Michael J Scotter
email: m.scotter@csl.gov.uk

Laurence Castle

Dominic P T Roberts

A method for the estimation of sucrose esters (E473) in foods using gas chromatography-mass spectrometry

Keywords: Bakery products, Confectionary, Processed foods

A method for the estimation of sucrose

mixtures that comprise this additive involved, selective solvent extraction of the intact esters 4 using a mixture of tetrahydrofuran and ethyl acetate, alkaline hydrolysis of the esters to 5 liberate sucrose, and then GC-MS measurement of the liberated sucrose using GC-MS after 6 acidic hydrolysis to glucose and fructose and then silylation. The method was developed to 7 aid future estimates of intake of this food additive. The method determines the total sucrose 8 esters content of a food sample and does not attempt to discrimination between individual 9 sucrose esters when present as a mixture in a food sample. A single (average) factor is used 10 to convert the liberated sucrose content into sucrose ester content. The method was applied to 11 analysis of 8 different food types (including bakery wares, sugar confectionery, dairy product, 12 margarine, meat pies and a sauce) spiked with 0.5 to 1% of a mixture of 3 sucrose esters that 13 spanned the hydrophilic/lipophilic balance (HLB) range 1-16. The limit of quantification was 14 around 50 mg/kg, which is more than adequate for these additives. The analytical recovery 15 was 73 -106% with an average of 91%. The precision of the method (RSD) was 6 -18% (n= 16 3 to 20 for each food type) with an average RSD of 11%. The main analytical uncertainty is 17 the conversion factor used to express sucrose ester content from the amount of sucrose 18 liberated. The method is also applicable to sucroglycerides (E474). 19 20 Keywords: food, additives, emulsifiers, analysis, sucrose esters, E473, E474, gas 21 chromatography, mass spectrometry, GC-MS, intake estimates. 22 Sucrose esters of fatty acids (SuE, E473) are a group of chemically similar additives 2 controlled in Europe by Directive 95/2/EC (European Parliament, 1995). They may be used 3 as emulsifiers in a wide range of foods, including bakery products, cakes, beverage whiteners, 4 desserts, confectionery, sauces and dairy-based drinks. Sucrose esters are defined in Directive 5 96/77/EC (as amended) as the mono-, di-and tri-esters of sucrose with fatty acids occurring in 6 food fats and oils (EU, 1998). They may be prepared from sucrose and the methyl and ethyl 7 esters of food fatty acids or by extraction from sucroglycerides. Sucroglycerides (E474) are 8 produced by reacting sucrose with edible fat or oil to produce a similar mixture of SuE with 9 fatty acids together with residual mono-, di-and tri-glycerides from fat or oil (Figure 1). For 10 the purposes of this work, E473 and E474 are considered to be chemically equivalent. 11 12 These two groups of emulsifiers comprise a range of products from stiff gels, soft solids to 13 white or slightly greyish-white powders. They are sparingly soluble in water but soluble in 14 ethanol and other organic solvents, mineral oil and vegetable oils. In a review of food additive intake in the European Union (EU, 2001), several additives 1 including E473 were highlighted as in need of refined intake estimates using actual 2 occurrence and consumption data. Therefore a method was needed to measure levels of E473 3 in foods. 4 5

Review of published analytical methods 6

The quantitative analysis of these emulsifiers in food is difficult, primarily because: (i) the 7 multiple components are similar in structure, (ii) most commercial sources of emulsifiers are 8 quite heterogeneous and (iii) emulsifiers can be difficult to extract from foods that contain 9 significant amounts of lipid, starch and/or protein. Several analytical strategies have been 10

