F Calbiani

M Careri

L Elviri

A Mangia

I Zagnoni

Validation of an ion-pair liquid chromatography-electrospray-tandem mass spectrometry method for the determination of heterocyclic aromatic amines in meat-based infant foods

Keywords: In-house validation, Method validation Heterocyclic aromatic amines, Ion-pair liquid chromatography-mass spectrometry, Validation, Robustness

Validation of an ion-pair liquid chromatography-electrospray-tandem mass spectrometry method for the determination of heterocyclic aromatic amines in meat-based infant foods

Lisa Elviri

To cite this version:

Lisa Elviri. Validation of an ion-pair liquid chromatography-electrospray-tandem mass spectrometry method for the determination of heterocyclic aromatic amines in meat-based infant foods. Food

Introduction

Heterocyclic aromatic amines (HAAs) are substances of great concern for food safety being recognized as potent mutagenic compounds to which humans are regularly exposed through diet and ambient environments [START_REF] Skog | Carcinogenic heterocyclic amines in model systems and cooked foods: a review on formation, occurrence and intake[END_REF]]. These compounds are formed in thermally treated protein-rich foods at the ng/g level, but they are also detected in several other environments, including cooking fumes, cigarette smoke, air, rain, and river water [Kataoka, 1997].

For the unambiguous identification and determination of these mutagenic and carcinogenic substances in different kinds of food commodities it is therefore necessary to develop and validate fast and reliable analytical methods. Due to the great complexity of food matrices and the low concentration levels of HAAs, their identification and determination require a comprehensive analytical approach involving the use of efficient sample preparation techniques and the employment of hyphenated analytical techniques. Solid-phase extraction (SPE) under cationic exchange and reversed-phase mechanisms have been described as the most widely used methods for their isolation and clean-up [START_REF] Gross | Quantitation of mutagenic/carcinogenic heterocyclic aromatic amines in food products[END_REF][START_REF] Toribio | Evaluation of different clean-up procedures for the analysis of heterocyclic aromatic amines in a lyophilized meat extract[END_REF]. High-performance liquid chromatography (HPLC), gas chromatography (GC), and capillary electrophoresis have been used for separation purposes [START_REF] Knize | Chromatographic methods for the analysis of heterocyclic amine food mutagens/carcinogens[END_REF][START_REF] Pais | Chromatographic and related techniques for the determination of aromatic heterocyclic amines in foods[END_REF]. At the present the most widely applied technique for HAA analysis is liquid chromatography coupled with mass spectrometry (LC-MS), since LC-MS-based techniques allow to avoid time-consuming derivatization procedures needed prior to GC-MS determination. In particular, LC coupled with tandem mass spectrometry (MS-MS) using both triple quadrupole and ion trap mass spectrometers have proved highly successful in the determination of HAAs [START_REF] Klassen | Heterocyclic aromatic amines in cooked hamburgers and chicken obtained from local fast food outlets in the Ottawa region[END_REF]Toribio, et al 2002;Barceló-Barrachina et al., 2004a;2004b]. The applicability of a LCelectrospray-quadrupole time of flight (ESI-QTOF)-MS and LC-ESI-QTOF-MS/MS techniques for the identification and determination of HAAs in foods has been also investigated using high mass accuracy measurements [Barceló-Barrachina et al., 2004b].

Very recently a low-time consuming solid-phase extraction procedure was developed together with a liquid chromatography-ion trap mass spectrometry method for their determination in griddled beef steak [Toribio, et al., 2006].

In our research work dealing with the investigation of the separation of HAAs (figure 1) by reversed-phase ion-pair LC-MS/MS [START_REF] Bianchi | Investigation of the separation of heterocyclic aromatic amines by reversed phase ion-pair liquid chromatography coupled with tandem mass spectrometry: the role of ion pair reagents on LC-MS/MS sensitivity[END_REF], we demonstrated that under gradient conditions the use of an eluent containing formate buffer at pH 2.8 proved more effective in terms of chromatographic behaviour and detectability of these analytes than the use of a mobile phase containing the acetate buffer, which is commonly used for the analysis of HAAs.

The need of a standard methodology and validated analytical methods for HAAs analysis is actually of great interest. In particular, interlaboratory exercises of the determination of HAAs in meat extract laboratory reference materials have been recently presented highlighting the importance to improve the quality of the analytical measurements [START_REF] Santos | Analysis of heterocyclic amines in food products: interlaboratory studies[END_REF].

In this context the aim of the present work was the single-laboratory validation of the previously developed chromatographic method to be applied to meat-based infant foods to measure their HAA content. A procedure entailing solvent extraction and clean-up by SPE was used. Besides to linearity, limit of detection (LOD), limit of quantificattion (LOQ), accuracy and recovery, attention was focused to the evaluation of method robustness towards both chromatographic and mass spectrometric parameters. Harman (1-methyl-β-carboline) and norharman (9H-pyrido[3,4-b] indole) were from Sigma-Aldrich (Germany). IQ (2-amino-3-methyl-3H-imidazo[4,5- Stock standard solutions of HAAs and internal standards at concentrations of 0.5 mg/mL were prepared in methanol and stored in the dark at 4°C. Working standard solutions were prepared daily for a better stability by diluting stock solutions with HPLC-grade water. Ammonium formate, formic acid, HPLC-grade acetonitrile and HPLC-grade water were from Carlo Erba (Milan, Italy).

Experimental

Chemicals

f]quinoline), MeIQ (2-amino-3,4-dimethyl-3H- imidazo[4,5-f]quinoline),

Liquid chromatography-mass spectrometry

An Alliance 2690 liquid chromatograph (Waters, Milford, MA, USA) equipped with a 120-vial capacity sample management system was used. Chromatographic separation was obtained using a TSKgel ODS-80TS (250 x 2.0 mm, 5µm) (TOSOH BIOSEP, Germany) column under gradient conditions. The mobile phase consisted of a mixture of ammonium formate-formic acid buffer (pH=2.8) (v/v) aqueous solution (eluent A) and acetonitrile (eluent B) delivered at a flow-rate of 0.2 mL min -1 . The gradient was as follows: at 0 min 5% B, in 20 min 54.5% B hold for 3 min, back to 5% B in 3 min and 10 min to re-equilibrate the column.

A Quattro LC triple quadrupole-mass spectrometer (Micromass, Manchester, UK) with a pneumatically-assisted electrospray interface was used. Data acquisition was performed in positive ion mode (ESI(+)). Interface parameters were set as follows: capillary voltage 2.8 kV, cone voltage 50 V (IQ, MeIQ, MeIQx, Norharman, Harman, PhIP) and 40 V (AαC), extractor lens voltage 2 V, source temperature 110°C, desolvation temperature 240°C, rf lens 0.1 V. The nebulizer and desolvation gases (nitrogen, 99.999 % high-purity) were delivered at 60 L hr -1 and 550 L hr -1 respectively.

Experiments for optimization of ESI interface parameters were performed by directly infusing solutions into the ESI-MS system at a flow-rate of 6 µL min -1 . Full-scan analyses were performed over the scan range m/z 150-300 using a step size of 0.1 Da and a rate of 0.4 scans/s. Operating in MS/MS mode, product-ion scan mass spectra of protonated molecules were acquired in the m/z 30- For data acquisition and processing the Masslynx v4.0 software was used.

Samples.

Solvent extraction and SPE procedure

Beef-based infant foods were purchased in big trades. Three homogenized and one lyophilized beefbased infant foods coming from different manufacturers were analyzed. Horse-, lamb-, rabbit-and chicken-based infant foods from three different manufactures were also analyzed.

To 1.0 g of homogenized sample weighed into a polypropylene graduated conical tube with cap 12 mL of acetone. After mixing for 5 min on a vortex, the extract was centrifuged at 4800 rpm (1350 g) for 5 min. Extraction was performed twice. The clarified solution was removed and stored in the freeze (-18°C) for one hour to allow protein to precipitate. The mixture was centrifuged at 4800 rpm (1350 g) for 5 min and 24 mL of the final extract were loaded on the SPE cartridges. Strong cation exchange (benzenesulfonic-SCX bonded silica) cartridges (300 mg, 6 mL) (Supelco) were used for concentration and clean-up purposes. Cartridges were first conditioned with 4 mL of methanol and 4 ml acetone, 24-mL sample were then loaded and eluted with 4-ml 2 M ammonium acetate aqueous solution/acetone (50/50, v/v). The volume of the final extract was then reduced to 2 mL in a graduate vial by evaporating acetone under a nitrogen stream. The sample was filtered and directly injected (injection volume 30 µl) onto the LC system. Sample treatment based on solvent extraction followed by SPE was carried out three times for each sample and three LC replicate analyses were run for each extract.

Recovery studies were carried out by spiking samples of lyophilized meat at two different concentration levels (10 and 50 ng/g) with appropriate volumes of the working mixtures of HAAs. The mixture was left to stand overnight at 4° C. The sample was equilibrated to room temperature before following the extraction procedures above described.

Validation of the LC-ESI-MS/MS method

Validation of the developed method was carried out following EURACHEM guidelines [START_REF] Anon | The Fitness for Purpose of Analytical Methods 1998: A Laboratory Guide to Method Validation and Related Topics[END_REF]] on a lyophilized meat matrix extracted and purified using the procedure described.

Detection limit (y D ) and quantitation limit (y Q ) were preliminarly calculated as signals based on the mean blank ( were verified to be absent was used as the blank matrix. y D and y Q were converted from the signal domain to the concentration domain to estimate limit of detection (LOD) and limit of quantitation (LOQ) respectively, using an appropriate calibration function. In order to satisfy basic requirements such as homoscedasticity and linearity, the Bartlett test and the Mandel's fitting test were performed at the 95% confidence level.

Linearity was established over two orders of magnitude of concentration calculating matrixmatched calibration curves. d 3 -IQ, 13 C 2 -MeIQx and d 3 -PhIP labelled internal standard was used. Six equi-spaced concentration levels were chosen and three replicated injections were performed at each level. Homoscedasticity test was run and the goodness of fit of the calibration curve was assessed by applying the lack-of-fit and Mandel's fitting tests [Funk et al., 1995]. A t-test was carried out to verify the significance of the intercept (confidence level 95%).

Accuracy was tested in terms of precision and trueness. Precision was calculated in terms of intraday and between-day repeatability as R.S.D.% at two concentration levels (10 and 50 ng/g).

Trueness was evaluated by calculating the recovery function [Funk et al., 1995], which allows to assess both constant and proportional systematic errors.

