<0.9	<0.9	not	not
								determined	determined
	6	38.6	0.7	53.8	1.1	<0.9	<0.9	not	not
								determined	determined
	10	43.4	1.1	65.4	1.7	<0.9	<0.9	<8.8	<8.8
	20	63.0	2.1	99.0	3.2	<0.9	<0.9	14.8	<8.8
	30	98.5	2.1	146.4	4.0	1.6	<0.9	21.2	<8.8

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Table 5 :

 5 Estimated maximum concentrations [ppm] of the investigated model compounds in the bottle wall of the test bottles corresponding to a migration of 10 µg l -1 in food simulant (contact conditions: 10 d at 40 °C)

					Page 22 of 22
	Surrogate	Estimated maximum concentrations [ppm] in the bottle wall
		non-coated bottle	SiO x coated bottle
		3% acetic acid 10% ethanol	3% acetic acid	10% ethanol
	Toluene	204	67	>204	>204
	Chlorobenzene 108	54	>250	>250
	Phenyl	>782	>782	>601	>601
	cyclohexane
	Benzophenone >974	>974	>883	>883

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Food Additives and Contaminants

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


Kevin Mark Cooper

Le Jian

Christine Kane

David Glenn Kennedy

Jian Le

D Glenn Kennedy
email: glenn.kennedy@afbini.gov.uk

A study

Keywords: Veterinary drug residues -antimicrobials, Veterinary drug residues -semicarbazide Nitrofurans, Nitrofurazone, Semicarbazide, Eggs, LC-MS/MS

  methanol (80 : 20 v/v, solvent A) and methanol (solvent B), 138 starting at 95% A/5% B and changing linearly to 25% A/75% B in 6 min. These 139 conditions were held until 11 min. Initial conditions were restored by 11.5 min. The 140 total run time was 20 min. The precursor/product ions monitored during analysis of 141 nitrofuran metabolites are listed in

  00 ± 0.02 g) were weighed into 30 ml glass tubes. Mixed internal 240 standard (50 µl of a 100 ng ml -1 solution of D 4 -AOZ, D 5 -AMOZ, 13 C 3 -AHD and 241 13 C 15 N 2 -SEM) was added to all samples, controls and standards. To all tubes were 242 added hydrochloric acid (0.1 M, 5 ml) and 2-nitrobenzaldehyde (150 µl, 50 mM in 243 DMSO). Calibration standards were prepared by addition of mixed standard solutions 244 to 30 ml glass tubes. Egg samples from nitrofuran-free chickens were fortified at 245 1.0 µg kg -1 with mixed standard (100 µl of a 10 ng ml -1 solution) to act as recovery 246 control samples. Blank egg samples were also included in every analytical run. 247 Tubes were capped, vortex mixed for 20 sec and incubated overnight (approximately 248 16 hr) in a water bath held at 37°C. 249 All tubes were then adjusted to pH 7.2 ± 0.2 with 0.1 M di-potassium hydrogen 250 orthophosphate (5 ml) and 1 M sodium hydroxide (0.4 ml). Liquid-liquid extraction 251 was carried out using ethyl acetate (2 x 6 ml, repeatedly inverting the tubes for 1 min 252 then centrifuging at 2000 rpm for 15 min) and the organic phase evaporated to 253 dryness at 45°C under a stream of nitrogen. Residues were re-dissolved by vortex 254 mixing for 1 min in methanol : water (50 : 50 v/v; 0.4 ml

Figure. 1 .Figure 2 .

 12 FIGURE CAPTIONS

Table 1 .

 1 The source settings were as follows:

	142
	143	capillary voltage 2.45 kV, RF lens 25.0 V, source temperature 120 °C, desolvation
	144	temperature	350	°C,	cone	nitrogen	gas	flow
		80 L hr -1 , desolvation gas flow 670 L hr -1 . Argon was used as the collision gas and

145

the multiplier was operated at 650 V. The cone voltage was 35 V and the collision 146 energy (7 to 16 eV) changed during analysis depending on the analyte. Sample 147 injection volume was 25 µl. LC-MS/MS conditions for nitrofurazone analysis were 148 as described previously

[START_REF] Mccracken | The detection, accumulation and 486 distribution of nitrofuran residues in egg yolk, albumen and shell[END_REF]

.

149 [INSERT TABLE 1 ABOUT HERE] 150

Treatment of laying hens with NFZ 151 Broiler breeder hens (30) in mid-lay, purchased from a local supplier, were housed in 152 wire-floored cages in five separate batteries of six birds. Hens were allowed ad 153 libitum access to fresh water and feed at all times. Commercial broiler breeder layer 154 crumb feed was used to prepare five medicated diets (one at therapeutic and four at 158 fed medicated diets for 16 days when, with the exception of the 300 mg kg -1 group, 159 the experiment was terminated. On day 16, diet of the birds fed nitrofurazone at a 168 the pooled samples were thawed to room temperature. Yolk and albumen pools were 169 diluted with further yolk and albumen from locally reared hens that had not received 170 NFZ. The whole egg pool was homogenised and divided into 1.0 kg aliquots and 171 refrozen. The egg albumen pool could not be homogenised mechanically without 172 dramatically changing its consistency, so it was stirred thoroughly for 2 h before 173 being divided into 1.0 kg aliquots and refrozen. Upon thawing, the consistency of the 174 egg yolk pool was too thick to allow homogenisation. Consequently, it was diluted 175 50:50 (w/w) with distilled water before being homogenised, divided into 0.8 kg residues, for the purposes of regulatory control, it was inappropriate to use that 234 analytical approach with eggs in the current study. It was essential to study the total 235 SEM concentration in eggs to enable a comparison to be made between total NFZ 236 concentration and total SEM content of the egg. Were tissue-bound SEM to be 237 measured, a poorer, and possibly erroneous, correlation may have been found between 238 NFZ and SEM.

