Anja Zülch

Otto Götz Piringer

O Piringer

Measurement and modelling of migration from paper and board into foodstuffs and dry food simulants

Keywords: Chromatographic analysis, Chromatography -GC/MS, Chromatography -HPLC, Extraction Additives/Contaminants: Food contact materials, Packaging paper and board, Packagingmigration modelling, Migration -diffusion Food Types: Biscuits, Confectionary food packaging, migration, modelling, diffusion coefficient, partition coefficient, paper, board

Measurement and modelling of migration from paper

and board into foodstuffs and dry food simulants

Introduction

During the last two decades many studies on the migration from paper and board into foodstuffs and food simulants have been published. Some of these publications are cited as examples in the following. Castle et al. (1997aCastle et al. ( , 1997b) performed a comprehensive compositional analysis including migration tests with several paper and board samples. The results of migration investigations with phthalates and alkylbenzenes from paper and board packaging in contact with foodstuffs and the simulant Tenax ® (modified polyphenylene oxide) were reported by [START_REF] Aurela | Phthalates in paper and board packaging and their migration into Tenax ® and sugar[END_REF][START_REF] Aurela | Migration of alkylbenzenes from packaging into food and Tenax[END_REF]. Boccacci [START_REF] Mariani | Potential migration of diisopropyl naphthalenes from recycled paperboard packaging into dry foods[END_REF] investigated the potential migration of diisopropyl naphthalenes from recycled paperboard packaging into dry foods and Summerfield and Cooper (2001) developed methods for rapid testing of mass transfer from paper and board packaging. Several studies demonstrate that Tenax ® is a suitable food simulant for the migration from paper and board (Summerfield and Cooper 2001[START_REF] Aurela | Phthalates in paper and board packaging and their migration into Tenax ® and sugar[END_REF], 2001[START_REF] Castle | Migration from recycled paper and board to dry foods. Research into the factors involved, leading to practical avoidance and amelioration measures[END_REF], Triantafyllou et al. 2007).

It was observed by Triantafyllou et al. (2007) that the migration from paper and board depends on the structure of the packaging samples, the chemical nature of the migrants and the time and temperature conditions of the migration tests.

Whereas in the last years diverse other migration investigations with paper and board in contact with foods or food simulants were described [START_REF] Anderson | Benzophenone in cartonboard packaging materials and the factors that influence its migration into food[END_REF][START_REF] Song | Effectiveness of polypropylene film as a barrier to migration from recycled paperboard packaging to fatty and high-moisture food[END_REF][START_REF] Jickells | Determination of the potential for transfer from secondary packaging to foods and development of guidelines to reduce transfer to levels of no concern[END_REF], Sturaro et al. 2006[START_REF] Nerin | Kinetic migration studies using Porapak as solidfood simulant to assess the safety of paper and board as food-packaging materials[END_REF][START_REF] Begley | Migration of flourochemical paper additives from food-contact paper into foods and food simulants[END_REF][START_REF] Pastorelli | Study of migration of benzophenone from printed paperboard packages to cakes through different plastic films[END_REF], up to now only few authors reported on diffusion coefficients derived from kinetic migration investigations or partition coefficients for paper and board [START_REF] Haack | Migrationsabschätzung für Kartonverpackungen[END_REF][START_REF] Aurela | Diffusion of volatile compounds in fibre networks: experiments and modelling by random walk simulation[END_REF], Triantafyllou et al. 2005).

The structure of paper can be described as a thin layer of mostly cellulosic plant fibres. With its polar hydroxyl groups natural cellulose is building hydrogen bonds between the individual fibres that account for the strength of the paper [START_REF] Anders | Zur chemischen Festigung von Papier in Zusammenhang mit der Papierentsäuerung[END_REF][START_REF] Holik | Paper and board today[END_REF].

Paper and board for food contact use is an inhomogeneous material and can be composed of virgin or recycled pulp (cellulose, hemicellulose and lignin), additives like fillers, sizes, starch, starch derivates, wet strength sizing agents etc. and pigment or polymer coatings [START_REF] Castle | Migration from recycled paper and board to dry foods. Research into the factors involved, leading to practical avoidance and amelioration measures[END_REF]).

Compared to plastic packaging materials, where the migrant is in general homogeneously distributed in the polymeric matrix, migrants in paper and board can be adsorbed on the cellulose fibres or other constituents employed for paper and board production. The mechanism of transport through the paper and board sample can be understood as a repeated process of desorption/evaporation and adsorption /condensation until thermodynamic equilibrium is reached in the system.

The overall aim of this study was to deepen the understanding of migration processes of migrants from paper and board into foodstuffs as well as dry food simulants and to explore migration modelling as a tool for compliance assessment of paper and board. The working strategy was to carry out migration investigations with different spiked and non-spiked paper and board food packaging materials in the temperature range between -18°C and 40°C. The target migrants were components and contaminants determined in the non-spiked paper and board samples or model substances used for spiking the samples with a low initial concentration of migrants. From the kinetic migration studies diffusion and partition coefficients were derived by using the software MIGRATEST © Exp. for modelling migration in multi-layer materials. On the basis of these results for the first time a model for the migration from paper and board into foods and simulants was developed by adapting the validated diffusion model for plastics.

Materials and methods

Materials

Paper and board

The paper and board samples used for the migration studies show a great variety of properties (Table 1). They were of food-grade quality, unprinted and supplied by members of the Confederation of European Paper Industries (CEPI) Food Contact group. An exception was the thermal fax paper which was purchased from the local market. According to their characteristics the samples were assigned to different foodstuffs as listed in Table 1.

For the migration investigations with sample 10 at a temperature of 22°C and 40°C blotting paper was used as receptor material like a solid food simulant as given in Table 1. This blotting paper with a paper grade of 1600 according to ISO 5269-1:1998 and a grammage of 250 g was purchased from Munktell Filter AB.

Insert Table 1.

Foodstuffs

For the migration studies with spiked paper and board samples the food (butter, ice cream and deep-frozen spinach) was purchased from the local market. Until the beginning of the migration investigations the food was removed from the original packaging, wrapped in aluminium foil or filled in glass containers and stored in the refrigerator or freezer.

For the migration studies with non-spiked paper and board samples one part of the food (rice, egg pasta, sponge finger biscuits and chocolate) was obtained from the food manufacture. The foods were sampled directly from the production line before packing and were placed in glass containers or wrapped in aluminium foil.

The other part (ice cream, wheat flour, tomatoes, deep-frozen puff pastry as well as deep-frozen straw berries and raspberries) was purchased in bulk packs from a local supermarket. To avoid contamination from the original packaging the food used for the migration experiments was preferably taken from the centre of the packaging and stored in glass containers until the beginning of the experimental studies.

Food simulant Tenax ® and chemicals

The model substances and solvents were of reagent grade or highest purity available purchased from Sigma Aldrich, Carl Roth, Fluka and VWR International Ltd. Tenax ® (60-80 mesh), a standard food simulant for dry fatty foods [START_REF] Piringer | Use of high temperature-resistant sorbents as simulants for testing[END_REF], was obtained from Grace Alltech Grom GmbH and cleaned up prior use by solvent extraction using acetone and ethanol.

Selection of model migrants for spiking

Three different board samples were spiked with model migrants and typical contaminants of paper and board as shown in Table 2. The target substances vary in their molecular weights (in the range of 178 -391 Dalton) as well as their chemical properties, especially in their polarity. Whereas diisobutyl phthalate (DiBP), di(2-ethylhexyl) phthalate (DEHP) and diisopropyl naphthalene (DIPN) represent the non-polar, hydrophobic compounds, benzophenone (BP) and triclosan (TRCL) are polar migrants.

Insert Table 2.

Selection of components and contaminants in the non-spiked samples

For the migration investigations with non-spiked paper and board samples the initial concentration of potential migrants in 15 different samples was determined by means of solvent extraction in conjunction with semi-quantitative gas chromatographic screening. Six adequate substances in 10 of the 15 samples were identified in higher concentrations and employed as target migrants as listed in Table 3.

Insert Table 3.

The assignment of the target migrants to the paper and board samples, the foods and the food simulants and the temperature conditions of the migration tests are given in Table 1.

Migration investigation

Spiking procedure for the paper and board samples For the migration tests with the spiked paper and board samples a special procedure for an interaction-free spiking was developed in order to avoid changes of the paper and board samples which might influence their migration behaviour. Blotting paper was spiked with a diethyl ether solution of the contaminants followed by the gentle evaporation of the solvent. The paper and board samples were sandwiched between two spiked blotting paper sheets and stored at a temperature of 60°C to induce migration of the contaminants from the blotting paper into the paper and board samples. After three days a constant concentration of the surrogates in the samples was obtained.

Test design

Depending on the chosen foodstuffs the migration investigations were performed at -18°C, 5°C, 22°C as given in the Table 1. With sample 10 additional tests with blotting paper as receptor material at the elevated temperature of 40°C were carried out. Tenax ® as food simulant for comparison with the migration into the foods was used for the migration investigations with the spiked sample 1, 2 and 3 as well as the nonspiked samples 9, 10 and 11. In order to derive the migration parameters time dependent migration investigations with a minimum of five time points were carried out at the temperature of 5°C, 22°C and 40°C. The chosen time steps are listed in Tables 456789101112. Migration tests at a temperature of -18°C with one or two time points were not used for modelling migration. The single-sided migration investigations were performed from the food contact side of the paper and board samples.

For the migration tests with sponge finger biscuits, chocolate and wheat flour, the following test design was applied. Sponge finger biscuits and chocolate were exposed to a piece of paper or board sample with a defined area. In order to avoid losses through evaporation the samples in contact with the food were wrapped in aluminium foil. An intimate contact between sample and food was produced by covering the food exposed to the sample with an adequate weight. For the migration test with paper sample 4 (Table 1) wheat flour was filled into the paper bag and stored at a temperature of 22°C.

The migration investigations with the other foodstuffs, Tenax ® and blotting paper as well as wheat flour in contact with paper and board sample 10 and 11 were carried out using Petri dishes or with glass migration cells and stainless steel migration cells developed by FABES Forschungs-GmbH and Gaßner.

Glass migration cell. The design and the application of the glass migration cell for migration tests are described by [START_REF] Franz | Migration of plastic constituents[END_REF]. The paper and board sample was fixed in the migration cell and exposed to the food or the simulant Tenax ® .

Stainless steel migration cell. For the migration tests with the stainless steel migration cell the paper and board sample was exposed to the receptor material blotting paper in a stack. This sandwich of two layers was fixed in the migration cell which was closed with a certain turning moment.

Petri dishes. The paper and board sample was placed in the Petri dish and exposed to the food or the simulant Tenax ® . In order to avoid losses through evaporation the Petri dishes were sealed with Parafilm ® M and wrapped in aluminium foil during the migration test.

After exposure the foodstuffs, Tenax ® and blotting paper were worked up as described below.

Analytical methods

Apparatus

The contaminant quantification from the migration investigations with the spiked paper and board samples was performed with a Thermo Electron Spectra System high performance liquid chromatograph equipped with a UV6000 series PDA diode array detector (HPLC-UV). The compounds were separated on a Phenomenex type Sphere Clone column (5 µm particle size ODS (2), 250 mm L × 4.6 mm ID) coupled with Security Guard Cartridges C18 (ODS, Octadecyl, 4 mm L × 3.0 mm ID). The injections of 20 µl were performed by a Thermo Electron AS 3000 series auto sampler. The columns were thermostated at 40°C, the total flow was 1 ml min -1 and the wave length was 230 nm. Water and methanol were used as eluents.