Robustness of the analytical method was tested using a two-level Plackett-Burman (PB) design. 1 and2). The three dummy variables included in the design were used to evaluate the variability of the procedure. The effects were calculated as follows:

2 / ) ( 2 / ) ( N Y N Y E X ∑ ∑ - - + =
where E X is the effect of factor X on response Y; Y(+) and Y(-) are the responses where X is at the level (+) and (-) respectively, N=12 is the total number of experiments.

The standard error (SE) e was evaluated using the dummy variables as follows:

dummy dummy n E e SE ∑ = 2 ) (
Significance of an effect was evaluated at both α = 0.05 and α = 0.01 significance levels. An effect was considered significant at a given α level if E X > E critical = t critical (SE) e , where t is a constant of the t-Student distribution (one-sided) depending on the confidence level and the degrees of freedom (ν=n-1, n=number of measurements). The matrix of experiments is illustrated in Table 3. The experiments were carried out in random order and for each experiment a single injection was performed. Two experiments at the nominal level (one before and one after the experimental design execution) were run in order to assess the absence of instrumental drift. Method robustness was evaluated at 100 µg/L level on standard solutions. A blank injection was performed prior to each analysis.

All statistical analyses and tests were performed by using the statistical package SPSS v. 9.0 for Windows (SPSS, Bologna, Italy).

Results and Discussion

LC-ESI(+)-MS and-MS/MS performances

The separation of the investigated analytes was performed in less than 20 min with good resolution [Toribio, et al., 2006] (figure 2).

Under ESI(+)-MS conditions, mass spectra of all compounds showed predominant [M+H] + ions without fragmentation. The description of the product ion MS/MS spectra as obtained by low-energy collision-induced dissociation of protonated HAA molecules were previously depicted [Toribio, et al., 2006]. HAA extraction was performed from meat samples by using acetone as already proposed by Felton et al. [1981;1984] who successfully applied this solvent for HAA separation from food by liquid-liquid extraction.

In the development of the SPE procedure for purification and concentration of HAAs in meat products, mixtures of acetone/aqueous solution of ammonium acetate at different concentrations (0.5, 2 and 3 M) (50/50) were evaluated as eluting solvents. The method was assessed on the basis of the qualitative and quantitative results obtained on the meat-based food used in this work. The best results were obtained by using 2M ammonium acetate aqueous solution/acetone (50/50, v/v). Under these conditions, recovery values ranging from 78±4% to 98±2% (RSD<12%, n=12) were obtained for the analytes. SPE extracts did not show the presence of any interference in correspondence to the transitions monitored for the amines. However, suppression effects were observed in correspondence to the peaks of all the analytes, suggesting the presence of not detected co-eluting matrix compounds in the extracts. On the basis of these findings, further investigation on matrix suppression effect was performed during method validation.

Calibration and method performances

To check method performance, quality parameters such as robustness, detection limit, quantification limit, linearity range, precision and trueness were studied in matrix. Since the LC-MS/MS method developed is intended for routine use, its robustness, i.e. the capacity to remain unaffected by small variations in nominal experimental conditions, was tested at the beginning of the validation procedure. For this purpose, a screening design such as the Plackett-Burman was used. Factors were selected among those that are most likely to be controlled when a method is transferred between laboratories and that could influence the responses of the method.

The results of the robustness test in terms of the effects of the different factors on the considered responses are shown in Table 4. The factors having a statistically significant effect on a response at significance level of 1 and 5% were indicated. Relative peak area resulted to be significantly affected both by chromatographic and MS/MS parameters. In particular, under the conditions used in this work, the analyte-internal standard area ratio resulted to be affected by mobile phase flowrate, ion pair reagent concentration, column temperature (only for MeIQx), cone voltage (only for AαC), collision energy (for all analytes except for MeIQx and IQ). These findings suggest that these parameters should be strictly controlled for a reliable quantification of HAAs. None of the factors resulted to have significant effect on resolution between IQ and MeIQ peaks. The results obtained for the other factors evidenced that mobile phase flow-rate was found to be statistically significant (α = 0.05) for the capacity factor of all analytes except for AαC peak. In addition, mobile phase pH resulted to be a critical parameter for the k' values of IQ, MeIQ and norharman.

Under the optimized chromatographic and MS/MS conditions, operating in SRM mode, LOD and LOQ of HAAs in lyophilized meat samples were at levels lower than 5 and 8 ng/g respectively, except for PhIP (Table 5).

Using external calibration, good linearity of the method was established over two orders of magnitude (r 2 = 0.998-0.999, n=18) on both solvent and matrix. Linearity was demonstrated by verifying homoscedasticity over the range tested (Bartlett test) and by applying the Mandel fitting test (Table 5. The latter showed that a quadratic regression did not provide a better fitting than the linear one.

Excellent precision in terms of intra-day repeatability was calculated, RSD% values ranging from 1 to 4% (n=5) for all the analytes. Between-day precision calculated over three days provided RSD values lower than 10% for IQ, MeIQx and PhIP, whereas greater variability was observed for the analysis of MeIQ and AαC, being calculated RSD of ca. 25% for these compounds. Finally, the calculation of the recovery functions to test trueness of the developed method was performed by spiking and analyzing lyophilized meat samples. The intercepts of the recovery functions calculated from these data were compared with 0 by means of a t-test. For all analytes the t values calculated for the intercepts were lower than the tabulated value at 95% confidence level, thus demonstrating that constant systematic errors are not present. Instead, the presence of proportional systematic errors was evidenced, since slope values of the recovery functions were demonstrated to be significantly different from 1 (p<0.05). These findings suggest that to attain accuracy in the quantitative analysis of HAAs in lyophilized meat samples the use of the internal labelled standard method is recommended.

Determination of HAAs in meat based infant foods using the LC-ESI-MS/MS method

The applicability of the LC-ESI-MS/MS method was then evaluated for the identification and determination of heterocyclic aromatic amines in meat-based infant foods.

With the aim to adding confidence to the identity of the analyte, three fragmentation transitions per compound present in the samples were monitored, thus complying with MS confirmation criteria as established by 1999/333/EG [20]. After confirmation, the quantitative assay of HAAs identified in the samples was performed using suitable calibration curves (Table 6). Among the samples investigated, norharman and harman were detected in all samples considered at concentrations up to 8.9±0.3 and 19±3 ng/g respectively, whereas MeIQx was detected only in one beef-based infant sample at a concentration value under the quantification limit. Figure 3 illustrates the LC-ESI(+)-MS/MS extract transition chromatograms of the HAAs investigated in one infant food sample.

Conclusions

This study validated the ion pair HPLC under gradient conditions and ESI(+)-MS/MS detection method for the simultaneous determination of a number of heterocyclic aromatic amines in beefbased infant foods. Even if lower detection limits are demanded in this kind of application, valuable information was achieved from the robustness study, which indicated those factors with statistical significance which influence chromatographic separation and response.

The evaluation of matrix effects by means of the recovery function allowed us to exclude the presence of constant systematic errors, not otherwise detectable by using a labelled internal standard. The validation data demonstrate that this method is convenient for routine analysis of For chromatographic and MS7MS conditions see Experimental.

Analyte

  quinoxaline), PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine), AαC (2-amino-9H-pyrido[2,3-b]indole), Isotopically labelled IQ-D 3 , 13 C 2 -MeIQx e D 3 -PhIP were from Toronto research Chemicals Inc. (Canada).

  300 range. Selected reaction monitoring (SRM) analyses were performed as follows: m/z 199→184 (collision energy, CE 26 eV), m/z 199→157 and m/z 199 →130 (CE 40 eV) for IQ, m/z 202→184 eV) for d 3 -IQ, m/z 213→198 (CE 27 eV), m/z 213→170 (CE 40 eV) and m/z 213→117 (CE 50 eV) for MeIQ, m/z 214→131 (CE 40 eV), m/z 214→199 (CE 28 eV) and m/z 214→77 (CE 60 eV) for MeIQx, m/z 215→200 (CE 30 eV) for 13 C 2 -MeIQx, m/z 169→115 (CE 32 eV), m/z 169→89 (CE 50 eV) and m/z 169→142 (CE 30 eV) for Norharman, m/z 183→115 (CE 32 eV), m/z 183→89 (CE 50 eV) and m/z 183→168 (CE 30 eV) for Harman, m/z 225→210 (CE 30 eV), m/z 225→140 and m/z 225→115 (CE 50 eV) for PhIP, m/z 228→210 (CE 32 eV) for PhIP-D 3 , m/z 184→140 (CE 30 eV), m/z 184→167 (CE 25 eV) and m/z 184→113 (CE 50 eV) for AαC. The dwell time and the interchannel delay were set at 0.25 s and 0.01 s respectively.

  b x ) and the standard deviation (s b ) of the blank signals as follows: where t is a constant of the t-Student distribution (one-sided) depending on the confidence level and the degrees of freedom (ν=n-1, n=number of measurements). Ten blank measurements were performed to calculate b x and s b . A lyophilized beef based sample extract in which the analytes

  Eleven factors (eight real factors and three dummy variables) were considered and 12 experiments were carried out. The PB design was applied in order to identify the critical factors influencing the following responses: relative peak area (analyte area/IS area) (IQ, MeIQ, MeIQx, Norharman, , AαC); resolution (Rs) (IQ, MeIQ); capacity factor (k') (IQ, MeIQ, MeIQx, Norharman, Harman, PhIP, AαC); asymmetry factor (T) (IQ, MeIQ, MeIQx, Norharman, Harman, PhIP, AαC). Eight critical factors which were believed to affect the chromatographic results were chosen, three of which referred to the mass spectrometer: the mobile phase flow-rate (ml/min), the percentage of organic modifier (acetonitrile) at the beginning and at the end of the gradient (%), the pH and the concentration of ammonium formate in the mobile phase, the column temperature (°C), the cone voltage (V), the capillary voltage (kV) and the collision energy applied in the collision cell of the mass spectrometer (eV). Experimental conditions were set selecting symmetrical values around the nominal level which reflected the variations that could be encountered in laboratory during the mobile phase preparation or due to instrumental fluctuations (9-20% of the nominal value) (Tables

  in meat products, since a good repeatability was shown and detection limits and quantification limits were in the low ng/g level. .; Moyano, E.; Puignou, L.; Galceran, M.T.; 2002. Ion-trap tandem mass spectrometry for the determination of heterocyclic amines in food J. Chromatogr. A 948: 267-281. . Toribio, B.F.; Busquets, R.; Puignou, L.; Galceran, M.T.; 2006. Heterocyclic Amines In Griddled Beef Steak Analysed Using A Single Extract Clean-Up Procedure. Food Chem. Toxycol.