Table 1 .

 1 LC-MS/MS precursor/product ion combinations (base peaks underlined)

	506
	507	monitored in MRM ESI positive mode for the analysis of the nitrophenyl (2-NP)
	508	derivatives of four nitrofuran metabolites.
	509
		Compound	Precursor ion (m/z) Product ions (m/z)

Table 2 .

 2 Summary of validation data for the determination of four nitrofuran

	510
	511	metabolites in whole egg homogenate.
	512
			Fortification	Overall	Overall	Within	Between	CCα α α α	CCß
			Level µ µ µ µg kg -1	Mean µ µ µ µg kg -1	recovery	run	run	(µg/kg)	(µg/kg)
				(n=24)	(%)	CV	CV
		AOZ	0.5	0.51	102	7.4	1.7
			1.0	1.03	103	4.5	1.6
			1.5	1.54	103	4.2	1.4
							AOZ	0.14	0.44
		AMOZ	0.5	0.53	105	3.0	2.9
			1.0	1.07	107	3.1	3.9
			1.5	1.60	107	3.5	1.5
							AMOZ	0.13	0.25
		AHD	0.5	0.52	103	5.8	11.5
			1.0	1.02	102	6.3	11.6
			1.5	1.57	104	7.1	4.5
							AHD	0.21	0.60
		SEM	0.5	0.52	104	5.5	1.6
			1.0	1.01	101	5.2	1.5
			1.5	1.47	98	3.9	1.9
							SEM	0.19	0.28

Table 3 .

 3 Partitioning of NFZ and SEM between egg yolk and albumen at different 513 dietary NFZ levels with no withdrawal period (se = standard error).

	514
	515
		Mean	se	NFZ	Mean	se	Total SEM	Molar % of
		NFZ	(n=5)	Partitioning	total SEM	(n=5)	Partitioning	SEM present
		µg kg -1		(%)	µg kg -1		(%)	as NFZ
	3 mg NFZ kg -1 feed
	Yolk	10.8	0.7	63	27.8	2.3	79	15
	Albumen	6.4	0.7	37	7.3	0.6	21	33
	30 mg NFZ kg -1 feed
	Yolk	81.6	16.2	57	224.0	29.2	80	14
	Albumen	61.9	11.3	43	56.8	7.3	20	41
	300 mg NFZ kg -1 feed
	Yolk 712.9	112.3	58	1441.8	192.0	71	19
	Albumen 515.2	58.7	42	575.1	77.8	29	34.

Table 4 .

 4 Distribution of tissue-bound and total SEM in whole egg homogenates at 516 two dietary NFZ levels (se = standard error).

		Food Additives and Contaminants			Page 28 of 33
	517
	518
	NFZ in feed	Mean total	se	Mean tissue-bound	se	% Tissue-	n
	mg kg -1	SEM µg kg -1		SEM µg kg -1		bound
	3	13.7	0.4	3.8	0.2	28	10
	30	113.5	12.4	35.1	4.0	31	5

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Table 5 .

 5 Effect of pasteurisation and spray drying on NFZ and SEM concentrations in eggs (se = standard error). Whole egg, yolk and albumen 519 pools were derived from different sample populations so concentrations cannot be compared across sample type.

	Page 29 of 33						Food Additives and Contaminants
	520
	521
		NFZ µg kg -1 dry weight				SEM µg kg -1 dry weight			Molar % of SEM present as NFZ
		Before	se	After	se	% loss	Before	se	After	se	% loss of	Before	After	n
		drying		drying		of NFZ	drying		drying		SEM	drying	drying
	Whole egg	502.1	23.7	177.6	35.4	65	676.5	25.8	330.7	30.0	51	28	20	3
	Yolk	267.6	15.1	151.9	3.6	43	608.7	17.7	507.6	3.7	17	17	11	4
	Albumen	1085.6	203.7	44.5	6.8	96	954.3	148.1	264.5	1.8	72	43	6	4

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Samples were then transferred to a water bath held at 67°C. Sample temperature was

Retention of some egg powder within the spray drier mechanisms prevented accurate 386 calculation of individual sample dry matter contents. However, the approximate

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Acknowledgements

Analytical method & performance characteristics 295 Summary method validation data for the analysis of nitrofuran metabolites in egg are 296 shown in Table 2. Decision limits (CCα) and detection capabilities (CCß) were 297 <0.6 µg kg -1 for AOZ, AMOZ, AHD and SEM. Within and between-run CVs were