The contaminant quantification from migration investigations with the non-spiked paper and board samples was performed with a Shimadzu gas chromatograph equipped with a QP5000 or QP2010 series quadrupole mass spectrometer (GC-MS). The compounds were separated on a Phenomenex type ZB-50 capillary column (30 m L x 0.25 mm ID; 0.15 µm film thickness). Helium was used as carrier gas and the injections of 1 µl were performed by a Shimadzu AOC-20 i series auto sampler in splitless mode.

Determination of the initial contaminant concentration in the paper and board samples 1.0 g of each paper and board sample was cut into small pieces, placed in a septum glass vial and extracted with ethanol 95% for three days at a temperature of 60°C. To guarantee an optimal extraction the cut sample had to be fully immersed in the solvent. The extraction procedure was repeated twice. The extracts were combined and concentrated by rotary evaporation. The solutions were filtered with a 0.2 µm pore-size PTFE syringe filter and analysed by HPLC-UV (spiked paper and board samples) or GC-MS in the single-ion-monitoring (SIM) mode (non-spiked paper and board samples). The contaminants were quantified by external calibration.

Determination of the contaminant concentration in the foodstuffs

After exposure the foodstuffs were homogenized and an aliquot of 10.0 g was taken for analysis. The level of the contaminants was determined by solvent extraction according to well defined methods as described for instance in the final report of the EU Project "Foodmigrosure" [START_REF] Paseiro | Compilation of analytical methods for model migrants in foodstuffs: collection of method descriptions. Project deliverable D2 of the EU-project[END_REF]Simoneau 2008). Depending on the matrix and the fat content the foods were extracted with acetonitrile (ice cream, spinach, chocolate), diethyl ether (rice, sponge finger biscuits), acetone (egg pasta), acetone/hexane (wheat flour), acetonitrile/hexane (butter, puff pastry) and ethanol 95%/diethyl ether (rasp berries, straw berries, tomatoes). Anhydrous sodium sulphate was used to remove water from the extraction solutions. Residues of fat were separated by solid phase extraction (SPE). After evaporation of the solvent the fatty residue was transferred into a SPE-column. The fat was adsorbed on the C18-matrial and the contaminants were eluted with acetonitrile. The extraction solutions were concentrated by rotary evaporation and filtered using a 0.2 µm pore-size PTFE syringe filter if necessary. The food extracts obtained from migration experiments with spiked paper and board samples were analysed by HPLC-UV. The contaminants were quantified by external calibration. The food extracts obtained from migration experiments with non-spiked paper and board samples were analysed by GC-MS. The contaminants were quantified by external calibration in the SIM mode.

Determination of the contaminant concentration in Tenax ®

After exposure Tenax ® was extracted using diethyl ether. The extraction solution was concentrated by gentle evaporation under a nitrogen stream and analysed by GC-MS in the SIM mode. For analysis of the contaminants by means of HPLC-UV the diethyl ether was evaporated and the extract was solved in ethanol 95%. The contaminants were quantified by external calibration.

Determination of the contaminant concentration in the blotting paper

After exposure the blotting paper was cut into small pieces, placed in a septum glass vial and extracted with dichloromethane for two days at a temperature of 40°C. The solutions were filtered with a 0.2 µm pore-size PTFE syringe filter and analysed by GC-MS. The contaminants were quantified by external calibration in the SIM mode.

Experimental results

Migration experiments with spiked paper and board samples

For the spiked samples the best kinetic results were obtained with sample 1 showing the smallest scatter of the experimental data points. Therefore, sample 1 was selected for the evaluation of the migration parameters (Table 1). Due to the great volume of data the results of the migration investigations with sample 2 and 3 are not presented. The experimental results with the spiked sample 1 are shown in the following Tables 456789.

Insert Table 456789.

Migration experiments with non-spiked paper and board samples

The migration of the target migrants from the non-spiked sample 1, 2, 5 and 8 into the foods was below the detection limit as well as the migration of the paper component abietic acid (Aa) from sample 6 into sponge finger biscuits (Table 1). The results of the migration experiments with the non-spiked samples 4, 6 and 7 are given in Table 10.

Insert Table 10.

The results of the migration of DiBP from the non-spiked samples 10 into dry and frozen foods, Tenax ® and blotting paper at -18°C, 22°C and 40°C are given in Table 11 a) andb).

Insert Table 11 a)/b).
The results of migration experiments with DiBP from the non-spiked samples 9 and with bisphenol A (BPA) and benzyl 2-naphthyl ether (NAP-2) from the non-spiked sample 11 are given in Table 12.

Insert Table 12.

Modelling migration

The mass transfer (migration) from plastic materials into a contact medium is a predictable physical process. Mass transport is understood as the molecular diffusion in, out and through plastic materials. For the mathematical description many analytical and numerical solutions of the diffusion equation ( 1) are known (Crank (1)

Modelling of potential migration based on these solutions is already used as an additional tool for quality assurance of food packaging materials (Directive 2002/72/EC).

Whereas the migrant in a plastic material is distributed in the polymer matrix like in a liquid phase, the migration from paper and board is a more complex phenomenon, due to the adsorption/desorption process of the migrant on the fibre surface. Therefore, the specific behaviour of these materials must be taken into account in application of modelling. Nevertheless, the transfer process can also be treated as a diffusion phenomenon.

There are two different choices for a description of diffusion. The more fundamental is based on Fick's second law of diffusion (Equation 1). The second approach involves a mass transfer coefficient, that means a type of reversible rate constant [START_REF] Cussler | Diffusion. Mass transfer in fluid systems[END_REF]. If, as an example, two reservoirs containing two different gases are connected with a long thin capillary, the change of concentration occurs direct proportional with time. This behaviour can be described easily by using a mass transfer coefficient. Nevertheless, the fundamental process is the molecular diffusion with a long period in a stationary state (Fick's first law), where the concentration changes proportional with time. With larger connecting tubes and smaller reservoirs the concentration in the second reservoir increases with the square route of time, as follows from the diffusion in the non-steady state regime.

Paper and board (B) can be compared formally with the first case, where two layers B1 and B2 with a thickness of d B1 and d B2 are responsible for the whole process of mass transfer. A large fraction of board B1 with the thickness d B1 contains the main part of the migrant which permeates through the thin layer B2 with the thickness d B2 at the interface to the food (2-layer approach). The rate of transfer is determined by the desorption rate of the migrant from the surface. It is a function of the interaction strength between the board and migrant and can be characterized in principle with a mass transfer coefficient, k. As mentioned above, the mass transfer from the board sample as a whole into foodstuffs can be also described with the law of diffusion. But as will be shown later, due to the complexity of the occurring processes, transitions between direct proportionality with time (conform with Fick's first law) and proportionality with square root of time (conform with Fick's second law) are observed with different migrants and at different conditions. In many cases the diffusion in B2 occurs much slower in comparison to B1. In such a situation the rate of mass transfer from paper and board into the food or food simulants is governed by the diffusion through the surface layer B2 and is proportional to the ratio D B2 /d B2 = k (cm s -1 ), between the diffusion coefficient D B2 in this layer and the thickness d B2 For migrants with a small molecular weight and polarity and for paper and board samples with high humidity a strong increase of the desorption rate occurs. The differentiation between the diffusion in B1 and B2 is then no longer necessary and

D B1 = D B2 = D B (1-layer approach).
With the above assumptions it is possible to use the modelling procedure as described for plastic materials also for modelling the mass transfer through paper and board into foodstuffs. By using the algorithms with numerical solutions for the diffusion equation the experimental results described in this publication were modelled with the FABES software MIGRATEST © Exp. for multilayer materials [START_REF] Mercea | User-friendly software for migration estimation[END_REF].

In order to characterize the migration behaviour in paper and board in a similar way as it was done for plastic materials, an equation for the estimation of the diffusion coefficient in paper and board D B similar to that developed for polymers has been used. The semi-empirical formula for the estimation of the diffusion coefficient in plastics D P [START_REF] Piringer | Evaluation of plastics for food packaging[END_REF][START_REF] Begley | Evaluation of migration models that might be used in support of regulations for food-contact plastics[END_REF]) contains the relative molecular weight M r of the migrant, the absolute temperature T and a polymer specific parameter A P :

D P = 10 4 exp(A P -0.1351 M r 2/3 + 0.003 M r -10454/T) (cm 2 s -1 ) (2)
with A P = A P '-τ /T.

A P ' is an athermal dimensionless parameter and τ a constant which together with the constant 10454 and the gas constant R give the activation energy of diffusion,

E A = (τ +10454) R.
A statistical evaluation of many experimentally obtained diffusion coefficients for different polymers used as food contact materials has shown that low density polyethylene (LDPE) gives the highest diffusion results and consequently the highest A P -values (Begley et al. 2005). With the mean value of A P = 10 and τ = 0, corresponding to a mean activation energy of E A = 87 kJ mol -1 in LDPE, Equation ( 2) is used as a reference for characterizing the diffusion in paper and board, with the following adaptation:

D B = 10 4 exp(10 + A B -0.1351 M r 2/3 + 0.003 M r -10454/T) (cm 2 s -1 ) (3)
where A B is the board specific parameter, either as A B1 for layer B1 or A B2 for layer B2. Whereas the A P -values in polymer matrices are more or less independent from the molecular weight and chemical nature of the migrant, the corresponding A B -value for paper and board results from much more complex interactions between migrant and fibre and depends also on the polarity of the migrant. This is the reason why A B is distinguished from the A P for plastics and referred to the reference value of A P = 10 for LDPE.

For small A B2 -values, where the diffusion in B2 determines the whole migration rate in the board sample, more or less independently from A B1 , the value A B1 = 6 for B1 is used. This provides the maximum diffusion rates obtained in paper and board with the 1-layer approach.

If paper or board is selected as food simulant (blotting paper), also A F = 6 is used because the adsorption rate in the paper is much higher in comparison with the desorption rate from the board sample.

In Table 13 the results obtained with the multilayer modelling are summarized. With the A B2 -values and Equation (3) the diffusion coefficients, D B2 (cm 2 s -1 ) in the surface layer B2 are obtained and shown as their logarithmic values. In the last columns of Table 13 the partition coefficients, K B2/F , of the migrants between the board layer B2 and food or food simulant (F) as well as the A F -values of the foods or simulant are listed.

Insert Table 13.

Recently a full theoretical derivation of the diffusion coefficients in polymer matrices has been published [START_REF] Piringer | A uniform model for prediction of diffusion coefficients with emphasis on plastic materials[END_REF]). In the corresponding equation only the relative molecular weight, M r,p , of the polymer and the melting temperature, T m,p , or glass point, T g of the polymer are used in addition to the molecular weight, M r , of the migrant, without further empirical parameter values as in Equation ( 3):

( ) ( ) RT R T ww j w w D D p m e j e p e i u i P / 2 14 14 . 0 exp , 3 / 2 , 3 / 2 , , , - + ⋅ - = (cm 2 s -1 ) (4)
with i = (M r -2)/14, w i,e = (1+2 π /i) i/e , j = (i 1/3 ), w j,e = (1+2π /j) j/e , p = (M r,p /14) 1/3 , w p,e = (1+2π /p) p/e , w = e 2 π /e and the unit D u = 1 m 2 s -1 .