Figure 1 .

 1 Figure 1. Chemical structures of heterocyclic aromatic amines investigated.

Figure 2 .

 2 Figure 2. LC-ESI(+)-MS/MS extracted ion chromatograms of a standard solution of HAAs (100 µg/l).

Figure 3 .

 3 Figure 3. LC-ESI(+)-MS/MS product ion mass spectra of a standard solution of HAAs (100 µg/l). For chromatographic and MS7MS conditions see Experimental.

Figure 4 .

 4 Figure 4. LC-ESI(+)-MS/MS extracted ion chromatograms of HAAs identified in an homogenized beef-based infant food sample extract.

Table 1

 1 Factors investigated in the Plackett-Burman design of robustness test

	Factors	Units	Level (-)	Nominal level	Level (+)
				(0)
	Mobile phase flow-rate	ml/min	0.18	0.2	0.22
	Ion pair reagent concentration	mM	2.73	3.03	3.33
	pH of the buffer	-	2.52	2.8	3.08
	Column temperature	°C	30	35	40
	Percentage organic modifier (%B) in	%	4.5	5	5.5
	the mobile phase at the start of the
	gradient
	Percentage organic modifier (%B) in	%	52	54.5	57
	the mobile phase at the end of the
	gradient
	Collision energy	eV	-*	0 *	+ *
	Cone voltage	V	-*	0*	+*
	*Different conditions were used for each analyte (see Table 3)

Table 2 .

 2 Cone voltage and collision energy values used for each analyte in the PB design.

Table 3

 3 Matrix of the Plackett-Burman design (-= low factor level, + = high factor level)

	Experiment No.	Factors a
		A	B	C	D b	E	F	G	H	I c	J	K
	1	+	+	-	+	+	+	-	-	-	+	-

Table 4

 4 Results of the robustness test in terms of the effects of the different factors on the considered responses

	Analyte				Factors
		A 1	B 1	C 1	D 1	E 1	F 1	G 1	H 1
	IQ	k'**							k'**
	MeIQ	k'**	As**	A/A IS ** k'**			k'*	k'**
								As**
	MeIQx	k'**	A/A IS **					A/A IS **
		A/A IS **
	Norharman	k'**		A/A IS **					k'**
		As**							As**
	Harman	k'**		A/A IS **
	PhIP	k'**		A/A IS **
		As**
	AαC			A/A IS **			A/A IS *

Table 6

 6 Determination of HAAs in infant meat based samples a

	Analyte	Beef a	Chicken a	Horse a	Lamb a	Rabbit a
				(ng/g)
	IQ	-b	-	-	-	-
	MeIQ	-	-	-	-	-
	MeIQx	-	-	-	-	-
	Norharman	8.9±0.3	6.3±0.5	7.4±0.3	8.7±0.3	5.4±0.2
	Harman	19±3	6.4±0.8	11.1±0.7	10.7±0.8	9.2±0.6
	PhIP	-	-	-	-	-
	AαC	-	-	-	-	-

a Three different brands. Number of samples for each brand: 3. Results not corrected by the recovery.

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

(A) mobile phase flow-rate; (B) ion-pair reagent concentration; (C) collision energy; (D) organic modifier percentage at the start of the gradient; (E) organic modifier percentage at the end of the gradient; (F) cone voltage; (G) column temperature; (H) pH of the buffer

Acknowledgements

This work was financially supported by MIUR (Ministero dell'Istruzione, dell'Università e della Ricerca, Italy), COFIN 2002 Project "Food safety. New analytical methods for the control of natural trace contaminants".

The values used for each analyte are reported in Table 2


Claire Weeks

Steven N Brown

Ignacio Vazquez

Kara Thomas

Malcolm Baxter

Paul Warriss

Toby Knowles

Thomas

Multi-element survey of allotment produce and soil in the UK

Keywords: metals, Metals Food Types: Fruit, Vegetables Allotment, arsenic, cadmium, copper, lead, mercury, metals, pollution, soft fruit, soil, vegetables

Introduction

The main purpose of this survey carried out in 2004, was to determine concentrations of metals and other elements in fruits and vegetables from 6 rural and 6 urban allotment sites situated in a variety of environments in Great Britain. An allotment is a piece of land, rented by an individual from a local authority, for growing vegetables and/or flowers. Allotments can be situated on sites that have been used previously for the disposal of industrial and/or domestic waste. In this survey, urban allotments were chosen to represent those allotments that might be located on polluted land. Urban allotments are often positioned close to roads and industrial sites. For this reason, the soil as well as the fruits and vegetables grown on allotments may contain higher levels of chemical contaminants than agricultural soils and agricultural produce. The levels of potentially harmful or beneficial elements in edible crops were measured. Levels of cadmium (Cd), chromium (Cr), copper (Cu), lead (Pb), mercury (Hg), nickel (Ni), tin (Sn), titanium (Ti) and zinc (Zn) were measured in both soil and produce. In addition, the levels of arsenic (As), manganese (Mn) and platinum (Pt) were measured in allotment produce and levels of magnesium (Mg, phosphorus (P) and potassium (K) were measured in soil. Soil pH was measured, as this can affect the solubility and uptake of mineral salts. For example at Shipham, UK, levels of cadmium in the soil are very high, yet the cadmium has low solubility owing to the high pH (7.7) and high calcium carbonate and hydrous oxide content of the soils [START_REF] Alloway | Metal availability[END_REF].

The produce samples were chosen to represent a range of edible parts and time to harvest as well as the principal classes of vegetables namely: brassicas, roots, legumes and other. Broccoli and Brussels sprouts were also chosen because they are in the ground for a relatively long period. The mineral content of agricultural crops varies according to species, variety cultivated and season e.g. cadmium [START_REF] Davis | Cadmium in agriculture, with special reference to the utilisation of sewage sludge on land[END_REF]. Some elements such as arsenic, cadmium, mercury and lead are potentially harmful to human health (MAFF 1998a,b). Levels of these were measured in soil to indicate contamination, and in produce to determine the levels that had accumulated in the plants. The presence of these elements may reflect industrial contamination or natural levels. For example, an increase in soil cadmium content may, depending on pH, result in an increased plant uptake of the metal. This has been demonstrated for soils with naturally elevated cadmium levels [START_REF] Lund | Occurrence of naturally high cadmium levels in soils and its accumulation by vegetation[END_REF] and those contaminated by non-ferrous metal mining [START_REF] Alloway | Metal availability[END_REF].

Other elements such as chromium, copper, manganese and zinc are beneficial to human health at trace levels, whereas the roles of nickel and tin in human metabolism are unclear, although they are not thought harmful at low levels [START_REF] Haas | Staying healthy with Nutrition[END_REF]. Manganese-based fuel additives might be used in the future as replacements for leaded fuel and so levels in this survey could provide baseline data.

Platinum levels may represent aerial contamination due to emissions from catalytic converters in car exhausts (Anon, 1998[START_REF] Farago | Platinum concentrations in urban road dust and soil, and in blood and urine in the United Kingdom[END_REF][START_REF] Hees | Distribution of platinum group elements (Pt, Pd, Rh) in environmental and clinical matrices: composition, analytical techniques and scientific outlook[END_REF][START_REF] Ravindra | Platinum group elements in the environment and their health risk[END_REF]. Platinum was therefore included in this survey to compare its levels of contamination in rural and urban produce.

Titanium was analysed as an indicator of soil contamination. Plants do not take up titanium internally, and so the presence of titanium in plant samples can be used to indicate the presence of soil or dust on the outer surface of the plant sample. Potassium and phosphorus levels were measured principally to indicate soil fertility, being major elements required for normal plant growth. Some previous surveys have measured levels in purchased commercially grown vegetables of the metals in the survey reported here and found them to be below statutory and guideline limits (MAFF, 1994(MAFF, , 1998c)). The survey in this paper report levels in a wide range of home-grown allotment produce in the UK.

Materials and methods

The survey was carried out on 12 allotment sites between June and December 2004. Both rural and urban allotment sites were sampled in three geographical areas: 1. North (southern Scotland and northern England), 2. Midlands (Wales and midlands of England), 3. South (southern England). A photographic record was taken of each site and the position of each sampled plot was recorded using global satellite positioning (GPS).

Produce samples

Six categories of produce (soft fruits, legumes, leafy greens, Brassicas, potatoes and onions) were collected from each site. The aim was to obtain all types of produce from three plots per allotment site but if necessary produce was sampled from plots adjacent or close to the principal sampling plots. A minimum of 200 g fresh weight was collected for each sample. Twice this amount was collected for potatoes, which were analysed both peeled and unpeeled. The number of produce samples taken totalled 251. Once collected, the fruit samples were washed with distilled water, dried using absorbent paper (Kimwipes Classic (Steel Blue), Kimberly-Clark) and then sealed in two plastic bags. The edible parts (e.g. stalks and outer leaves removed) of vegetable samples were washed then rinsed in distilled water and shaken vigorously before being homogenised into small pieces from which random samples were taken for analysis.

Soil samples

A sample of the soil substrate was collected from each plot from which produce was collected. Each plot soil sample comprised up to 15 sub-samples taken over the whole plot to a depth of 15 cm, using a soil auger. The 51 soil samples were placed in double sealed, labelled, plastic sample bags.

Analysis and quality control

Soil samples were analysed in duplicate for As, Cd, Cr, Cu, Pb, Mg, Hg, Ni, Sn, Ti, Zn and pH by Direct Laboratory Services Ltd (now Eurofins). Following air drying to constant weight at 30 o C +/-2 o C, each soil sample was initially ground and passed through a 2 mm sieve and were analysed for N, P and K to pass values onto the allotment holders and thus are not reported here. The pH was determined using a combined glass electrode on the suspension of 10 ml of soil in 25 ml of deionised water shaken for 15 min at 20 o C. Mg was extracted from 10 ml of sieved soil by shaking with 50 ml of ammonium nitrated for 30 min at 20 o C and then determined on the extract filtered through Whatman no 2 filter paper using inductively coupled plasma optical emission spectroscopy (ICP-OES). Methodology was as indicated in MAFF (1986). The other elements were analysed following further grinding to pass through a 0.5 mm sieve.

For As analysis sample (1 g) was heated in the presence of concentrated nitric acid to destroy any organic matter present. Magnesium nitrate was added to prevent losses from volatile species. The extract was then ashed for a minimum of 16 hours at 520 °C. The ash produced was dissolved with heating in 50ml HCl and arsenic determined by hydride-generation assisted atomic fluorescence.