If LDPE with a mean relative molecular weight, M r,p = 25000 Dalton and T m,p = 363 K (90°C) is used as a reference polymer matrix for all polymers, than the following equation can be used for prediction of D B -values in paper and board, which is equivalent to Equation (3):

( ) ( ) T w j A w D e j B e i i P / 3662 2 14 9 . 0 exp 10 3 / 2 , 3 / 2 , 4 , - + - + = (cm 2 s -1 ) (5)
with i = (M r -2)/14, w i,e = (1+2 π /i) i/e , j = (i 1/3 ), w j,e = (1+2 π /j) j

In Figure 1 the graph of Equation (3) (trace 1) for LDPE (A B = 0) is compared with the graph for Equation (5) (trace 5), also for LDPE (A B = 0) until relative migrant masses M r = 400 Dalton at 25°C. By using Equation (5) the only empirical parameter is A B and for A B = 0 the diffusion in a defined type of LDPE as reference matrix results.

Insert Figure 1.

Discussion

From the variety of the results obtained with different migrants, foodstuffs, simulants and different temperatures the following trends can be extracted.

With A B2 < -2 the modelling of the migration kinetic can only be done correctly with the 2-layer approach. The diffusion coefficients in the large reservoir layer B1 are in these cases significantly higher than in the thin layer B2 and are modelled with a defined, constant value A B1 = 6. With A B2 ≥ -2 the modelling is done with the 1-layer approach, where A B2 = A B1 = A B ≤ 6.

The partition coefficients of the migrants between the two paper and board layers are in general considered K B1/B2 = 1, with the exceptions mentioned later in the conclusions.

For estimation of the diffusion in the foods or the food simulants (F), Equation (3) or Equation ( 5) can also be used. Instead of the A B -value an A F -value in the range of 3 -7.5 is applied (except for egg pasta with an A F -value of -2), which means that the diffusion in the foods or simulants is significant higher than in the B2 layer (Table 13).

The upper curve in Figure 1 (trace 2) is obtained with the 1-layer approach with A B = 6. This represents the maximum diffusion values obtained for high humidity in the fibres, for volatile, non-polar migrants and at high temperatures. The typical migration behaviour in dry paper and board samples is obtained with the 2-layer approach between the lowest curve with A B2 = -11 (trace 3) and the curve with A B2 = -2 (trace 4), below the reference curve obtained with Equation (3) (trace 1) or with Equation (5) (trace 5), both with A B = 0.

In the Figure 2 (a) and (b) for the migration of DiBP from the non-spiked sample 10 into Tenax ® at 22°C the differences between the modelled migration curves obtained with the 2-layer approach and the 1-layer approach are compared.

The significant difference appears at the beginning of the migration where the concentration increases approximately linear with time. If one considers this initial stage of the kinetic it is not possible to model the whole process with a single layer, although a partition coefficient K B2/F << 1 is used, which means a high solubility of the migrant in the food or simulant (Figure 2 A direct consequence of the rate determining desorption process at the paper surface is the dependency of the activation energy, E A , of diffusion in the B2-layer from the humidity in the paper and board and from the polarity of the migrant. Similar as for plastics, the A B for paper and board depends in general on the temperature:

A B = A B '-τ /T.
From the A B2 -values obtained at two different temperatures (Table 13) the athermal value A B2 ' and τ can be calculated and the corresponding

E A = (τ +10454) R results.
For DiBP in dry paper and board between 5°C and 40°C a value E A = 145 kJ mol -1 is obtained in the B2-layer. At 5°C and 22°C similar low diffusion coefficients in dry paper have been determined with the migrant phenanthrene (Ph). That means that dry paper and board is a relative good barrier for the diffusion of polar migrants at low temperatures. With increasing humidity, as occurred during the contact with butter, a much higher diffusion rate and a lower activation energy of about 87 kJ mol -1 has been obtained between 5°C and 22°C for DiBP, DIPN, DEHP, TRCL and a much lower value of 31 kJ mol -1 for BP.

In order to compare the migration behaviour between paper and board and the reference LDPE the following case study has been modelled: a 0.5 mm thick dry board sample with the density 0.8 g cm -3 has been compared with a 0.5 mm thick LDPE film. The initial concentration of a migrant with M r = 300 Dalton in both samples is assumed 200 mg kg -1 . A value A B2 = -5 has been assumed and the same partition coefficient, K B2/F = K LDPE/F = 1 between the packaging sample and the food simulant Tenax ® with A F = 6. With the 2-layer approach for board and by using the software MIGRATEST © Exp. for multilayers the following migration kinetic curves resulted at 40°C during 10 days (Figure 6). A significant difference between the two packaging materials can be observed. From the specific desorption behaviour of the board a more linear increase of the migration rate at the beginning occurs.

Insert Figure 6.

Conclusions

A general conclusion obtained from the systems studied is the principal possibility of modelling migration kinetics from paper and board. The matrix of paper and board behaves different than plastic materials due to the adsorption/desorption processes occurring at the fibres surface.

A characteristic behaviour of the migration in paper and board systems is the dependency of the A B2 -values from the polarity of the migrant. This follows from the property of the fibres with their polar hydroxyl groups on the surface. Consequently low values of A B2 for polar substances result. In addition in some cases a partition coefficient K B1/B2 < 1 was derived as for example for the migrants BP and Aa.

Like in plastic materials the diffusion rate decreases with increasing molecular weight of the migrants.

With increasing humidity a rapid displacement of the polar migrants from the active sites on the fibre surface occurs, resulting in a high diffusion rate and higher A B2values (Table 13). Hence the A B2 -value is also influenced by the water activity a w of the exposed foodstuff. The a w -value is defined as the vapour pressure of water above the food divided by that of pure water at the same temperature. With an increasing a w -value of the foods the migration rate and the A B2 -value in the paper and board are increasing as well. This could be observed for the migration of DiBP from sample 1 and 10 into foods with higher a w -values like butter, wheat flour and chocolate at a temperature of 22°C (Table 13).

As shown in Figure 1 and Table 13 the typical values for A B2 in dry paper and board systems are < 0 and consequently lower diffusion coefficients result than for the reference polymer LDPE. Dry paper and board are relative good barriers for polar migrants, especially at low temperature. As shown for example in Table 11 (a) a decrease of temperature from 22°C to -18°C leads to a drastic decrease of migration into the foods or simulants.

As given in Table 13 the migration rate of DiBP from the spiked sample 1 into Tenax ® at a temperature of 22°C is higher (A B2 = -1.3) than the migration from the non-spiked sample 10 (A B2 = -3.9) under the same conditions of use. Both samples have a similar thickness and grammage (Table 3). Possibly there is a stronger retention of substances which remained in the paper and board matrix after the recycling process than of substances artificially spiked into the paper and board matrix.

For polar migrants higher values for the partition coefficients K B2/F are observed in comparison to the non-polar compounds, as far as the paper and board matrix is dry.

In summary it can be concluded that the migration rates from paper and board into dry foods and simulants at a temperature below room temperature and under dry conditions are small compared to the migration rates from plastic materials. For a correct modelling the paper and board must be regarded as a 2-layer system. Above room temperature and/or higher humidity diffusion increases. Therefore paper and board can be treated as a 1-layer system and the mass transfer can modelled in the same way as monolayer plastic packaging materials. 5) and A B2 = 0. Figure 2. Comparison of (a) 2-layer approach (A B2 = -3.9; A B1 = 6) with (b) 1-layer approach (A B2 = A B1 = A B = -2.7) for paper and board: migration of DiBP from non-spiked sample 10 into Tenax ® 60 days at 22°C (experimental data points and modelled data curve). Figure 3. Comparison of (a) 2-layer approach (A B2 = -3.4; A B1 = 6) with (b) 1-layer approach (A B2 = A B1 = A B = -3.3) for paper and board: migration of DEGDB from non-spiked sample 7 into chocolate 60 days at 22°C (experimental data points and modelled data curve). Figure 4. Comparison of (a) 2-layer approach (A B2 = -3.3; A B1 = 6) with (b) 1-layer approach (A B2 = A B1 = A B = -2.4) for paper and board: migration of DiBP from non-spiked sample 10 into blotting paper 13 days at 22°C (experimental data points and modelled data curve). Figure 5. Comparison of (a) 2-layer approach (A B2 = -3.7; A B1 = 6) with (b) 1-layer approach (A B2 = A B1 = A B = -2.0) for paper and board: migration of DiBP from non-spiked sample 10 into blotting paper 10 days at 40°C (experimental data points and modelled data curve). Figure 6. Migration kinetics: (a) from the reference LDPE and (b) from board into a food simulant at 40°C during 10 days. Same values for initial concentration, molecular weight, partition coefficients, film thickness and surface/volume ratio were used. Whereas LDPE is treated as single layer, the board is treated with the 2-layer approach, with A B1 = 16 and A B2 = -5. This situation is typical for dry board samples and polar migrants. 1 Names and properties of the target migrants are listed in Table 2 and Table 3. 2 Migration of target migrants into foods was below the detection limit.

F

Figure captions

Because of interferences with the chromatographic fingerprint of the foodstuff a higher detection limit for DiBP between 30 µg/dm² and 90 µg/dm² was determined. The detection limit for Aa was between 1 µg/dm² and 7 µg/dm². 3 Migration experiments at a temperature of -18°C with only one or two time points were not modelled. n.s. not specified Whereas LDPE is treated as single layer, the board is treated with the 2-layer approach, with A B1 = 6 and A B2 = -5. This situation is typical for dry board samples and polar migrants.

  D is the diffusion coefficient and the concentration of the migrant c is a function of time t and position along the x-axis.

  (b)). The same behaviour was observed for the migration of diethylene glycol dibenzoate (DEGDB) from the non-spiked sample 7 into chocolate at 22°C shown in Figure 3 (a) and (b) and for the migration of DiBP from the non-spiked sample 10 into blotting paper at 22°C shown in Figure 4 (a) and (b). In contrast the migration of DiBP from the non-spiked sample 10 into blotting paper at the elevated temperature of 40°C with A B2 = -2 is best modelled with only one paper and board layer as shown in Figure 5 (b). Insert Figure 2 -5.These different kinetic forms are the consequence of the complex phenomenon mentioned above, where both, the linear dependence resulting with the transport constant D B2 /d B2 = k and the square root dependence on a D B for the whole paper and board, are occurring with different degrees.

Figure 1 .

 1 Figure1. Diffusion coefficients, D B2 (cm 2 s -1 ), as function of the relative molecular weight M r , calculated with Equation (3): trace 1 with A B2 = 0; trace 2 with A B2 =6 (upper dots); trace 3 with A B2 = -11 (lower dots); trace 4 with A B2 = -2 and trace 5 with Equation (5) and A B2 = 0. Figure2. Comparison of (a) 2-layer approach (A B2 = -3.9; A B1 = 6) with (b) 1-layer approach (A B2 = A B1 = A B = -2.7) for paper and board: migration of DiBP from non-spiked sample 10 into Tenax ® 60 days at 22°C (experimental data points and modelled data curve). Figure3. Comparison of (a) 2-layer approach (A B2 = -3.4; A B1 = 6) with (b) 1-layer approach (A B2 = A B1 = A B = -3.3) for paper and board: migration of DEGDB from non-spiked sample 7 into chocolate 60 days at 22°C (experimental data points and modelled data curve). Figure4. Comparison of (a) 2-layer approach (A B2 = -3.3; A B1 = 6) with (b) 1-layer approach (A B2 = A B1 = A B = -2.4) for paper and board: migration of DiBP from non-spiked sample 10 into blotting paper 13 days at 22°C (experimental data points and modelled data curve). Figure5. Comparison of (a) 2-layer approach (A B2 = -3.7; A B1 = 6) with (b) 1-layer approach (A B2 = A B1 = A B = -2.0) for paper and board: migration of DiBP from non-spiked sample 10 into blotting paper 10 days at 40°C (experimental data points and modelled data curve). Figure6. Migration kinetics: (a) from the reference LDPE and (b) from board into a food simulant at 40°C during 10 days. Same values for initial concentration, molecular weight, partition coefficients, film thickness and surface/volume ratio were used. Whereas LDPE is treated as single layer, the board is treated with the 2-layer approach, with A B1 = 16 and A B2 = -5. This situation is typical for dry board samples and polar migrants.