Hg was determined using the Advanced Mercury Analyser (Leco instruments). A sample of known weight (approximately 100mg) was placed in a sampling boat, which was introduced into a decomposition tube. By controlled heating of the decomposition furnace, the sample was first dried and then thermally decomposed. The decomposition products of the sample were passed through a gold amalgamator for the selective trapping of Hg, which was released by subsequent heating of the amalgamator and determined by cold vapour Atomic Absorption Spectrometry.

The remaining metals (Cd, Cr, Cu, Ni, Pb, Sn, Ti, and Zn) were determined by ICP-OES. 1 g of 0.5 mm ground sample was heated under reflux with 15ml of aqua regia at 130°. The extract was then diluted to 50ml with 10% nitric acid and metals determined on the extract by ICP-OES.

The produce samples were prepared and analysed raw by CSL for As, Cd, Cr, Cu, Pb, Mn, Hg, Ni, Pt, Sn, Ti, and Zn. Millipore ® (18.2 MΩ) water and high purity grade reagents were used throughout. Aliquots of each test sample (3 to 4 g) plus certified reference materials (0.5 g) were digested under high pressure in quartz vessels using a nitric/hydrochloric (4:1) acid mixture (5 ml), then diluted to 10 ml with water. Aliquots (1 ml) of sample digest and also acid matched standards were diluted with internal standard solution (4 ml) containing both indium and rhodium prior to quantification by inductively coupled plasma-mass spectrometry (ICP-MS). Reagent blanks and a reagent blank spiked with a known amount of each analyte were analysed with the test samples for recovery estimate purposes. Chromium and arsenic were measured by high resolution ICP-MS to remove severe polyatomic interferences associated with the use of hydrochloric acid (ClO and ArCl respectively).

Both laboratories are UKAS accredited to ISO 17025 for a wide range of determinations in analytical chemistry and further details of analytical and quality control procedures are discussed elsewhere [START_REF] Baxter | Quality control in the multi-element analysis of foods using ICP-MS. Plasma source mass spectrometry: developments and applications[END_REF], Food Standards Agency 2004a,b) and are similar to those used previously for fungi and blackberry samples [START_REF] Weeks | Multi-element survey of wild edible fungi and blackberries in the UK[END_REF]. The limits of detection (LOD) of the elements analysed in the produce and soil are shown in Table 1. Note that in subsequent tables of results LODs were corrected for the actual weight of sample analysed.

Data handling

We have used upper bound data and unless stated otherwise, in order to include all data, where values were below the LOD, the LOD has been used to compute means and medians in tables and for graphical representations of ranges. These represent the "worst case scenario" as actual values would have been between zero and the LOD. For statistical modelling we have used zero for samples where values were below the LOD partly because of the confounding effects of different LODs for different sample sizes. For most metals the assays were very sensitive, giving small LODs, so that the results are not substantially affected by the minima used.

Statistical multi-level modelling was used to examine the extent to which levels of metals in soil were associated with levels in different types of produce. The use of multilevel models was appropriate as they account for the structures within the data of: sample within plot, within allotment site. The program MLwiN (Centre for Multilevel Modelling, University of Bristol) was used to fit the models. The effect of soil pH and the clustering effect of plots and allotment sites were built into the model. Data were initially matched at plot level and then to obtain reasonable sample sizes, the following eight categories of produce were included in the model, with unpeeled potatoes as the baseline for comparison: peeled potatoes, onions, cauliflower, cabbage, broccoli, Brussels sprouts, beans (runner, french, dwarf and green) and soft fruits (gooseberrries, raspberries, redcurrants, blackcurrants and blackberries). Grapes, peas, spinach and curly kale were left out of the models as too few samples were obtained within these categories. Natural logarithms of the data values were used in the models to achieve a normal distribution of errors and homogeneity of variance.

Results and discussion

The overall mean, median, maximum and minimum concentrations of metals in the fruits and vegetables sampled are summarised in Table 2, which shows that minimum levels were often below the LOD. Levels of the metals were similar to those found in the total diet study, in which samples were obtained through the usual retail outlets (Food Standards Agency 2004c). The range of levels measured is illustrated in Figure 1 according to concentration levels and it should be noted that the scale of the y axis is different for each graph.

Lead, cadmium, mercury and arsenic

None of the samples analysed was found to be above the corresponding regulatory limits, which are detailed in Table 3. The highest concentrations of these elements were found in individual samples of blackcurrant (0.16 mg/kg of lead), spinach (0.04 mg/kg of cadmium), and raspberries (0.03 mg/kg of arsenic). Although not a direct comparison, strawberries analysed in a Finnish study [START_REF] Tahvonen | Lead and Cadmium in some Berries and Vegetables on the Finnish Market in 1991-1993[END_REF] had a mean cadmium concentration of 0.007 mg/kg, which is similar to the overall mean concentration (Table 1) in this survey. None of the samples had a mercury concentration that was greater than the limit of detection. Table 4 summarises the results for these metals by produce, where it is important to note sampling frequency in interpretation.

Chromium, manganese, nickel, copper and zinc

None of the samples analysed were above the corresponding guideline limits for Cu or Zn. There are no guideline limits for Cr, Mn or Ni levels in vegetables or fruits; however, the levels of these metals were similar to those found in the total diet study (Food Standards Agency 2004 c) and are not considered to pose a risk to consumer safety. The highest concentrations of these metals were found in individual samples of cabbage (0.08 mg/kg of Cr) and spinach (11.65 mg/kg of Mn). This highest level of Mn was similar to the median levels in wild blackberries that we previously reported [START_REF] Weeks | Multi-element survey of wild edible fungi and blackberries in the UK[END_REF] and could be associated with use of artificial fertilisers. As shown in summary Table 5, a single sample of peas had the highest levels for Ni (0.52 mg/kg), Cu (2.27 mg/kg) and Zn (17.92 mg/kg).

Titanium, platinum and tin

There are no regulatory limits for titanium or platinum. The corresponding regulatory limit for tin in canned foods is 200 mg/kg. The highest concentrations of these metals were found in individual samples of spinach (0.45 mg/kg of titanium) and gooseberries (0.02 mg/kg of tin). Levels of platinum in the analysed samples were extremely low (generally below the LOD). Values for all produce are summarised for these three metals in Table 6, where, as in Tables 4 and5, more weight should be given to results from produce with more samples.

Rural and urban site and regional variation

In non-parametric (Kuskal-Wallis) statistical comparisons, produce grown on urban allotments had higher median levels of manganese (p=0.048) but lower levels (p=0.004) of zinc contaminants. Table 7 summarises the rural/urban variations in metal concentrations in allotment soil. There were no significant differences in levels of metals found in produce when comparing rural sites with urban sites. As the purpose of sampling from different regions was to obtain a good geographical distribution of sites, regional differences are more likely to be linked to site, however, analysis indicated (p≤0.001) lower levels of Ni and Pb in the North, higher levels of As and Sn in the Midlands and higher levels of Zn in the South. This analysis, necessary because these data did not fit the assumptions necessary for parametric statistics even after transformation, does not take into account the clustering of data by allotments. Levels of contamination were, however, low in produce from all areas and so the UK Food Standards Agency deemed there to be no food safety concern [START_REF] Fsa | Food Survey Information Sheet 01/06 Multi-element survey of allotment produce[END_REF].

Variation of levels of metals in different crops

There was both inter-and intra-produce variation in levels of metals. It is usual to find mineral content varying with crop, climate and soil [START_REF] Fageria | Growth and Mineral Nutrition of Field Crops Marcel Dekker Inc[END_REF]. Table 8 indicates for each metal those types of produce with significantly lower or higher concentration than in unpeeled potatoes, which were used as a baseline reference in the statistical model. No clear patterns emerge and neither the levels of metal in the soil nor soil pH had a detectable effect on the levels found in the produce. The majority of soil samples in the study had a pH close to neutral (median 6.90, range 5.8 to 7.8). There was a tendency for those crops with a longer growing period before harvest and permanent crops such as fruit bushes to accumulate higher levels of metals.

Conclusions

The overall accumulation of heavy metals in allotment produce was relatively low and consistent with levels reported by others in commercially produced vegetables and fruit. The variation in soil pH and soil heavy metal content within the survey was small and no significant association was detected with the levels of metals present in crops.

F

  in box and whisker plots. Metals are grouped

  Note that most minima were below the LOD. Where values were below the LOD, the LOD was used to compute means and medians which therefore are probable overestimates (see text). Shaded cells indicate means or medians thereby calculated as equal to the LOD.Note Where values were below the LOD, the LOD was used to compute means and medians which therefore are probable slight overestimates (see text). Shaded cells indicate means or medians thereby calculated as equal to the LOD.NoteWhere values were below the LOD, the LOD was used to compute means and medians which therefore are probable slight overestimates (see text). Shaded cells indicate means or medians thereby calculated as equal to the LOD.NoteWhere values were below the LOD, the LOD was used to compute means and medians which therefore are probable slight overestimates (see text). Shaded cells indicate means or medians thereby calculated as equal to the LOD.