Figure 1 .Figure 2 .

 12 Figure 1. Diffusion coefficients, D B2 (cm 2 s -1 ), as function of the relative molecular weight M r , calculated with Equation (3): trace 1 with A B2 = 0; trace 2 with A B2 = 6 (upper dots); trace 3 with A B2 = -11 (lower dots); trace 4 with A B2 = -2 and trace 5 with Equation (5) and A B2 = 0.

Figure 3 .

 3 Figure 3. Comparison of (a) 2-layer approach (A B2 = -3.4; A B1 = 6) with (b) 1-layer approach (A B2 = A B1 = A B = -3.3) for paper and board: migration of DEGDB from non-spiked sample 7 into chocolate 60 days at 22°C (experimental data points and modelled data curve).

Figure 4 .

 4 Figure 4. Comparison of (a) 2-layer approach (A B2 = -3.3; A B1 = 6) with (b) 1-layer approach (A B2 = A B1 = A B = -2.4) for paper and board: migration of DiBP from non-spiked sample 10 into blotting paper 13 days at 22°C (experimental data points and modelled data curve).

Figure 5 .

 5 Figure 5. Comparison of (a) 2-layer approach (A B2 = -3.7; A B1 = 6) with (b) 1-layer approach (A B2 = A B1 = A B = -2.0) for paper and board: migration of DiBP from non-spiked sample 10 into blotting paper 10 days at 40°C (experimental data points and modelled data curve).

Figure 6 .

 6 Figure 6. Migration kinetics: (a) from the reference LDPE and (b) from board into afood simulant at 40°C during 10 days. Same values for initial concentration, molecular weight, partition coefficients, film thickness and surface/volume ratio were used. Whereas LDPE is treated as single layer, the board is treated with the 2-layer approach, with A B1 = 6 and A B2 = -5. This situation is typical for dry board samples and polar migrants.

  B2 remains constant, as can be observed experimentally. In order to obtain diffusion coefficients D B2 which are reproducible and comparable for different systems, a hypothetical thickness, d B2 = 10 µm has been defined which represents a value within the range, where the ratio D B2 /d B2 remains constant.

	the ratio D B2 /d
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y

. The value of d B2 is an unknown parameter and is also a function of D B2 . The consequence of this is that within a certain range of d B2 -values

Table captions Table 1 .

 captions1 Properties of paper and board samples, exposed foods and simulants, temperature of migration tests and modelling of experimental data. Table2. Model migrants and contaminants used for spiking of paper and board samples. Table 3. Components and contaminants in the non-spiked paper and board samples. Table 4. Results of migration experiments with benzophenone from the spiked sample 1. Table 5. Results of migration experiments with diisobutyl phthalate from the spiked sample 1. Table 6. Results of migration experiments with diisopropyl naphthalene from the spiked sample 1. Table 7. Results of migration experiments with di(2-ethylhexyl) phthalate from the spiked sample 1. Table 8. Results of migration experiments with phenanthrene from the spiked sample 1. Table 9. Results of migration experiments with triclosan from the spiked sample 1. Table 10. Results of migration experiments with abietic acid, diisobutyl phthalate, diethylene glycol dibenzoate and dipropylene glycol dibenzoate from the non-spiked samples 4, 6 and 7.Table 11 (a). Results of migration experiments with diisobutyl phthalate from the non-spiked sample 10 in contact with foodstuffs. Table 11 (b). Results of migration experiments with diisobutyl phthalate from the non-spiked sample 10 in contact with Tenax ® and blotting paper. Table 12. Results of migration experiments with diisobutyl phthalate, bisphenol A and benzyl 2-naphthyl ether from the non-spiked samples 9 and 11. Table 13. Results of modelling of the migration investigations.

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

Table 1 .

 1 Properties of paper and board samples, exposed foods and simulants, temperature of migration tests and modelling of experimental data.

	Properties of paper and board samples
	ID Description	Grammage	Thickness	Percentage of	Spiking	Target	Exposed	Temp.	Modelling of
		(g/m²)	(µm)	virgin/recycled		migrants 1	foods/simulant	(°C)	data
				material
	1 Food board, one-	300	460	100% virgin	No	Aa,	Rice	22	No 2
	side pigment					DiBP
	coated				Yes	BP, TRCL,	Butter	-18/5/22	-18°C: No 3
						DiBP,	Tenax ®	-18/5/22	5°C: Yes
						DIPN,			22°C: Yes
						DEHP, Ph
	2 Food board, one-	300	480	100% virgin	No	Aa	Ice cream	-18	No 2
	side pigment coated				Yes	DiBP, BP, TRCL,	Spinach Ice cream	-18/5/22 -18/5/22	No
						DIPN,	Tenax ®	-18/5/22
						DEHP, Ph
	3 Virgin kraft liner 200	220	100% virgin	Yes	BP, TRCL,	Spinach	-18/5/22	No
						DiBP,	Tenax ®	-18/5/22
						DIPN,
						DEHP, Ph
	4 MG paper bag	40	60	100% recycled No	Aa	Wheat flour	22	Yes
	5 Food board, one-	320	395	100% recycled No	Aa,	Egg pasta	22	No 2
	side pigment					DiBP
	coated
	6 Food board, one-	300	400	95% recycled	No	Aa,	Sponge finger	22	Aa: No 2
	side pigment					DiBP	biscuits		DiBP: Yes
	coated
	7 Chocolate	200	3300	approx. 20%	No	DPGDB,	Chocolate	22	Yes
	padding board			recycled		DEGDB
	8 White top	950	6461	100% recycled No	Aa,	Straw berries	5	No 2
	corrugated board					DiBP	Tomatoes	5
	9 White top	422	2882	n.s.	No	DiBP	Raspberries	-18	No 3
	corrugated board						Tenax ®	-18
	10 Board, one-side	350	448	n.s.	No	DiBP	Wheat flour	-18/22	-18°C: No 3
	pigment coated						Chocolate	-18/22	22°C: Yes
							Egg pasta	22	40°C: Yes
							Sponge finger	22
							biscuits
							Puff pastry	-18
							Tenax ®	-18/22
							Blotting paper	22/40
	11 Thermal fax	52	64	n.s.	No	BPA,	Wheat flour	-18	No 3
	paper					NAP-2	Chocolate	-18
							Tenax ®	-18

Table 2 .

 2 Model migrants and contaminants used for spiking of paper and board samples.

	Substance	CAS Registry	Abbreviation	Category	Molecular weight
		Number			(Dalton)
	Benzophenone	119-61-9	BP	impurity from printing inks in recycled fibres	182
	Diisobutyl phthalate	84-69-5	DiBP	impurity from adhesives in recycled fibres	278
	Diisopropyl naphthalene	24157-81-1	DIPN	impurity from recycled fibres	212
	Di(2-ethylhexyl) phthalate	117-81-7	DEHP	impurity from recycled fibres (plasticizer)	390
				model migrant for
	Phenanthrene	85-01-8	Ph	polycyclic aromatic	178
				hydrocarbons
	Triclosan;
	5-Chloro-2-(2.4-	3380-34-5	TRCL	model migrant for biocides	290
	dichlorophenoxy)phenol

Table 3 .

 3 Components and contaminants in the non-spiked paper and board samples.

	Substance	CAS Registry	Abbreviation	Category	Molecular weight
		Number			(Dalton)
	Abietic acid	514-10-3	Aa	rosin component	303
	Benzyl 2-naphthyl ether	613-62-7	NAP-2	component in fax paper	234
	Bisphenol A	80-05-7	BPA	component in fax paper	228
	Diisobutyl phthalate	84-69-5	DiBP	impurity from adhesives in recycled fibres	278
	Diethylene glycol dibenzoate	120-55-8	DEGDB	impurity from adhesives	314
	Dipropylene glycol dibenzoate	27138-31-4	DPGDB	impurity from adhesives	342

Table 4 .

 4 Results of migration experiments with benzophenone from the spiked sample 1.

	Food/Simulant Temperature	Time	Migration	Initial	Quotient
			point		concentration	Migration/Initial
					after spiking	concentration
		(°C)	(d)	(µg/dm²)	(µg/dm²)	(%)
	Butter	-18°C	30	x	552	X
			30	x		X
			60	15		2.7
		5°C	0.33	x	552	X
			1	15		2.7
			2	28		5.1
			4	84		15.2
			10	212		38.4
			10	195		35.3
		22°C	0.33	x	552	X
			1	68		12.3
			2	156		28.3
			4	275		49.8
			10	473		85.7
			10	528		95.7
	Tenax ®	-18°C	30	n.d.	552	<0.4
			30	n.d.		<0.4
			60	n.d.		<0.4
		5°C	0.33	n.d.	552	<0.4
			1	n.d.		<0.4
			2	n.d.		<0.4
			4	n.d.		<0.4
			10	n.d.		<0.4
			10	n.d.		<0.4
		22°C	0.33	n.d.	552	<0.4
			1	n.d.		<0.4
			2	n.d.		<0.4
			4	2.2		0.4
			10	4.7		0.9
			10	4.9		0.9
	x contaminant could not be detected because of interferences with the chromatographic fingerprint of
	the foodstuff (food blank)
	n.d. not detectable (detection limit: 2,2 µg/dm²)

Table 5 .

 5 Results of migration experiments with diisobutyl phthalate from the spiked sample 1.

	Food/Simulant Temperature	Time	Migration	Initial	Quotient
			point		concentration	Migration/Initial
					after spiking	concentration
		(°C)	(d)	(µg/dm²)	(µg/dm²)	(%)
	Butter	-18°C	30	83	422	19.7
			30	69		16.4
			60	111		26.3
		5°C	0.33	47	422	11.1
			1	69		16.3
			2	71		16.8
			4	69		16.4
			10	100		23.7
			10	115		27.3
		22°C	0.33	106	422	25.1
			1	87		20.6
			2	121		28.7
			4	178		42.2
			10	161		38.2
			10	187		44.3
	Tenax ®	-18°C	30	3.3	422	0.8
			30	2.5		0.6
		5°C	0.33	n.d.	422	<0.5
			1	5.3		1.3
			2	6.3		1.5
			4	16		3.8
			10	22		5.2
			10	23		5.5
		22°C	0.33	17	422	4.0
			1	27		6.4
			2	35		8.3
			4	50		11.8
			10	61		14.5
			10	52		12.3
	n.d. not detectable (detection limit: 2,2 µg/dm²)

Table 6 .

 6 Results of migration experiments with diisopropyl naphthalene from the spiked sample 1.

	Food/Simulant Temperature	Time	Migration	Initial	Quotient
			point		concentration	Migration/Initial
					after spiking	concentration
		(°C)	(d)	(µg/dm²)	(µg/dm²)	(%)
	Butter	-18°C	30	34	438	7.8
			30	34		7.8
			60	36		8.2
		5°C	0.33	43	438	9.8
			1	64		14.6
			2	94		21.5
			4	133		30.4
			10	228		52.1
			10	237		54.1
		22°C	0.33	103	438	23.5
			1	137		31.3
			2	206		47.0
			4	211		48.2
			10	296		67.6
			10	326		74.4
	Tenax ®	-18°C	30	16	438	3.7
			30	41		9.4
		5°C	0.33	15	438	3.4
			1	25		5.7
			2	28		6.4
			4	51		11.6
			10	69		15.8
			10	80		18.3
		22°C	0.33	42	438	9.6
			1	61		13.9
			2	94		21.5
			4	157		35.8
			10	196		44.7
			10	196		44.7

Table 7 .

 7 Results of migration experiments with di(2-ethylhexyl) phthalate from the spiked sample 1.

	Food/Simulant Temperature	Time	Migration	Initial	Quotient
			point		concentration	Migration/Initial
					after spiking	concentration
		(°C)	(d)	(µg/dm²)	(µg/dm²)	(%)
	Butter	-18°C	30	12	214	5.6
			30	9.3		4.3
			60	5.8		2.7
		5°C	0.33	14	214	6.5
			1	18		8.4
			2	23		10.7
			4	19		8.9
			10	20		9.3
			10	23		10.7
		22°C	0.33	33	214	15.4
			1	23		10.7
			2	29		13.6
			4	26		12.1
			10	21		9.8
			10	29		13.6
	Tenax ®	-18°C	30	n.d.	214	<1.0
			30	n.d.		<1.0
		5°C	0.33	n.d.	214	<1.0
			1	n.d.		<1.0
			2	n.d.		<1.0
			4	n.d.		<1.0
			10	n.d.		<1.0
			10	n.d.		<1.0
		22°C	0.33	n.d.	214	<1.0
			1	n.d.		<1.0
			2	3.3		1.5
			4	8.1		3.8
			10	15		7.0
			10	12		5.6
	n.d. not detectable (detection limit: 2,2 µg/dm²)

Table 8 .

 8 Results of migration experiments with phenanthrene from the spiked sample 1.

	Food/Simulant Temperature	Time	Migration	Initial	Quotient
			point		concentration	Migration/Initial
					after spiking	concentration
		(°C)	(d)	(µg/dm²)	(µg/dm²)	(%)
	Butter	-18°C	30	23	515	4.5
			30	37		7.2
			60	21		4.1
		5°C	0.33	14	515	2.7
			1	28		5.4
			2	39		7.6
			4	79		15.3
			10	190		36.9
			10	183		35.5
		22°C	0.33	73	515	14.2
			1	133		25.8
			2	186		36.1
			4	270		52.4
			10	363		70.5
			10	394		76.5
	Tenax ®	-18°C	30	3.0	515	0.6
			30	10.6		2.1
		5°C	0.33	5.3	515	1.0
			1	8.0		1.6
			2	8.6		1.7
			4	13		2.5
			10	17		3.3
			10	17		3.3
		22°C	0.33	10	515	1.9
			1	13		2.5
			2	24		4.7
			4	36		7.0
			10	42		8.2
			10	42		8.2

Table 9 .

 9 Results of migration experiments with triclosan from the spiked sample 1.

	Food/Simulant Temperature	Time	Migration	Initial	Quotient
			point		concentration	Migration/Initial
					after spiking	concentration
		(°C)	(d)	(µg/dm²)	(µg/dm²)	(%)
	Butter	-18°C	30	38	571	6.7
			30	32		5.6
			60	29		5.1
		5°C	0.33	44	571	7.7
			1	56		9.8
			2	66		11.6
			4	57		10.0
			10	67		11.7
			10	70		12.3
		22°C	0.33	85	571	14.9
			1	92		16.1
			2	81		14.2
			4	84		14.7
			10	84		14.7
			10	95		16.6
	Tenax ®	-18°C	30	n.d.	571	<0.4
			30	n.d.		<0.4
		5°C	0.33	n.d.	571	<0.4
			1	n.d.		<0.4
			2	n.d.		<0.4
			4	n.d.		<0.4
			10	6.9		1.2
			10	5.3		0.9
		22°C	0.33	2.3	571	0.4
			1	7.3		1.3
			2	14		2.5
			4	29		5.1
			10	44		7.7
			10	33		5.8
	n.d. not detectable (detection limit: 2,2 µg/dm²)

Table 10 .

 10 Results of migration experiments with abietic acid, diisobutyl phthalate, diethylene glycol dibenzoate and dipropylene glycol dibenzoate from the non-spiked samples 4, 6 and 7. Table 11 (a). Results of migration experiments with diisobutyl phthalate from the non-spiked sample 10 in contact with foodstuffs.

	Food/Simulant Temperature	Time	Migration	Initial	Quotient
			point		concentration	Migration/Initial
					after spiking	concentration
		(°C)	(d)	(µg/dm²)	(µg/dm²)	(%)
	Sample 4 (Abietic acid)
	Wheat flour	22°C	1	2.9	437	0.7
			2	4.6		1.1
			4	5.8		1.3
			4	5.3		1.2
			15	14		3.2
			30	21		4.8
			30	22		5.0
			60	33		7.5
			60	27		6.2
	Sample 6 (Diisobutyl phthalate)
	Sponge finger	22°C	2	6.2	158	3.9
	Biscuits		4	11		7.0
			4	14		8.9
			15	28		17.7
			15	31		19.6
			30	36		22.8
			30	43		27.2
			60	55		34.7
			60	60		38.0
	Sample 7 (Diethylene glycol dibenzoate)
	Chocolate	22°C	2	16	3359	0.5
			4	23		0.7
			4	24		0.7
			15	59		1.8
			15	63		1.9
			30	103		3.1
			30	107		3.2
			60	144		4.3
			60	148		4.4
	Sample 7 (Dipropylene glycol dibenzoate)
	Chocolate	22°C	2	12	1709	0.7
			4	20		1.2
			4	20		1.2
			15	41		2.4
			15	42		2.5
			30	64		3.7
			30	66		3.9
			60	78		4.6
			60	83		4.9

Table 11 (

 11 b). Results of migration experiments with diisobutyl phthalate from the non-spiked sample 10 in contact with Tenax ® and blotting paper.

	Food/Simulant	Temperature	Time	Migration	Initial	Quotient
			point		concentration	Migration/Initial
					after spiking	concentration
		(°C)	(d)	(µg/dm²)	(µg/dm²)	(%)
	Sample 10 (Diisobutyl phthalate)
	Tenax ®	-18°C	90	7.2	182	4.0
			90	6.9		3.8
		22°C	2	13.6	182	7.5
			2	13.3		7.3
			6	23.7		13.0
			6	19.4		10.7
			15	47.0		25.8
			15	56.1		30.8
			30	67.7		37.2
			30	74.0		40.7
			60	79.5		43.7
			60	80.3		44.1
	Blotting paper	22°C	1	9.4	182	5.2
			2	15.2		8.4
			2	15.1		8.3
			5	26.2		14.4
			6	35.6		19.6
			8	38.8		21.3
			10	40.5		22.3
			12	47.2		25.9
			12.9	48.9		26.9
		40°C	0.6	25.3	182	13.9
			1	33.5		18.4
			2	49.2		27.0
			3	54.8		30.1
			5	71.4		39.2
			10	88.0		48.4

Table 12 .

 12 Results of migration experiments with diisobutyl phthalate, bisphenol A and benzyl 2-naphthyl ether from the non-spiked samples 9 and 11.

	Food/Simulant	Temperature	Time	Migration	Initial	Quotient
			point		concentration	Migration/Initial
					after spiking	concentration
		(°C)	(d)	(µg/dm²)	(µg/dm²)	(%)
	Sample 9 (Diisobutyl phthalate)
	Raspberries	-18°C	90	n.d.	303	<0.9
			90	n.d.		<0.9
	Tenax ®	-18°C	90	6.3	303	2.1
			90	6.9		2.3
	Sample 11 (Bisphenol A)
	Wheat flour	-18°C	90	1.1	2469	0.04
			90	1.1		0.04
	Tenax ®	-18°C	90	1.6	2469	0.07
			90	2.5		0.1
	Sample 11 (Benzyl 2-naphthyl ether)
	Wheat flour	-18°C	90	15.9	5006	0.3
			90	15.6		0.3
	Chocolate	-18°C	90	19.9	5006	0.4
			90	16.5		0.3
	Tenax ®	-18°C	90	24.9		0.5
			90	20.3		0.4
	n.d. not detectable (detection limit: 2,6 µg/dm²)

Table 13 .

 13 Results of modelling of the migration investigations.

	Migrants	A B2	log D B2	K B2/F	A F
	Sample 1 (spiked) with Tenax ® : 10days, 5°C
	Benzophenone	-		-	-
	Diisobutyl phthalate	-2.8	-11.3	29.0
	Triclosan Phenanthrene	--2.0	-10.5	-	-95.0	6
	DIPN	-1.0	-10.2	1.0
	Di(2-ethylhexyl) phthalate	-		-	-
	Sample 1 (spiked) with Tenax ® : 10days, 22°
	Benzophenone	-10.2	-13.1	10.0
	Diisobutyl phthalate	-1.3	-9.7	17.0
	Triclosan Phenanthrene	-5.0 -4.6	-11.4 -10.7	10.0 43.0		6
	DIPN	-1.0	-9.3	1.0
	Di(2-ethylhexyl) phthalate	-4.0	-11.4	15.0
	Sample 1 (spiked) with butter: 10days, 5°C
	Benzophenone	-4.8	-11.7	17.0
	Diisobutyl phthalate	6.0	-7.9	20.0
	Triclosan Phenanthrene	6.0 -2.2	-7.6 -10.6	55.0 3.5		7.5
	DIPN	1.2	-9.1	0.01
	Di(2-ethylhexyl) phthalate	6.0	-8.0	65.0
	Sample 1 (spiked) with butter: 10days, 22°C
	Benzophenone	-6.2	-11.4	1.0
	Diisobutyl phthalate	6.0	-6.6	11.0
	Triclosan Phenanthrene	6.0 -0.45	-6.6 -8.9	40.0 1.0		7.5
	DIPN	1.0	-8.4	3.0
	Di(2-ethylhexyl) phthalate	6.0	-7.1	50.0
	Sample 4 (non-spiked) with wheat flour: 60days, 22°C
	Abietic acid	-10.2	-13.7	100.0	6
	Sample 6 (

non-spiked) with sponge finger biscuits: 60days, 22°C

	Diisobutyl phthalate	-3.8	-10.8	18.5	4
	Sample 7

(non-spiked) with chocolate: 60days, 22°C

	Diethylene glycol dibenzoate Dipropylene glycol dibenzoate -2.0 -3.4	-10.8 -10.3	13.8 1.5	3

Sample 10 (non-spiked) with Tenax ® : 60days, 22°C

	Diisobutyl phthalate	-3.9	-11.3	4.1	6
	Sample

10 (non-spiked) with blotting paper: 13days, 22°C

	Diisobutyl phthalate	-3.3	-11.0	2.4	6
	Sample 10 (non-spiked) with blotting paper: 10days, 40°C
	Diisobutyl phthalate	-2.0	-9.6	0.6	6
	Sample 10 (

non-spiked) with wheat flour: 60days, 22°C

	Diisobutyl phthalate	-2.0	-10.5	15.0	6
	Sample 10 (non-spiked) with egg pasta: 60days, 22°C
	Diisobutyl phthalate	-4.5	-11.6	0.6	-2
	Sample 10 (non-spiked) with sponge finger biscuits: 60days, 22°C
	Diisobutyl phthalate	-3.9	-11.3	33.0	6
	Sample 10 (non-spiked) with chocolate: 60days, 22°C
	Diisobutyl phthalate	-3.3	-11.0	6.5	4
	-Migration of target migrants was below the detection limit.