	Food Additives and Contaminants Food Additives and Contaminants Food Additives and Contaminants		Page 14 of 16 Page 16 of 16
	Metal Mean Median Minimum Maximum Produce No. of As Cd Produce No. of Cr Cu Produce No. of Pt	Sn	Hg	Mn	Ti	Pb	Ni	Zn
	o r P e e r R e v i e w O n l y F o r P e e r R e v i e w O n l Metal Produce LOD (mg/kg) Soil LOD (mg/kg) As 0.0005 0.02 Cd 0.0006 0.08 Cr 0.006 0.2 Cu 0.006 0.12 Hg 0.0006 0.0008 Mn 0.01 n/a Ni 0.006 0.4 Pb 0.0006 1.0 Pt 0.0003 n/a Sn 0.001 0.4 Ti 0.03 0.3 Zn 0.04 0.4 Note:Values shown are based on a typical sample weights of 0.4 g (fresh weight) for produce and 1.0 g (dry matter) for soil F o r P e e r R e v i e w O n l As 0.0025 0.0016 <0.0005 samples Mean Median Max Mean Median Max Mean Median Max Mean samples Mean Median Max Mean Median Max Mean Median Max samples Mean Median Maximum Mean Median Maximum Mean Median Maximum Median Max Mean Median Max Metal Whole potatoes peeled potatoes onions cauliflower cabbage broccoli Brussels sprouts legumes 0.0253 Cd 0.0066 0.0052 <0.0006 0.0391 Cr 0.0081 0.0060 <0.006 0.0808 Cu 0.6092 0.5456 0.1193 2.2709 Hg 0.0007 0.0006 <0.0006 0.0027 Mn 1.5322 1.3406 0.2363 11.6533 Ni 0.0432 0.0247 <0.006 0.5224 Pb 0.0097 0.0040 <0.0006 0.1637 Pt 0.0003 0.0003 <0.0003 0.0004 Sn 0.0017 0.0010 <0.001 0.0200 Ti 0.0744 0.0512 <0.03 0.4537 Zn 3.5263 2.8986 0.6921 17.9246 F o r P e e r R e v i e w O n l Metal Foods Maximum permitted Level (mg/kg) Lead Potatoes and onions 0.1 Brassicas 0.3 Small fruits and berries 0.2 Potatoes and onions 0.1 Cadmium Brassicas 0.2 Small fruits and berries 0.05 Arsenic All produce 1.0 F o r P e e r R e v i e w O n l y Blackberries 2 0.0017 0.0017 0.0021 0.0040 0.0040 0.0065 0.0008 0.0008 0.0010 0.0079 0.0079 0.0095 Blackcurrants 3 0.0063 0.0074 0.0093 0.0015 0.0012 0.0022 0.0014 0.0011 0.0024 0.0726 0.0396 0.1637 Broccoli 9 0.0017 0.0020 0.0037 0.0077 0.0073 0.0137 <0.0006 <0.0006 <0.0006 0.0115 0.0089 0.0271 Brussels Sprouts 10 0.0009 0.0005 0.0020 0.0099 0.0087 0.0244 0.0007 0.0006 0.0014 0.0016 0.0006 0.0057 Cabbage 34 0.0019 0.0009 0.0129 0.0048 0.0037 0.0177 0.0008 0.0006 0.0023 0.0065 0.0010 0.0868 Cauliflower 13 0.0018 0.0012 0.0057 0.0037 0.0035 0.0062 0.0006 0.0006 0.0008 0.0134 0.0091 0.0657 Curly kale 2 0.0055 0.0055 0.0068 0.0265 0.0265 0.0365 0.0026 0.0026 0.0027 0.0182 0.0182 0.0215 Dwarf beans 6 0.0007 0.0007 0.0011 0.0023 0.0020 0.0048 0.0007 0.0006 0.0008 0.0071 0.0058 0.0137 French beans 3 0.0012 0.0005 0.0026 0.0025 0.0015 0.0051 0.0007 0.0006 0.0008 0.0096 0.0015 0.0260 Gooseberries 19 0.0016 0.0015 0.0035 0.0014 0.0011 0.0052 0.0006 0.0006 0.0008 0.0078 0.0043 0.0566 Grapes 1 0.0009 0.0009 0.0009 <0.0006 <0.0006 <0.0006 <0.0006 <0.0006 <0.0006 0.0115 0.0115 0.0115 Green beans 1 0.0044 0.0044 0.0044 0.0014 0.0014 0.0014 <0.0006 <0.0006 <0.0006 0.0638 0.0638 0.0638 Onions 37 0.0037 0.0035 0.0096 0.0102 0.0084 0.0258 0.0006 0.0006 0.0008 0.0074 0.0032 0.0461 Peas 1 0.0010 0.0010 0.0010 0.0059 0.0059 0.0059 0.0008 0.0008 0.0008 0.0008 0.0008 0.0008 Potatoes 36 0.0026 0.0023 0.0073 0.0093 0.0083 0.0244 0.0008 0.0008 0.0012 0.0133 0.0058 0.0609 Potatoes (peeled) 36 0.0014 0.0009 0.0069 0.0088 0.0080 0.0223 0.0008 0.0008 0.0008 0.0047 0.0009 0.0593 Raspberries 10 0.0101 0.0087 0.0253 0.0044 0.0045 0.0072 0.0008 0.0006 0.0017 0.0192 0.0089 0.0867 Redcurrants 1 0.0033 0.0033 0.0033 0.0032 0.0032 0.0032 <0.0006 <0.0006 <0.0006 0.0189 0.0189 0.0189 Runner beans 26 0.0018 0.0011 0.0094 0.0017 0.0015 0.0046 0.0006 0.0006 0.0008 0.0072 0.0046 0.0304 Spinach 1 0.0031 0.0031 0.0031 0.0391 0.0391 0.0391 0.0020 0.0020 0.0020 0.0286 0.0286 0.0286 F o r P e e r R e v i e w O n l y Blackberries 2 0.0107 0.0107 0.0113 0.8264 0.8264 0.9527 2.2611 2.2611 3.1774 0.0466 0.0466 0.0778 2.1525 Mean Blackcurrants 3 0.0136 0.0072 0.0277 0.9677 0.8576 1.2581 1.4710 1.5221 1.6192 0.0778 0.0473 0.1451 3.0052 Broccoli 9 0.0069 0.006 0.0107 0.6688 0.6149 0.8604 2.1933 2.2311 3.0931 0.0669 0.0555 0.1797 9.0588 Brussels Sprouts 10 0.0095 0.006 0.0381 0.6516 0.6283 0.8047 2.6595 2.6763 3.1737 0.0732 0.0598 0.1673 6.4515 Cabbage 34 0.0104 0.006 0.0808 0.3272 0.3005 0.7299 1.3203 1.2652 2.4153 0.0379 0.0250 0.1988 3.3552 Cauliflower 13 0.0067 0.006 0.0093 0.3963 0.4026 0.5727 1.4764 1.3668 1.9996 0.0209 0.0184 0.0553 4.7850 Curly kale 2 0.0137 0.0137 0.0190 0.6416 0.6416 0.7785 3.3958 3.3958 3.8675 0.0626 0.0626 0.1018 6.0348 Dwarf beans 6 0.0073 0.0070 0.0099 0.6245 0.6075 0.8684 2.1835 2.0117 3.7799 0.1343 0.0967 0.3526 3.9694 French beans 3 0.0111 0.006 0.0212 0.5038 0.5303 0.5748 1.7390 1.8494 1.9116 0.0817 0.0821 0.0830 3.6595 Gooseberries 19 0.0075 0.006 0.0117 0.4692 0.4303 0.7909 1.1636 1.1371 2.7409 0.0285 0.0278 0.0474 1.2500 Grapes 1 0.0112 0.0112 0.0112 0.5721 0.5721 0.5721 0.2463 0.2463 0.2463 0.0106 0.0106 0.0106 0.8491 Green beans 1 <0.006 <0.006 <0.006 0.4542 0.4542 0.4542 0.9068 0.9068 0.9068 0.0325 0.0325 0.0325 3.3112 Onions 37 0.0066 0.006 0.0174 0.4077 0.3724 0.7705 1.2278 1.1114 2.0885 0.0193 0.0134 0.0701 3.3135 Peas 1 0.0080 0.0080 0.0080 2.2709 2.2709 2.2709 3.1820 3.1820 3.1820 0.5224 0.5224 0.5224 17.9246 17.9246 17.9246 Median Max soft fruit 2.1525 2.1678 3.1693 3.7798 8.6071 12.8027 6.1703 9.6206 3.0239 14.2421 4.7594 8.8250 6.0348 9.4480 3.8338 5.5080 3.0865 4.9831 1.1974 1.7532 0.8491 0.8491 3.3112 3.3112 2.8274 10.3716 Potatoes 36 0.0082 0.0080 0.0192 0.9020 0.8461 1.5663 1.1900 1.1692 1.9857 0.0211 0.0162 0.0774 2.8308 2.7105 6.8063 Potatoes (peeled) 36 0.0078 0.0080 0.0165 0.9050 0.8864 1.6054 1.2214 1.1894 1.8704 0.0186 0.0143 0.0828 2.8379 2.6314 5.8020 Raspberries 10 0.0094 0.0064 0.0261 0.9745 0.9538 1.4999 2.0924 2.0459 3.4286 0.1088 0.0930 0.2044 3.7840 3.7120 4.9332 Redcurrants 1 0.0123 0.0123 0.0123 0.9111 0.9111 0.9111 1.4799 1.4799 1.4799 0.0552 0.0552 0.0552 3.3329 3.3329 3.3329 Runner beans 26 0.0067 0.0060 0.0162 0.3305 0.2974 0.6021 1.8224 1.7864 3.4905 0.0774 0.0407 0.4448 3.2108 2.9283 5.5275 Spinach 1 0.0137 0.0137 0.0137 1.3921 1.3921 1.3921 11.6533 11.6533 11.6533 0.0358 0.0358 0.0358 5.4313 5.4313 5.4313 F o r P e e r R e v i e w O n l y Blackberries 2 <0.0003 <0.0003 <0.0003 0.0025 0.0025 0.0030 0.0621 0.0621 0.0641 Blackcurrants 3 <0.0003 <0.0003 <0.0003 0.0073 0.0050 0.0157 0.1664 0.1175 0.2786 Broccoli 9 <0.0003 <0.0003 <0.0003 0.0014 0.001 0.0028 0.1237 0.1337 0.1728 Brussels Sprouts 10 <0.0003 <0.0003 <0.0003 0.0016 0.001 0.0071 0.1707 0.1375 0.4395 Cabbage 34 0.0003 0.0003 0.0004 0.0016 0.001 0.0043 0.0919 0.0737 0.2859 Cauliflower 13 0.0003 0.0003 0.0004 0.0014 0.001 0.0033 0.0877 0.0720 0.1992 Curly kale 2 <0.0003 <0.0003 <0.0003 0.0021 0.0021 0.0024 0.2475 0.2475 0.3141 Dwarf beans 6 0.0003 0.0003 0.0004 0.0023 0.0015 0.0064 0.0635 0.0562 0.1121 French beans 3 0.0003 0.0003 0.0004 0.0016 0.0011 0.0026 0.0529 0.0474 0.0864 Gooseberries 19 <0.0003 <0.0003 <0.0003 0.0031 0.0012 0.0200 0.0348 0.0283 0.0895 Grapes 1 <0.0003 <0.0003 <0.0003 0.0020 0.0020 0.0020 0.0501 0.0501 0.0501 Green beans 1 <0.0003 <0.0003 <0.0003 0.0054 0.0054 0.0054 0.0678 0.0678 0.0678 Onions 37 0.0003 0.0003 0.0004 0.0013 0.001 0.0081 0.0584 0.0469 0.2309 Peas 1 0.0004 0.0004 0.0004 0.0017 0.0017 0.0017 0.0437 0.0437 0.0437 Potatoes 36 0.0004 0.0004 0.0004 0.0012 0.001 0.0042 0.0623 0.0330 0.2401 Potatoes (peeled) 36 0.0004 0.0004 0.0004 0.0012 0.001 0.0036 0.0434 0.0330 0.2420 Raspberries 10 <0.0003 <0.0003 <0.0003 0.0033 0.0026 0.0077 0.0985 0.0776 0.1687 Redcurrants 1 <0.0003 <0.0003 <0.0003 0.0023 0.0023 0.0023 0.1200 0.1200 0.1200 Runner beans 26 0.0003 0.0003 0.0004 0.0016 0.001 0.0041 0.0613 0.0394 0.2164 Spinach 1 <0.0003 <0.0003 <0.0003 0.0021 0.0021 0.0021 0.4537 0.4537 0.4537 F o r P e e r R e v i e pH As Cd Cr Cu Hg Ni Pb Sn Ti Zn Rural 6.90 11.47 0.83 35.63 40.90 0.83 32.99 152.19 12.05 232.00 194.70 Urban 7.04 13.15 1.11 28.88 86.25 1.79 29.10 464.72 13.56 295.09 338.90 ALL 6.97 12.33 0.97 32.19 64.02 1.32 31.01 311.52 12.82 264.56 268.20 Soil LOD (mg/kg) 0.02 0.08 0.2 0.12 0.0008 n/a 0.4 1 n/a 0.4 0.3 As -6.068(0.397) -0.686(0.227)** -0.989(0.327)** -0.839(0.365)* -2.355(0.431)*** -0.770(0.227)*** Cd -0.019(.0007) -006(0.001)*** -0.006(0.001)*** -0.005(0.001)*** -0.008(0.001)*** -0.007(0.001)*** Cr -9.568(0.464) +1.293(0.586)* +2.423(0.407)*** Cu -0.036(0.169) -0.783(0.074)*** -0.733(.108)*** -1.059(0.076)*** -0.259(119)* -0.334(0.074)* -0.409(0.103)*** Hg -8.957(0.128) +0.848(0.284)** +0.525(0.195)** Mn +0.114(0.249) +0.251(0.108)* +0.604(0.120)*** +0.865(0.144)*** +0.425(0.074)*** Ni -5.065(0.712) +2.271(0.567)*** +1.414(0.650)* + 1.978(0.349)*** +1.354(0.456)** Pb -3.907(0.764) -1.733(0.283)*** -1.579(0.291)*** -4.528(0.574)*** Sn -8.358(0.366) +0.718(0.323)* +1.858(0.382)*** Ti -2.348(1.074) -1.066(0.450)* +1.505(0.765)* Zn +0.990(0.245) +0.540(0.084)*** +1.127(0.138)*** +0.531(0.165)*** +0.196(0.085)*** -0.430(117)*** F o r P F o r P e e r R e v i e w e e r O n l y R e v i e w w O n O l n l y
				y y y y