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

Acknowledgements

The authors gratefully acknowledge the financial support of this work by the Confederation of European Paper Industries (CEPI). The findings and conclusions in this paper are the responsibility of the authors alone and should not be taken to represent the opinion of CEPI.


Mark Cronly

Patrice Behan

Barry Foley

Edward Malone

Sheila Martin

Michael Doyle

Liam Regan

Rapid multi-class multi-residue method for the confirmation of 1 chloramphenicol and eleven nitroimidazoles in milk and honey by 2 liquid chromatography tandem mass spectrometry 3 4

Keywords: Nitroimidazoles, chloramphenicol, milk, honey, LC-MS/MS, validation 6 7

Rapid multi-class multi-residue method for the confirmation of chloramphenicol and eleven nitroimidazoles in milk and honey by liquid chromatography tandem mass spectrometry

5-Nitroimidazoles are primarily used for the prophylactic and therapeutic treatments 10 of diseases such as histominiasis and coocidiosis in poultry, hemorrhagic enteritis in 11 pigs and genital trichomoniasis in cattle. Recently, reports from China suggest that the 12 use of nitroimidazoles in beekeeping is being practiced [Jinhui [START_REF] Zhou | Simultaneous 27 Determination of Nitroimidazole Residues in Honey Samples by High-Performance 28 Liquid Chromatography with Ultraviolet Detection[END_REF]. [START_REF] Thompson | Improved screening 14 method for the detection of a range of nitroimidazoles in various matrices by optical 15 biosensor[END_REF] Nitroimidazoles may be used to prevent and control Nosema apis in hives. Nosema 14 Apis is a microsporidian pathogen that is commonly found in Apis mellifera 15 throughout the beekeeping world. [Official Method (2003)] 16 17 Nitroimidazoles(NMZs) are believed to be carcinogenic and mutagenic to humans and 18 as a consequence are placed in the group A6 (prohibited) substances and their use in 19 food producing animals within the European Union is not permitted under Regulation 20 2377/90 [5]. They are also prohibited for use in the U.S.A and China. The analysis of 21 these compounds is required under Council Directive 96/23/EC. The Community 22 Reference Laboratory (CRL) has suggested a recommended level (RL) of 3 µg L -1 / 23 µg kg -1 .There is a need for rapid multi-residue analytical methods that have the 24 capability to include a wide rage of these analytes in all matrices where abuse may be 25 found. The structure of these 5-nitroimidazoles and their metabolites are seen in table 26 1. 27 [START_REF] Sheridan | Analysis and Occurrence of 29 14 Sulfonamide Antibacterials and Chloramphenicol in Honey by Solid-Phase 30 Extraction Followed by LC/MS/MS Analysis[END_REF] The CRL in Berlin has carried out homogeneity and stability studies on NMZs in 29 various matrices. [START_REF] Polzer | Treatment of turkeys with nitroimidazoles 4 Impact of the selection of target analytes and matrices on an effective residue control[END_REF][START_REF] Polzer | Nitroimidazoles in turkey muscle-the influence of 8 sampling conditions on measurement results in residue control[END_REF]) These studies showed that 30 there is not a homogenous distribution of these analytes in turkey muscle and they 31 also observed a rapid degradation in analyte concentration stored for prolonged 32 periods above 4 °C. In contrast it was discovered that for plasma, retina and egg 33 3 samples; the analytes were stable during storage under the same conditions which 1 resulted in stable concentrations and allowed detection of these compounds for longer 2 periods after medication had been halted. Therefore, it is advised that plasma, retina 3 and eggs be used as target matrices for the residue control of NMZs (Polzer et al. 4 2004, [START_REF] Polzer | Nitroimidazoles in turkey muscle-the influence of 8 sampling conditions on measurement results in residue control[END_REF]. 5 6 As a result of these published studies there has been an increase in the methods being 7 developed for the analysis of these compounds in matrices such as egg (Mottier et While suitable methods now exist for monitoring these analytes, no suitable methods 27 in matrices such as milk and honey exist for the comprehensive confirmatory analysis 28 of NMZs. The CRL for NMZs has suggested that honey be tested to ascertain any 29 possible misuse and if non-compliant results are found then this matrix should be 30 included in monitoring plans. They also state that countries with high milk production 31 should also analyse for these analytes in milk as their possible misuse in this matrix 32 cannot be discounted. From investigation of literature only two method could be 33 found that allows for the analysis of NMZs in honey. analysis of veterinary residues in honey. While the method was confirmatory for eight 6 nitroimidazole compounds it could only analyse to 10 µg kg -1 which is three times 7 higher than the RL for these compounds. 8 9

In relation to the analysis of nitroimidazoles in milk there are a limited number of 10 published methods available. (Ortelli et al. 2009, Stolker et al.2008, Thompson et al. 11 2007). These methods are all screening methods using either optical biosensor 12 (Thompson et al. 2007) [START_REF] Boyd | Development of an improved method for trace analysis of chloramphenicol 14 using molecularly imprinted polymers[END_REF], Rejtharova et 4 al. 2009). An investigation into the methods used to analyse for CAP found that it is 5 often analysed singly in several matrices including milk and honey. Ronning et al. 6 developed a method that anaylse for CAP residues in meat, seafood, egg, honey, milk, 7 plasma and urine with liquid chromatography-tandem mass spectrometry. Samples 8 were extracted with acetonitrile and chloroform was added to remove water. Extracts 9 were then evaporated to dryness, reconstituted and filtered before injection. 10 Rejtharova et al. described a method for the analysis of CAP in urine, feed water, milk 11 and honey samples by GC-MS-NCI using molecular imprinted polymer clean-up. [START_REF] Aerts | Liquid-chromatographic 3 multicomponent method for determination of residues of ipronidazole, ronidazole, and 4 dimetridazole and some relevant metabolites in eggs, plasma, and feces and its use in 5 depletion studies in laying hens[END_REF] On examining published literature it is quite clear that the confirmatory analysis of 13

NMZs in milk and honey has not been examined to date. No confirmatory methods 14 could be found for the analysis of these analytes in the matrices of honey and milk. [START_REF] Ronning | Determination of chloramphenicol 16 residues in meat, seafood, egg, honey, milk, plasma and urine with liquid 17 chromatography-tandem mass spectrometry, and the validation of the method based 18 on 2002/657/EC[END_REF] With growing concerns from China about the use of NMZs in honey the CRL having 16 recommended that this matrix be examined. The CRL have also recommended that 17 countries with high milk production should analyse for these compounds in milk. 18 Therefore there is a need for a comprehensive method to allow for the confirmatory 19 analysis of both these matrices. In addition to this, while the analysis of CAP in all 20 matrices is being performed it is often performed using single analyte methods which 21 contain time consuming SPE clean-up steps. From literature investigation no method 22 was found that was capable of analysing for CAP and the eleven NMZs listed in this 23 paper. The sample preparation described in this study is less time consuming than 24 previously published methods. The milk method has a sample size of 1ml which 25 allows for fast extraction times. The majority of methods for the analysis of CAP 26 incorporate the use of an SPE clean up. The method presented here omits this step and 27 still achieves satisfactory results. [START_REF] Sheridan | Analysis and Occurrence of 29 14 Sulfonamide Antibacterials and Chloramphenicol in Honey by Solid-Phase 30 Extraction Followed by LC/MS/MS Analysis[END_REF] 29

Materials and Methods 30

Materials and Reagents 31

CAP and CAP-d5 were purchased from Sigma (St. Louis, MO) and Dr. Ehrenstorfer 32 GmbH (Germany) respectively. CNZ, TNZ, TRZ, ORZ were provided by the CRL 33 Water is of LC-MS grade (Fluka). All other solvents were of LC grade and purchased 3 from Reagecon (Clare, Ireland). Sodium chloride was AnalaR grade and purchased 4 from VWR (Poole, England). Individual stock standards of each analyte at 1mg ml -1 5 in ethanol were prepared and stored at 4˚C for 1 year. Individual intermediate 6 standard solutions (10,000 and 100 ng ml -1 ) in methanol were prepared. Two working 7 standards solutions (mixture of analytes) were prepared in methanol containing all 8

NMZs at levels of 300ng ml -1 and 100ng ml -1 respectively (for honey) and CAP at 9 levels of 30ng ml -1 and 10ng ml -1 respectively(for milk). Deuterated standards were 10 prepared similarly except the mixed standard contained 200ng ml -1 deuterated NMZs 11 and 20ng ml -1 deuterated CAP. 12 13

LC-MS/MS Instrumentation 14

The LC-MS/MS system is an AB Sciex Triple Quad 5500 couple to Shimadzu UFLC 15 XR LC system. The instrument is controlled by Analyst Software 1.5 and operated in 16 positive and negative electrospray ionisation (ESI +/-). Separation was achieved 17 using a 100x2mm, 1.8 micron particle size Zorbax Eclipse Plus C18 column supplied 18 by Agilent Technologies (Santa Clara, CA). The column oven temperature was set at 19 45 ºC. The chromatographic separation was achieved using gradient mode consisting 20 of water acidified with 0.1% acetic acid (mobile phase A) and acetonitrile acidified 21 with 0.1% acetic acid at flow rate 0.5ml min -1 . The gradient is as follows; 95% A for 22 the first 1.5 min. Then this changes to 5% A from 1.5-3.0 min and maintained for 2.0 23 min. The conditions then return to the initial 95% A in 1.0 min (5-6 min) and remain 24 the same till the end of the run of 9.0 min. A divert valve is utilised to help remove 25 any matrix impurities from entering the MS/MS. The LC flow is diverted for the first 26 minute and the last three minutes of the method. The ionisation mode used was 27 positive electrospray ionisation for the NMZs and negative electrospray ionisation for 28 CAP. The MS/MS method was segmented in order to obtain enough data points on 29 each peak. The first three minutes is run in positive mode and from then on it is run 30 with positive and negative switching. A cone temperature of 350ºC with a spray 31 voltage of 4500V was used to produce parent to product ions. The individual 32 precursor and products ions for each analyte with their respective collision energies 33 are listed in table1 34

Milk and Honey Samples 2

Milk and honey were obtained and stored at -20 ºC. Portions of these samples were 3 analysed and those found to contain no detectable residues of the analytes of interest 4 were used as blanks for the validation study. Chromatograms of blank milk and honey 5 samples can be seen in figure 1

Milk Extraction 17

Milk (1mL) was pipetted into polypropylene centrifuge tubes (15mL). These were 18 fortified with mixed internal standard (30 µL) which corresponded to 6 ng mL -1 of 19 deuterated NMZs and 0.6 ng mL -1 deuterated CAP. Acetonitrile (2mL) was added and 20 vortexed. NaCl (0.5g) was added to this slurry which was shaken (30 secs) and then 21 centrifuged (4350 x g for 10min). The top organic layer from each sample was then 22 transferred to amber vials (5mL) and evaporated (50ºC) to dryness under a stream of 23 nitrogen. The extracts were reconstituted in Water: Acetonitrile (95:5, 200µL) and 24 filtered through 0.2µm PVDF syringe filters. An aliquot (10µL) was injected onto the 25