Formatted Table http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants

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


John Andrew Sidwell

Bryan G Willoughby

used in contact with food, for potential migrants

Keywords: Ion exchange resin, food contact, styrene-divinyl benzene resin, extractable substances, LC-MS, thermal desorption GC-MS, two-dimensional gas chromatography time of flight mass spectrometry (GCxGC-TOF-

The nature of extractable substances from five types of styrene-divinylbenzene ion exchange resins used in the preparation of foodstuffs was investigated. Strong acid cation resins, strong and weak base anion resins and an active carbon replacement resin were examined. These resins are used for a variety of purposes including water softening, decalcification of sugar syrups, demineralisation, removal of nitrate ions from water and decolourisation. Analysis was carried out using electrospray LC-MS and GC-MS based methodologies. Extractable substances from new resins were identified as mainly being by-products of the resin manufacturing process. Levels of extractable substances decreased with washing.

Introduction

Ion exchange resins are very high surface area materials that have the potential to contaminate food. Annex 1 to EC Regulation No 1935[START_REF] Ec | [END_REF][START_REF] Ec | [END_REF], the revised and up-dated food-contact framework regulation, now includes ion exchange resins in the list of groups of materials and articles that may be covered by specific harmonised measures.

A Council of Europe (1997) resolution on ion exchange resins (AP 97/1) includes an inventory list of substances used in the manufacture of ion exchange resins and a migration limit of 1mg/L total organic carbon in the 5 th bed volume (water) rinse solution.

In the USA, a list of ion exchange resins authorised for food-contact use, together with some restrictions, are given in the FDA regulations Title 21, Section 173.25. A number of successful food contact notifications have been granted for applications of ion exchange resins with specific functionality. [START_REF] Miers | Regulation of ion exchange resins for the food, water and beverage industries[END_REF] has reviewed US regulations on ion exchange resins. [START_REF] Utsunomiya | Government regulations on the use of ion exchange resins for the processing of potable water, food products and pharmaceuticals in Japan[END_REF] has reviewed requirements in Japan.

In this paper we report the chemical nature, extractability and source of potential extractable substances from a range of styrene divinylbenzene ion exchange resins used in contact with food, as received from the manufacturer. No pre-washing of resins as would be recommended before first-use, was undertaken.

Conceptually, ion exchange materials are insoluble acids or bases which, when converted to salts, remain insoluble.

Cation exchange materials contain fixed electronegative charges (associated with counter ions), e.g. RSO 3 -Na + , and anion exchange materials analogously have fixed electropositive charges, e.g. RN + Me 3 Cl -. Strong-acid cation exchange materials are usually based on sulphonic acids. Strongbase anion exchange materials may be quaternary ammonium hydrochlorides and weaker base types may be the tertiary amines themselves. Most ion exchange resins can be regenerated with appropriate counter ions. It is not uncommon for resin beds to be continually used and regenerated over periods of months/years before total resin replacement. Some resins are operated at elevated temperatures e.g. 70 o C.

The main applications of ion exchange resins in food processing include the treatment of water added to or used with food products, sugar decolourisation/demineralisation, isolation of proteins and enzymes from milk products and wine/spirit treatments. Of these applications, treatment of water for use with food products is the largest application.

Potential migrants from ion exchange resins include both organic and inorganic species.

The latter includes the inorganic ions involved in the process of ion exchange or of resin regeneration. The type and amount of these will be dependent on the resin types used and the manner and sequence of operation. Given the nature of these operations, and the copious use of water, it is likely that any problems from ionic impurities will be transient ones.

Organic contaminants are potentially more persistent. Organic contaminants in output streams will include those input contaminants incompletely removed by the resin and those contributed by the resin itself. The latter potentially includes: input contaminants accumulating on the resin and being re-released under breakthrough conditions, products of thermal or oxidative oxidation of the resin and residues of the chemistry of manufacture of the resin. Whilst the first two above are artefacts of the process and the conditions of operation, and are amenable to control in a properly run process, the third is outside the control of the process operators. Studies of water deionisation for the electronics industry [START_REF] Gottlieb | New advances in the production of ultra low TOC effluents with virgin resins[END_REF] have found that this third source (residues of manufacture) is the largest source of organic impurities. Such leachable organic impurities are at their highest level in brand-new resins, and gradually rinse out with time.

Materials and Methods

The ion exchange resins examined were suitably functionalised styrene-divinylbenzene resins.

Cation exchange styrene-divinylbenzene resins are acid or acid-salt functionalised. For these resins, the initial step is sulphonation of the aromatic ring, e.g.:

∼C 6 H 5 + SO 3 → ∼C 6 H 4 SO

Weak base anion exchanger

Resin C was a macroporous poly(vinylbenzyl) tertiary amine exchanger of moderate porosity, specially developed for use in the demineralization of juices from the beet, cane and liquid sugar industries.

Strong base anion exchanger

Resin D was a macroporous strong base anion resin that is specially designed for the removal of nitrates from water for potable processes. Details of the functionality of the resins examined are shown in Figure 1.

The studies undertaken on potential migrants and their extraction behaviour included an examination of: volatiles, water-soluble material from the resins, methanol soluble organic material and extractables into the EU food simulant 10% ethanol under various extraction conditions. The 10% ethanol simulant was considered to be more extractive than using distilled water alone and is recommended by the FDA for evaluating ion exchange resins used in contact with milk and milk products. A limited examination of air-dried aged resins was also undertaken.

Examination of volatile species

Volatile species from the resins (0.3g) were analysed using thermal desorption GC-MS.

A The major volatile component from the resins as received was water. Although largely removed by the cold trap, water presented some problems in the dynamic headspace work.

Examination of water and methanol soluble material

In a preliminary examination of potential migrants, 1g of each resin was immersed in 2 ml of purified water (LC-MS grade) or methanol and subjected to 30 min ultrasonic agitation. Water will extract soluble ionic and polar organic species from the resins.

Methanol was selected as being a good solvent for low molecular weight non-ionic extractable resin components. Resulting extracts were then examined by LC-MS under conditions developed using the model compounds toluene sulphonic acid and its sodium salt (for cation resins) and benzyl trimethyl ammonium chloride (for anion resins). After obtaining information on the nature of extractable species soluble in water and methanol, further migration testing with the food simulant 10% ethanol was undertaken.

A quantity of each resin (20ml) was placed in a chromatography column and immersed in 10% ethanol. The resin/10% ethanol in the column was stored for 24 hours at 40 o C.

The simulant was then drained and retained for analysis. To examine the ease of removal of extractable species by washing, each drained resin was successively washed four times with 1 bed volume (20ml) of 10% ethanol (at 40 o C) and washings retained for analysis. The 24-hour extraction period was selected to mimic a foodstuff left in contact with the resin for a prolonged period e.g. overnight.

To investigate rate of extraction of species with time, portions of each resin (20g) were stored in contact with 40 ml of 10% ethanol at 70 0 C for up to 13 days. After various time periods, aliquots of solution were removed for analysis. The temperature of 70 o C was selected as being a typical elevated resin use temperature. Additionally, to examine possible effects of ageing, portions of each resin (10g) in loosely capped 40ml glass vials were heated in an air circulating drying oven at 70 o C for 10 days. After the 10 days, 20ml of 10% ethanol was added and the solutions stored for 24 hours, after which the extract solutions were examined for the presence of any new migrants.

Extract analysis

Portions of all of the 10% ethanol extract solutions were examined by LC-MS under the same conditions as detailed for the water and methanol extracts. Five ml of each test solution was also partitioned with 2 ml of dichloromethane. Dichloromethane partition extracts of the solutions were examined by GC/GC-TOFMS (5ml of test solution partitioned with 2 ml of dichloromethane over 2 min with vigorous shaking). An Agilent

Results

Resins A and B Volatile species

Dynamic thermal desorption in helium (150°C/20 min) generated no significant detectable organic material from either resin A or B.

Water and methanol extractables

The major ionised species detected in water and their concentrations were similar to those found in the more detailed studies with 10% ethanol. For the methanol extracts, no retained non-ionic species were observed under the gradient elution conditions employed. For resin B, with GC/GC-TOF/MS, trace quantities of aromatic species were detected after 13 days contact at 70 o C with 10% ethanol. Reasonably confident assignments could be made to the three most abundant of these, and these are listed in Table 3. It is unclear whether these peaks are associated with ageing or slow rate of release from the resin. However, amounts detected were all close to the limit of detection of the instrumentation.

10% ethanol extractables

Resin C Volatile substances

For resin C, a range of volatile species was detected (Figure 3). In view of the compositional complexity of these peaks, no attempts at quantification were made. The early peaks were incompletely resolved. Carbon dioxide dominated, providing the peak at 1.85 minutes and tailing into the later peaks. Both styrene and chlorostyrene were detected at similar levels. The presence of chlorostyrene highlights the complexity of the chloromethylation reaction during functionalisation of this anionic resin, which involves Coupling may well occur here, although the excess or polymer-supported aromatic rings will ensure that the such coupling effectively serves to bind such by-products into the polymer.

In the case of styrene, the chloromethylation reaction is potentially polymerising if the ArCH 2 + cation initiates a vinyl polymerisation. Furthermore, if the cation is ortho to the vinyl group, an internal cyclisation is possible. With elimination of a proton, the two-ring aromatic indene (benzocyclopentadiene, C 9 H 8 ) would be obtained.

The trace component at 8.90 min is an aromatic of MW 116. The mass spectrum is shown in Figure 4. This might correspond to either methylphenylacetylene, CH 3 C≡CC 6 H 5 , or to indene). A feasible route to the acetylene cannot be formulated, and hence the assignment given here is to indene.

Chlorinated species are particularly easy to recognise by their isomeric distributions.

Methanol extractables

Several UV absorbing extractable constituents were detected under the 50% acetonitrile/50% 0.1% formic acid in water to 100% acetonitrile gradient conditions (Figure 6). Fragmentation of the main m/z 220 ion from the peak eluting at the solvent front showed a loss of 45 associated with NH(CH 3 ) 2 . This loss was also seen in the peak eluting at 1.1 minutes. No ESI response was found for UV absorbing peaks between 1.6 and 4.8 min indicating that they are unlikely to be very polar or ionic in nature.

10% ethanol extractable substances

For resin C, low levels of extractable species were observed by LC-MS, even after 13 days at 70 o C (much lower than Resins A and B). No species were detected by LC-MS in the wash solutions. Some extractable components were observed by GCxGC-TOFMS (Figure 7). The largest peak, at 620 sec, gave a mass spectrum identical to that seen at 11.82 min in the resin volatiles (Figure 5). This spectrum shows the spectral features of a dichloro-compound. The assumption made previously was that this was a molecule based on an HCl/formaldehyde adduct. Whilst a precise structure cannot be determined, it is interesting to note that the detection here indicates stability to hydrolysis or ethanolysis. The amount present in the extract was estimated to be at the low mg/kg level.

Ethanolysis of chloroformals would be expected to give rise to structures of the type it is tempting to speculate on whether an increase of 18 Daltons on 124/126 might be accountable as stable hydrate, it seems doubtful that such a hydrate would survive the gas chromatography.

Resin D Volatiles

The chromatogram obtained (Figure 8) shows a number of peaks with the same mass spectra. For example, three closely eluting peaks had the mass spectrum of carbon dioxide, whilst the peaks from 3.37 min upwards had the mass spectrum of triethylamine.

Although amine chromatography is not straightforward, it is not clear why such widely separated peaks should all analyse as triethylamine. Indeed the similarity amongst the spectra for the peaks from 3.37 min upwards was particularly noteworthy. None of the spectra showed any ions at higher m/z than for the molecular ions (M, M+1 and M+2) of triethylamine.

It may be significant that ethyl chloride was also found (1.95 and 1.99 min) -hence the possibility of some association in the cold trap of the dynamic headspace unit. Any delay in thermal dissociation raises the prospect of staggered injection, although the number of discrete peaks is difficult to rationalise.

Water and methanol extractables

No significant extractable species were detected in the short-term tests.

10% ethanol

By LC-MS, no abundant extractable species were detected in the 24 hr 40 o C extract from resin D. On prolonged contact at 70 o C, the concentration of extracted species increased with contact time. Three main extractable species were detected (Figure 9) The extractable substance at 6.0 min is also not chlorinated. The parent ion is at m/z 234.2 -i.e. 2 Daltons lower than for the component at 2.9 min. The fragment ions occurred at m/z 133.0, 100.1and 86.2 (consecutive losses of 101, 33, 14). The similarity with the peak at 2.9 min, coupled with difference of 2 Daltons suggests an ion related to vinylbenzytriethylammoniun ion but with two hydrogens less. By reference to the earlier These assignments are summarised in Table 5.

F
By GC/GC-TOF/MS the only main extractable substance detected was ethyl benzoateagain a possible product of ethanolysis. An increasing concentration of ethyl benzoate with storage time was noted.

Resin E Volatiles

Possible traces of acetone were seen at 2.2 min (Figure 10). No other identifications were made.

Water, methanol and 10% ethanol

Very little extractable material was observed by LC-MS in the tests at 40 o C and 70 o C. No significant extractable components were detected in the dichloromethane partition solutions. No observable differences between the control and the aged sample extractions were observed.

Conclusions

Extractable substances from the strong acid cation resins with sulphonate functionality (resins A and B) were identified as being reaction products of styrene and/or the resin backbone with sulphuric acid during manufacture of the resin. The major extractable species was thought to be the product of sulphuric acid attack on both the ring and vinyl group on styrene monomer to give a hydroxyethylsulphonic acid, CH 3 CH(OH)C 6 H 4 SO 3 H. For the weak base anion exchanger (resin C), a wide range of aromatic, aliphatic and chlorinated volatile species were found. Extractable substances into 10% ethanol included several species from the chloromethylation step of the manufacturing process.

For the strong base anion exchanger (resin D), extractable substances included an adduct of chlorostyrene with triethylamine. The expected by-product of aminolysis of chloromethylstyrene was also found as the hydrated vinylbenzyltriethylammonium ion

[CH 2 =CHC 6 H 4 CH 2 NEt 3 ] + . Other reaction products of styrene chloromethylation include a possible internal cyclisation to indene or derivatives.

The Macronet resin E showed a particularly low level of extractable substances.

In the ageing tests undertaken, no new products that could be unambiguously assigned to oxidation were detected.

Possible implications of the data on food safety

With only a few exceptions, the extractable species from the ion exchange resins tested have not been given specific migration limits or allowable daily intakes by the European Food Safety Authority (EFSA). There is also limited information available on their toxicity.

Identified species are in general predictable bye-products of the manufacturing process.

Although here the extractable species have been studied from non-washed resins as supplied by the manufacturer, it is evident that continual washing or repeat use of the resins with water or foodstuffs will reduce the level of the reported extractable species.

The Council of Europe 1997 Resolution on ion-exchange resins requires levels of total organic carbon to be less than 1 mg/kg in the 5 th bed volume water rinse, this being consistent with Industrial practice for washing resins with four bed volumes of potable water after installation or after regeneration. FDA requirements for ion-exchange resins (Title 21 Section173.25) incorporate a larger 20-bed volume rinse wash prior to testing.

All of the resins tested, as supplied, are understood to meet one or both of these test requirements.

In the resin wash tests undertaken at 40 o C, data obtained on the 5 th bed volume rinse for resins B-E indicates that migrant levels have been reduced to low µg/kg levels. For resin A, the major extractable species was determined as 0.4 mg/kg in the 5 th wash.

Therefore, data obtained on specific migrants is generally consistent with overall limits specified by the Council of Europe. Further studies are required to establish whether or not any of the identified migrants can be detected in food processed using these types of ion exchange resins.

ACKNOWLEDGEMENT

This study was funded by the UK Food Standards Agency (Project A03042: Substances migrating from ion exchange resins). Permission to publish the data is gratefully acknowledged.

F

  The LC-MS instrument used was an Agilent 1100 Series LC/MSD Trap SL. Extractable materials were examined using an Aqua C18 (Phenomenex) 3µm 125A o pore size,150 x 2.00mm column at a column oven temperature of 45 o C. The mobile phase was acetonitrile 5%, 0.1% formic acid 95% at 0.5ml/min and injection volume 5µl. Detection was by electrospray ionisation (ESI) +ve and -ve, and UV absorbance 210nm and 280nm. ESI drying gas temperature was 350 o C, nebulizer pressure was 40 psi and drying gas flow 10 L/min.For the examination of the methanol extracts for non-ionic extractables, gradient elution was employed (mobile phase 50% acetonitrile/50% 0.1% formic acid in water to

  with a LECO Pegasus III GC/GC-TOF/MS instrument was employed. The primary column was a J and W Scientific DB-5, 10m x 0.180mm, 0.18µm film thickness. The secondary column was a SGE BPX-50, 2m x 0.10mm, 0.10µm film thickness. Split injection (10:1) at 310°C was used with 1µl sample injection. The primary oven program was 40°C for 2.5 minutes, 10°C/min to 300°C and held at 300°C for 5 minutes. The secondary oven program was 75°C for 2.5 min, 10°C/min to 335°C and held at 335°C for 5 min. Mass spectra were collected in the range m/z 25-650 at 70 spectra/sec.

  and (commonly) Lewis acid catalysts. Substitution by chlorine becomes a viable side reaction under these conditions. Other side reactions of chloromethylation include those of the intermediate ArCH 2 + cation, which may itself undergo electrophilic substitution. For example in the case of reaction on low MW aromatics, coupled products of the form ArCH 2 Ar are obtained.

  C volatiles, this may be an indene derivative. The MW corresponds to the hydrated indenyltriethylammonium ion [C 9 H 7 NEt 3 .H 2 O} + .

  acid were reduced during repeated washing of the resin.

Figure

  Figure 7 Resin C, reconstructed first dimension total ion chromatogram of the dichloromethane partition solution of 7 day 10% ethanol extraction

Figure

  Figure 9 Resin D, +ve APCI chromatogram, 13 days 10% ethanol 70 o C extract

  Two styrene divinyl benzene cation resins with sulphonate functionality (Resins A and B) were analysed. Resin A complied with European standards for use in potable water applications and was in compliance with the U.S. Food and Drug Administration Code of Federal Regulations section 21, paragraph 173.25, for use in the treatment of foods for human consumption. This gel type resin is used for water softening and has ~10% cross-linking. Resin B was a macroporous cation exchange resin. It is used in the decalcification of sugar syrups during isolation of sugar from sugar beet.