LC column. 26 27

Honey Extraction 28

Honey (3 g) was weighed into polypropylene centrifuge tubes (50 mL). These were 29 then placed in an oven at 50 ºC for 30 min to soften. The samples were then fortified 30 with mixed internal standard (50 µL) which correspond to 2 µg kg -1 of deuterated 31 NMZs and 0.2 µg kg -1 deuterated CAP. Water (5ml) was then added to each sample 32 and these are then placed back in the oven for a further 10 min. The samples were 33 then thoroughly vortexed until the honey was fully dissolved in the water. To this 34 acetonitrile (10 mL) was added and the tubes were vortexed (20secs). NaCl (2 g) was 1 added to this slurry which was then shaken (30 secs) and centrifuged (4350 x g for 2 10min). The top organic layers were then transferred to polypropylene tubes (15 mL) 3 and evaporated (50ºC) to 6mL under nitrogen. Hexane (5 mL) was added and this was 4 vortexed (30secs). The hexane layer was then discarded and the extracts were 5 evaporated to dryness at 50ºC under a nitrogen stream. They were then reconstituted 6 in Water:ACN (200 µL of 95:5 and filtered through 0.2 µm PVDF syringe filters. An 7 aliquot (10 µL) was injected onto the LC column. 8 9

Matrix Matched Calibration Curves 10

Quantification was carried out using matrix-matched calibration curves. Blank honey 11 and milk samples were used. These samples were fortified with mixed working 12 standard and submitted to the full extraction procedure of the method. A matrix 13 matched calibration curve is performed with every batch. Six samples are fortified 14 with internal standard and mixed working standard for a calibration range of 0 to 20 15 ng mL -1 (µg kg -1 Honey) for the NMZs and a range of 0-2 ng mL -1 (µg kg -1 Honey) for 16 CAP. Calibration curves were prepared by plotting the response factor (the ratio of 17 transitions. This allows for clear identification and quantification of all analytes. To 4 investigate the linearity of the method, matrix-matched calibration curves were 5 prepared and run with each of the validation batches to give 6 point calibration curves 6 ranging from 0 to 20µg L -1 / µg kg -1 for the NMZs and 0 to 2µg L -1 / µg kg -1 for CAP. 7

Since no certified reference materials were available for the analytes and matrices of 8 interest, the recovery from fortified negative samples was measured as an alternative 9 to trueness. The recovery and precision were determined through the analysis of 10 negative milk and honey samples fortified in six replicates at 1, 1.5 and 2 times the 11 RL and the MRPL of 3 and 0.3 µg L -1 / µg kg -1 for NMZs and CAP respectively. Six 12 replicate test portions at each of the three fortification levels (n = 18) were analysed 13 on three separate days over a period of two weeks. Samples were fortified with NMZs 14 at 3, 4.5 and 6 µg L -1 / µg kg -1 and with CAP at 0.3, 0.45 and 0.6 µg L -1 / µg kg -1 by 15 adding mix working standard solution (30, 45 and 60 µL) and analysed. To determine 16 any matrix effects caused by biological variations arising from various milk and 17 honey samples a fourth day analysis was carried out. For each matrix two sets of ten 18 different samples were analysed. The first set was fortified with only internal 19 standard, and the second set was fortified with both internal standard and with the 20 analytes at a concentration equivalent to 4.5µg L -1 / µg kg -1 of NMZs and 0.45µg L -1 / 21 µg kg -1 of CAP. From these four separate validation days an estimation of recovery, 22 precision (repeatability and within-laboratory reproducibility) and analytical limits 23 (decision limit CCα, and detection capability CCβ) were determined. 24 25

Results and Discussion 26

Method Optimisation 27

The method was developed to provide confirmatory data for the analysis of CAP and 28 11 NMZs in milk and honey. The method was developed from an existing method 29 used in the lab to analyse for NMZs in plasma and eggs (Cronly et al. 2009). The LC-30 MS/MS instrument used for this method was different than the one used previously so 31 fragmentation conditions were investigated and collision energies were optimised for 32 each individual compound to give best response. For a method to be deemed 33 points. In this method a precursor ion (parent mass) and two product ions 2 (corresponding to quantifier and qualifier ion) are monitored for each analyte (Table 3 1). This yields 4 identification points (1 for the precusor ion and 1.5 for each product 4 ion) hence it can be deemed a confirmatory method. In addition to this relative 5 retention times and ion ratios are tracked for each compound and ensured that they are 6 within acceptable ranges stated in EC 2002/657. The LC gradient was also optimised 7 in order to have a quick run time but also have enough data points for each peak. For a 8 method to achieve reliable quantitation each analyte peak should have at least 10-12 9 data points. As this method involved positive and negative ionisation switching the 10 MS/MS method had to be segmented. This along with altering the LC gradient 11 allowed for the analysis of all 12 analytes in a complete run time of less than 9 min. 12 13 In developing the milk method; the sample size was reduced to as low as possible to 14 allow for a more efficient extraction method with reduced extraction costs as 1ml of 15 milk was extracted with 2ml of acetonitrile with the addition of 0.5g of NaCl. The 16 extract was then evaporated to dryness and reconstituted in initial mobile phase. The 17 samples were filtered and run on the LC-MS/MS. The instrument was sensitive 18 enough to see all the analytes at low concentrations with a sample size of only 1ml. 19

The matrix of honey is more complex and not much work had been carried out 20 previously on it. The majority of methods use a SPE clean-up and this was overcome 21 by adapting the previous methods used for aqueous based matrices (eggs and plasma). 22

The honey sample was first diluted in water before extraction with acetonitrile and the 23 addition of NaCl. Initial studies saw that the honey was quite difficult to dissolve in 24 the water so the honey was first softened in an oven before the addition of water and 25 this allowed the honey to dissolve fully. It was also seen that when acetonitrile was 26 added to this solution that two layers formed after shaking. It was decided to take top 27 organic layer and investigate if the analytes had been extracted into this layer. It was 28 clear upon evaporation of this layer that some honey had been taken into the layer and 29 therefore could not be reconstituted. This did not occur when NaCl (2g) was added to 30 the mixture and shaken. The extract was hexane washed and evaporated to dryness. 31

The samples were reconstituted and filtered before been run on the LC-MS/MS. This 32 is a much less involved extraction than used previously with these analytes in honey 33 but results achieved were still satisfactory. 2 for these criteria. The ruggedness of the 6 method is demonstrated on an ongoing basis through the use of it to analyse National 7

Residue Control Plan milk and honey samples in Ireland. The criteria of relative 8 retention times (RRT) and ion ratios were monitored for all analytes in the four 9 validations days. The values identified for these were all within European 10 requirements. The RRT tolerance of 2.5% was adhered to when standards were 11 compared to samples in the validation runs. Two transition ions were monitored for 12 each of the twelve analytes. The most intense was used for quantitation. All ion ratios 13 of samples were within tolerances as set out by European criteria when compared with 14 standards used during validation. 15 16

Specificity 17

The technique of LC-MS/MS itself offers a great deal of specificity and selectivity. 18

To establish the specificity and selectivity of the method blank milk and honey 19 samples and samples fortified with all 12 analytes were analysed over the 3 validation 20 days. On the fourth day 10 different types of milk and honey samples were also 21 analysed. Blank samples showed no interfering peaks in the area of interest for any of 22 the analytes. Chromatograms of blank milk and honey and milk and honey fortified at 23 the RL and MRPL for each of the analytes are seen in figure 1 and2. 24 25 Linearity 26

The linearity of the chromatographic response was tested with matrix matched 27 calibration curves using six calibration points in the range of 0-20 µg L -1 / µg kg -1 28 NMZs and 0-2.0 µg L -1 / µg kg -1 for CAP. The regression coefficients for all the 29 analytes on each of the validation days in both matrices were greater than 0.99. The accuracy (trueness) of the method was determined by fortifying 6 replicate milk 1 and honey samples at 1, 1.5 and 2 times the analytes respective RL or MRPL on three 2 separate days. Mean corrected recovery (n=6) of the analytes, determined in the three 3 separate validation batches are shown in table 2 range between 90.8 and 108.9% for 4 the twelve analytes in both matrices. No absolute recovery was determined as the use 5 of internal standards means that each sample is individually corrected for. 6 7 Precision 8 Satisfactory values for inter-assay precision expressed as %CV values for the within 9 lab reproducibility (table 2) were achieved for all analytes in both matrices. According 10 to Commission Decision 2002/657/EC this coefficient of variance for the repeated 11 analysis of fortified material under reproducible conditions shall not exceed the level 12 calculated by the Horwitz equation. For a concentration of 100 µg L -1 / µg kg -1 this 13 equation gives a value of 23%. However when concentrations go under this value the 14 equation gives unacceptably high results. Therefore it's stated in Commission 15 Decision 2002/657/EC that %CV should be kept as low as possible. Results achieved 16 range from 3.5 to 15.3% for all analytes in both matrices. These acceptable results can 17 be attributed to the availability of 7 deuterated analogues to use as internal standards. 18 HMMNI-d3 was used as an internal standard and worked well for compounds with 19 no deuterated internal standards such as CNZ, ORZ, TRZ and TNZ. 20 21

CCα α α α and CCβ β β β

22 CCα is defined as the limit above which it can be concluded with an error probability 23 of α, that a sample contains the analyte. For prohibited substances an α value equal to 24 1 % is applied. CCβ is the smallest content of the substance that may be detected, 25 identified and quantified in a sample, with a statistical certainty of 1-β, where 26 β = 5 %. CCα and CCβ were calculated using the calibration curve procedure in 27 accordance with ISO 11843. After identification, the signal is plotted against the 28 added concentration. The corresponding concentration at the y-intercept plus 2.33 29 times the standard deviation of the within-laboratory reproducibility of the intercept 30 equals the CCα. CCα values of for the 12 compounds in honey and milk are listed in 31 Table 2 and are all below 1.55 µg L -1 / µg kg -1 for the NMZs and below 0.08 µg L -1 / 32 µg kg -1 for CAP. CCβ is the concentration corresponding to the signal at CCα + 1.64 33 times the standard error of the intercept (i.e. the intercept + 3.97 times the standard 1 error of the intercept). CCβ values for all compounds in honey and milk are listed in 2 table 2 and are all below the RL of 3ng mL -1 and MRPL of 0.3 µg kg -1 for NMZs and 3 CAP respectively. 4 5 Measurement Uncertainty 6 The measurement uncertainty (MU) was estimated by taking into account the 7 within laboratory reproducibility over days 1, 2 and 3 as well as considering the 8 repeatability on day 4 due to matrix effects caused by various honey and milk 9 samples. These two variability's were combined and multiplied by a coverage factor 10 of three to give an overall figure for the MU. This approach of using the within 11 laboratory reproducibility as a good estimator of measurement of uncertainty is taken 12 from the SANCO/2004/2726rev4 document. It recommends using the within 13 laboratory reproducibility and using a coverage factor of 2.33 to estimate expanded 14 uncertainty, however it was felt that not all the environmental factors that could be 15 varied over the course of the validation were examined. Therefore a coverage factor 16 of 2.33 may underestimate the true uncertainty of the method and instead a value of 3 17 was chosen to give a more realistic value for the true uncertainty. Values for 18 MU are seen in Table 2 and lie between 23 and 69% for all compounds in milk and 19 between 24 and 90% for all analytes in honey. 20 21 Higher MUs are seen in milk for some compounds with no deuterated analogues to 22 use as internal standards which is expected. Honey results in general display higher 23 MUs. High MU estimates are again seen for some of the compounds with no 24 deuterated analogues to use as internal standards in particular ORZ (81%) and CRZ 25 (90%). Their MU estimates in honey are the highest due to large values for 26 reproducibility due to matrix as a result of a variation between matrices used in the 27 day 4 experiment and the lack of suitable internal standards. [START_REF] Sheridan | Analysis and Occurrence of 29 14 Sulfonamide Antibacterials and Chloramphenicol in Honey by Solid-Phase 30 Extraction Followed by LC/MS/MS Analysis[END_REF]

29

Conclusions 30

The objective of this work was to develop a rapid multi-class multi-residue 31 confirmatory method capable of identifying, confirming and quantifying eleven NMZ 32 compounds and CAP in milk and honey at µg L -1 and µg kg -1 levels and to validate 33 This study shows that the developed method meets the required sensitivities of 3 µg L - 11 1 / µg kg -1 for NMZs and 0.3µg L -1 / µg kg -1 for CAP which are the RL and MRPL 12 used for these compounds. The CCα and CCβ values determined for each analyte are 13 lower than this level. The method performs very well in terms of accuracy and 14 repeatability for each of the analytes due to the utilisation of seven different 15 deuterated internal standards. The values achieved for accuracy, %CV and 16 measurement of uncertainty all fall within acceptable ranges. The applicability of the 17 method for use on various types of milk and honey samples was demonstrated by the 18 satisfactory results obtained from the Day 4 analysis of different species.The reduced 19 number of analytical steps within the method makes it very amenable for high 20 through-put regulatory monitoring of these compounds. 21 22 From examination of published literature no method was found that was capable of 23 the sensitive confirmatory analysis of CAP and eleven NMZs in milk and honey. 24 Methods published on these matrices analysed at most seven analytes and in the case 25 of CAP often analysed as a single analyte method. The method developed in this 26 study allows for improvement on any existing method as it allows for the analysis of 27 an increased number of analytes in matrices that have been previously overlooked. It 28 also allows for reduced sample preparation times as SPE clean-up has been omitted. 29

In the case of the extraction protocol for milk time and solvent usage is greatly 30 reduced compared to other published methods as a result of reduced sample size of 31 1mL 32

  al. 8 2006, Mohammed et al. 2008, Xia et al. 2006, Daeseleire et al. 2000, Cronly et al. 9 2009) and plasma (Aerts et al. 1991, Fraselle et al. 2007, Thompson et al. 2009, 10 Cronly et al. 2009). The majority of methods published for the analysis of eggs 11 involve extraction with acetonitrile and the addition of NaCl. Purification 12 methodologies of these extracts varied. Solid Phase Extraction (SPE) such 13 Hydrophilic-lipophilic balance (HLB) cartridges (Mottier et al. 2006) or Molecular 14 imprinted (MIPs) SPE cartridges (Mohamed et al. 2008) were used. In some methods 15 the samples were just filtered before injection and satisfactory results were still 16 achieved (Xia et al. 2006, Daeseleire et al. 2000, Cronly et al. 2009). Methods for the 17 analysis of these compounds in plasma are varied in their extraction protocols. 18 Extraction solutions such as aqueous buffer (Aerts et al 1991), NaCl/potassium 19 phosphate buffer (Fraselle et al. 2007) and acetonitrile (Thompson et al. 2009, Cronly 20 et al. 2009) have been used in the extraction of NMZs from plasma. SPE catridges of 21 Extrelut (Aerts et al.1991) and XTR kieselguhr (Fraselle et al. 2007) have been used 22 in the sample purification in this matrix. Cronly et al. omits the use of SPE and clean 23 up is achieved by purifying samples with the use of a hexane wash step and filtering 24 before inject. 25 26

  analysis of 5 NMZs in honey by HPLC-UV. Samples were extracted 1 with ethyl acetate and evaporated. The residue containing the NMZs was dissolved in 2 ethyl acetate-hexane and subjected to solid-phase extraction cleanup by amino 3 extraction columns. The eluent was evaporated, reconstituted and injected onto the 4 column. The second Mol et al., 2008, was a multi-class multiresidue method for the 5

  , RNZ-d3 were purchased from WITEGA Laboratorien (Berlin, Germany). 2

  peak area analyte over peak area of internal standard) against analyte concentration. 18 Seven deuterated internal standards are used; d 3 -DMZ, d 3 -RNZ, d 3 -HMMNI, d 2 -19 MNZ-OH, d 3 -IPZ, d 3 -IPZ-OH and d 5 -CAP. For those compounds with no deuterated 20 analogues; MNZ, TRZ, TNZ, ORZ and CRZ, d 3 -HMMNI is used as an internal 21standard. For each analyte calibration curves were linear in the given range with a 22 correlation coefficient of at least 0.99.23 24Method Validation 25The LC-MS/MS method was validated according to 2002/657/EC guidelines. The 26 same validation protocol was used for both the honey and milk matrix. LC-MS/MS 27 identification criteria were verified throughout the validation study by monitoring 28 relative retention times, ion detection (signal-to-noise ratio (S/N)) and relative ion 29 intensities. LC-MS/MS identification criteria as set out in the legislation were verified 30 throughout the validation of the method. 31 32 Several method validation parameters were determined including linearity, specificity, 33 recovery, precision (repeatability and within-laboratory reproducibility) and analytical 34 limit CCα and detection capability CCβ). Specificity was determined 1 by analysing 10 different blank milk and honey samples sourced from different 2 suppliers. No interfering peaks were observed at the retention time for any of the 3

  Decision 2002/657/EC it must yield 4 identification 1

2

 2 Validation is carried out in accordance with the procedures outlined in Commission 3Decision 2002/657/EC covering specificity, calibration curve linearity, accuracy, 4 precision, decision limits (CCα), decision capability (CCβ) and measurement 5 uncertainty. Results are seen in table

2 38

 2 requirements in Commission Decision 2002/657/EC [33]. This was 1 successfully completed.The method can be considered as rapid, as it utilises an efficient extraction protocol 4 without the use of SPE. It also utilises chromatography which separates all analytes in 5 a total run time of only 9 minutes. The method includes the confirmatory analysis of 6 CAP and 11 NMZs in milk and honey which has not been seen before. 7The obtained data fulfils the requirements laid down in Commission Decision 9 2002/657/EC and allows the calculation of all relevant performance characteristics. 10

Figure 1 : 26 Figure 2 :

 1262 Figure Headings 2223Figure 1: Chromatogram of blank milk(A) and milk(1ml) fortified at 2.5 µg L -1 for 24 NMZs and 0.25 µg L -1 for CAP (B) 25 26 Figure 2: Chromatogram of blank honey(A) and honey (3g) fortified at 2.5 µg kg -1 for 27 NMZs and 0.25 µg kg -1 for CAP (B) 28 29 30

  or time of flight mass spectrometers (Ortelli et al. 2009, 13 Stolker et al. 2008). From investigation of literature there are no methods for the 14 confirmatory analysis of NMZs in milk at the levels desired.

	1	A common trend in analysis of CAP in any matrix seems to be the used of an SPE
	2	clean-up. Common cartridge chemistries used in the analysis of CAP are HLB (Shen
	3	et al 2009), Mixed Cation Exchange (MCX) (Zhang et al. 2008) and the selective
		technique of Molecular Imprinted Polymers (MIPs)
		F o r
		P
		e
	15	e r
	16 17	Chloramphenicol	R
	18	Chloramphenicol (CAP) is a broad spectrum bacteriostatic antibiotic that exerts its e
	19 20 21 22	effect by inhibiting bacterial protein synthesis. The structure of CAP is seen in table 1. Research carried out on CAP has shown that it has adverse toxicological affects in human such as aplastic anaemia or grey-syndrome. Due to these health concerns it has v i e been classified as an A6 compound in annex IV of council regulation 2377/90 which w
	23 24 25 26 27	means it is prohibited for use in food producing species. CAP has been issued a minimum required performance limit (MRPL) of 0.3 µg L -1 / µg kg -1 which means all methods should be able to at least see to this level. As a result of this low level; CAP is often analysed in single analyte methods (Rejtharova et al., 2009, Ronning et al., O n l 2006) although some multi amphenicol methods do exist (Zhang et al.2008, Shen et y
	28	al, 2009). In addition to this there are two other multi-class methods which include the
	29	analysis of CAP (Lopez et al., 2008 and Sheridan et al., 2008.) These analyse for CAP
	30	with compounds that are not prohibited for use such as sulfonamides and
	31	tetracyclines.

  and 2. For the day four validation studies of variability 6 due to matrix variances a wide range of milk and honey samples were obtained. Milk 7 samples comprised of high fat, low fat, organic milk and also milk with added extra 8 calcium, folic acid and vitamins A, B, D and E, a sample of goat's milk was also 9 examined. Honey samples comprised of the following types Kapetanios Pure Greek; 10

	11	Rowse Australian Eucalyptus; Capilano Australian Organic Blended; Tropical Forest
	12	Ltd. Ethiopian Forest; Marks and Spencer's New Zealand Clover; Famille Michaud-
	13	Lavender Honey, Provence, France; New Zealand Manuka Honey, Irish Honey; Irish
	14	Honeycomb, Dublin; De Rit Blended Flower, Holland.
	15
	16	Methods

Table 1 :

 1 Structures, Retention time(Rt), Precursor and Product ions and typical ion ratios for all 12 analytes

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

Table 2 :

 2 Validation Results for Milk and Honey; Coefficient of Variance (%CV), Accuracy, Decision Limits (CCα) and Detection Capabilities (CCβ), Measurement Uncertainty (MU) and Correlation Coefficients (R 2 )

	Page 25 of 25
	Analytes	CV%	Accuracy %	CCα µg kg -1	CCβ µg kg -1	MU %	R 2 Value
		Milk Honey Milk Honey Milk Honey Milk Honey	Milk	Honey
	MNZ-OH 4.5	4.1	92.0 103.8 0.43 0.39 0.73 0.67	27	25	>0.99
	HMMNI	4.1	4.6	94.5 104.2 0.42 0.42 0.71 0.71	27	29	>0.99
	MNZ	7.8	6.7	92.6 108.9 0.65 0.72 1.10 1.22	30	51	>0.99
	RNZ	4.3	3.5	94.3 102.4 0.41 0.38 0.70 0.66	23	24	>0.99
	DMZ	9.2	9.4	96.2	94.7	1.08 0.73 1.83 1.24	40	42	>0.99
	TRZ	5.4	9.0	93.9 102.0 0.53 0.78 0.91 1.34	23	56	>0.99
	TNZ	15.3	12.4	90.5 104.4 1.55 1.16 2.64 1.98	61	64	>0.99
	IPZ-OH	4.8	6.2	94.2 100.8 0.46 0.64 0.78 1.10	20	39	>0.99
	ORZ	11.4	11.3	90.8 100.7 1.08 1.00 1.85 1.71	69	81	>0.99
	CRZ	10.8	10.7	91.7 101.0 1.23 0.95 2.09 1.62	40	90	>0.99
	CAP	7.5	8.0	95.1 100.7 0.07	0.08	0.11	0.13	32	31	>0.99
	IPZ	4.7	4.3	95	97.8	0.52	0.40	0.88	0.68	25	35	>0.99

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