	Strong acid cation exchange resins
	F o
	r
	P
	e e r
	∼C 6 H 5 + CH 3 OCH 2 Cl → ∼C 6 H 4 CH 2 Cl + CH 3 OH R e v i e w
	This is then followed by amination: ∼C 6 H 4 CH 2 Cl + NHR 2 → ∼C 6 H 4 CH 2 NHR 2 Cl ∼C 6 H 4 CH 2 Cl + NR 3 O n → ∼C 6 H 4 CH 2 NR 3 Cl Resins were analysed as supplied by the manufacturer. l y
	5

3 H Styrene-divinylbenzene anion exchange resins are obtained via chloromethylation. The reaction is complex and may be represented in a number of ways. Conceptually it is a reaction of chloromethyl ether. Chloromethyl ether is created in situ by formaldehyde/HCl reaction.

  For these resins, the principal extractable components in 10% ethanol, identified from their mass spectra and MS 2 fragmentations are given in Table1.

	components were observed. All major peaks showed UV absorption. No extractable
	oxidation products were detected in aged dried resin.
	The major extractable component observed CH 3 -CH(OH)-C 6 H 4 -SO 3 -(m/z 201) is Partitioning of the 10% ethanol extracts with dichloromethane gave solutions for more
	believed to be the product of sulphuric acid attack on both the ring and the vinyl group in detailed GC analysis. In this case, GC/GC analysis was used in conjunction with time-
	F o r the monomer (styrene) during resin manufacture. CH 2 =CHC 6 H 5 + H 2 SO 4 → CH 3 CH(OH)C 6 H 4 SO 3 H of-flight mass spectrometry (TOF/MS). By this technique, for resin A, no observable F differences between the dichloromethane control and the sample dichloromethane o r partition solutions were observed after both 13 days extraction at 70 o C or after 10 days
	air ageing at 70	P P
		e e r e e r
	R e v i e w The concentrations of the most abundant extract peak CH 3 -CH(OH)-C 6 H 4 -SO 3 -of resin 1 R e v i e w
	O n l y and 2 (calculated as sodium p-toluene sulfonate by initial comparison of UV 211nm peak areas) in the different washes are given in Table 2. Levels reduced significantly with O n washing. l y
	UV Chromatograms for the 10% ethanol extracts for resins A and B after 24 hours Aged resins
	contact at 40 o C showed similar extractable species with overall levels being lower for In 13 days contact of resins A and B with 10% ethanol at 70 o C, only a small increase in
	Sample B. The UV chromatogram (210nm) for Sample A is shown in Figure 2. concentration of extractables was observed with storage time. Extractable species
	detected by LC-MS were as found in the tests at 40 o C. No new significant extractable
		10 11

It was more abundant in the extract from the gel resin A than from the macroporous resin B. Other species found include the sulphonated products of ethyl and propylbenzene (ethylbenzene and cumene are anticipated impurities in the monomer). Sulphonation is a very aggressive reaction, and any low molecular weight aromatic components of the original resin are likely to be encountered in their sulphonated forms after functionalisation. o C. No new oxidation or degradation species were observed.

  The isotopes 17 Cl 35 and 17 Cl 37 should be present in the ratio of 3:1. Thus, chlorostyrene

	reference spectrum was found for this molecule, although the base peak at m/z 45 is
	seen in related species chloromethyl derivatives, including chloromethyl ether.
	Dichloro-species have three principal molecular ions, i.e: M, M+2, M+4 -in the
	abundance ratios 3:2:1. Thus BCME (bis-chloromethyl ether) would have molecular ions
	F o r at m/z 114, 116 and 118. No BCME was found in resin C. F o One component, which does show this characteristic dichloro-pattern, is that at 11.82 r
	P e e r min (Figure 5). This shows the 3:2:1 ratio at m/z 213/215/217, which is repeated at m/z P values 183/185/187, 153/155/156 and 124/126/128. There is also what appears to be a monochloro-fragment at m/z 89 and 91 (i.e. loss of Cl from 124/126/128). It is not easy to invoke any specific structure here. If the molecular ion pattern is 213/215/217 then e e r
	R e v i e w the initial fragmentation is likely to be the sequential loss of two molecules of formaldehyde. Again this appears to be another molecule based on an HCl/formaldehyde adduct. The spectrum for the major peak at 7.89 min gives a reasonable fit for a C4-substituted R e v i e w
	O n l y cyclohexane (C 10 H 20 , MW 140) or dimedone (5,5-dimethyl-1,3-cyclohexadione, C 8 H 12 O 2 , MW 140). What appear to be other cyclohexyl derivatives are seen at 8.18 min and O n 10.06 min. Traces of solvents such as tetrachloroethylene (possibly used to swell the polymer prior to derivatisation) and decane/undecane (possibly an extender used in cell l y
	has two principal molecular ions (at m/z 138 and 140) in roughly the ratio 3:1. The large structure development) were found, but the apparent presence of a range of
	peak at 7.26 min has molecular ions in this ratio. They are at m/z 110 and 112 and there cyclohexane derivatives remains a mystery. The solvent composition for the
	is a significant fragment at m/z 75 (M -Cl). The base peak is at m/z 45, which is chloromethylation and the amination manufacturing steps is unknown. A summary of the
	possibly CH 3 OCH 2 assignments for the volatiles from resin C is given in Table 4.
	13 14

+

. In this case, a credible assignment is chloromethyl methyl formal, CH 3 OCH 2 OCH 2 Cl. This is an adduct of HCl and two molecules of formaldehyde. No

  The peak at 0.9 min is characterized by molecular ions at m/z 239 and 241 in abundances characteristic of a monochloro-compound. Fragmentation of the 239 ion, produced an ion of mass 102 (loss of 137). This suggests that this peak is an adduct of chlorostyrene (mol wt 138/140) with triethylamine (MW 101). By analogy with other quaternary ammonium salts, this might be envisaged as the salt [C 8 H 7 NEt 3 ] + [Cl] -, however, full dissociation is unlikely as the C-Cl bond, when the chlorine is bound to an aromatic carbon, is too strong for ionisation. So the adduct is probably better described as [CH 2 =CHC 6 H 4 Cl][NEt 3 ] -polar enough to be extracted by the aqueous ethanol, but not CH 2 =CHC 6 H 4 CH 2 Cl CH 2 =CHC 6 H 4 CH 2 Cl + NEt 3 → [CH 2 =CHC 6 H 4 CH 2 NEt 3 ] + Cl -

		o
		r
	truly ionic.	P
	e The peak at 2.9 min was not chlorinated. The 236.1 ion observed, fragmented into ions e r of m/z 135.0, 100.1, 86.2 (consecutive losses of 101, 35, 14). This suggests that this
	peak could be the hydrated ion of the triethylammonium salt of methylstyrene R [CH 2 =CHC 6 H 4 CH 2 NEt 3 ] + (i.e. the hydrated vinylbenzytriethylammoniun ion) with loss of e v triethylamine (m/z 101) giving the m/z 135 fragment. This is the expected by-product of i w e aminolysis of chloromethylstyrene.
	CH 2 =CHC 6 H 5 + CH 2 O/HCl →	n O
			l y
		16

Table 2 Effect of washing on levels of the main extractable species from resins A and B

 2

	o
	r
	P
	e e r
	R e v i e w
	O n
	l y
	20

Table 3 Extractable components from Resin B detected by GCxGC-TOFMS Retention time (sec)

 3

	Peak assignment
	594.83 , 2.53 butylphenol
	899.74 , 2.66 1,1,3-trimethyl-3-phenylindane (alpha-methylstyrene dimer)
	1189.64 ,
	4,4'-dihydroxydiphenylpropane
	3.75

Table 5 . Extractables from resin D after 7 days in 10% ethanol Retention time (min)

 5

		Peak assignment
	0.9	triethylamine-chlorostyrene adduct
	2.9	hydrated vinylbenzylriethylammonium ion [C 9 H 9 NEt 3 .H 2 O} +
	6.0	hydrated indenyltriethylammonium ion [C 9 H 7 NEt 3 .H 2 O} +

UV chromatogram (210nm) of 10% ethanol extract (24 hours at 40 o C) Page 27 of 35 http

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	AK0461-018															Scan EI+
	100	1.85														7.76e6 TIC
		2.10
					7.89
	%	F o r 2.17 7.26 2.54 9.10 8.18 10.06 10.16 F o 8.27 r	11.82
	0	2.00	4.00	6.00 6.79 5.89	8.00	P 10.00 12.00 12.54 P	14.00	16.00	18.00	20.00	22.00	24.00	26.00	28.00	Time
	e e r Figure 3 Resin C -Total ion chromatogram of volatiles e e r
									R e v i e w R e v i e w
															O n O n
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29 Figure 4 Resin C volatiles -Mass spectrum of small peak at 8.9 min

	AK0461-018 1168 (8.905) Cm (1167:1169-(1164:1166+1170:1173))																							Scan EI+
	100								115																									9.37e4
										116
	%
					63
					62			89
			39	50
	0	27	38 28	49	58	74 73 65	75 85	90	113 98 99	117 118	125 142	147	165	170	184	190	202	209 228	231	245	260	273 275	295	305	314	324 332		348 356	371	386	395	405	420	434	445
	20		40		60		80		100	120	140		160		180		200	220		240	260	280	300	320	340	360		380	400	420	440

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30 Figure 5 Resin C Volatiles -Mass spectrum of peak at 11.82 min

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	Intens.
	m AU
		4
	AK0461-018 1528 (11.819)	Scan EI+
	100		61	5.86e5
		2
	%	0	53 F o
			r
	20 0 27 29 31 40 39 45 51 -2 0 Figure 6 Resin C 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 P 89 88 75 62 77 119 91 103 105 124 126 183 128 155 153 157 185 213 215 253 251 325 310 292 258 399 339 353 383 368 418 424 e 2 4 6 8 10 12 14 e r	16	m/z	18	T im e [m i n]
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UV chromatogram (280nm) of methanol extract Page 31 of 35 http

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	Intens.
	AK0461-021 7 x10															Scan EI+
	100 1.50			3.38													TIC 9.29e6
	1.25
	1.00
	% 0.75		1.80	3.73 o F	10.82
	0.50					r
			2.12				10.37
	0.25						8.92 P 7.31
	0 0.00	0	2.00	4.00	1	6.00	8.00 2	10.00 e 12.00 14.00 r 16.00 3 4 e	18.00	5	20.00	22.00	6	24.00	26.00 7	28.00	Time 8	9	T im e [m in]
								R e v i e w
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