employed for the analysis of SuE as such, but there are very few literature references to their 11 determination in foods. 12 13 Analysis of the intact esters 14 Since SuE are liposoluble their measurement in foods requires differentiation of SuE from 15 other lipids/liposoluble materials. The JECFA specifications describe an assay by 16 solubilization in tetrahydrofuran followed by gel permeation chromatography with refractive 17 index (RI) detection (JECFA, 1995). This method is however applicable only to relatively 18 pure and concentrated SuE additive formulations. SuE can be determined by reversed-phase 19 (RP) high-performance liquid chromatography (HPLC) although interferences from fatty acid 20 esters of glycerol were reported (Brauen, Davidson and Salminen, 1990). Sucrose polyesters 21 are a related class of emulsifiers that are synthesized by esterifying 6 to 8 of the hydroxyl 22 groups of sucrose with fatty acids and they exhibit similar physicochemical properties to SuE. [START_REF]Food 4 Additives. Sucrose esters of fatty acids[END_REF] Methods of analysis for sucrose polyesters based on the direct analysis of the liposoluble 24 fraction have been developed using supercritical fluid chromatography (Chester, Innis and 25 Chase, Akoh and Eitenmiller, 1995). Thin layer chromatography (TLC) has been used (Xie et 2 al., 2000) but RP-HPLC is reported to be superior. RP-HPLC-UV was used for the separation 3 and determination of 18 different SuE and sucrose polyesters, unreacted sugar and other fatty 4 acid esters (Yang et al., 2000). RP-HPLC with light-scattering detection and LC-MS 5 confirmation was used for the separation of SuE isomers (Moh, Tang and Tan; 2000). 6 7 Specific methods for the analysis of mono-and di-SuE have been developed, notably that 8 reported by Murakami, Marayame and Niiya (1989) who determined mono-and di-SuE in 9 food by HPLC with UV detection. Samples were extracted with THF, the extracts purified by 10 chromatography on silica gel and derivatised with dinitrobenzoyl chloride. SuE were reported 11 to be detected without interference. Recoveries were 75-80% and the detection limit was 10 12 mg/kg for each ester. High temperature (400°C) GC-FID has been used to analyse SuE as 13 trimethylsilyl derivatives (Karrer and Herberg, 1992). The method performance was checked 14 by supercritical fluid chromatography (SFC) and direct chemical ionisation MS of standard 15 compounds, and allowed high molecular weight carbohydrate derivatives (1500 Da) to be 16 determined whilst maintaining high separation efficiency. 17 the mono-SuEs were characterised using 6 fragment ions. The analyte peaks were clearly 1 separated from acetylated derivatives of sucrose, tocopherols, diglycerides and triglycerides. 2 3 None of these direct methods were considered here to be suitable for the routine analysis of 4 SuE in different types of foods. 5 6

Analysis following hydrolysis 7

Other workers have used indirect approaches following hydrolysis. The liposoluble fraction of 8 foodstuffs was extracted with petroleum ether, which was then saponified by methanolic 9 KOH (Drake, Nagel and Swanson, 1984). The liberated sucrose was extracted with water and 10 quantified colorimetrically using a phenol-sulfuric acid test. Koh et al. (1997) determined the 11 composition of commercial SuEs using TLC and gas chromatography (GC) and subsequently 12 developed an indirect method for their analysis in certain foods (crackers, sausage and ice 13 cream) using ion-exchange HPLC with pulsed amperometric detection (PAD) and RI 14 detection of free sucrose following alkaline hydrolysis. The liposoluble fraction of the foods 15 was extracted with a selective mixed solvent of ethyl acetate (EtOAc) and tetrahydrofuran 16 (THF) (7:3 v/v), and purified using diol SPE cartridges prior to hydrolysis. The recovery was 17 86-99% and the limit of detection for sucrose using PAD was 4 mg/kg. Such methods 18 however did not discriminate between the different mono-, di-and tri-esters nor did any 19 allow for identification of the fatty acid residues associated with the SuE. 20 21 For the purposes of estimating concentrations of SuE in a wide range of foods for intake 22 estimates, it was considered that the degradation/analysis approach was likely to be the most 23

Materials and methods 1

Reagents were of recognized analytical grade unless specified otherwise. Tri-Sil ® Z was obtained 2 from Perbio Science UK Ltd., Cramlington, UK. Fourteen sucrose ester materials were obtained 3 from a European (4 samples) and a Japanese (10 samples) manufacturer. These comprised 4 samples with hydrophilic-lipophilic balance (HLB) values ranging from 1 to 16 (Table 1). 5 6 Extraction 7

Food samples were homogenised prior to weighing 1.0 g (or 0.25 g for margarine) portions 8 directly into a 40 ml glass vial. Samples containing high amounts of gelatin and/or starch were 9 mixed with 2 mL of water prior to solvent extraction. Mixed extraction solvent (tetrahydrofuran: 10 ethyl acetate , 7:3 v/v, 10.0 mL) was added. The mixture was blended using an Ultra Turrax  11 model T-25 probe at 4,000 to 10,000 rpm for 30 seconds. The vial was placed on a horizontal 12 shaker for 30 minutes, then centrifuged at 700-1000 x g at 20°C for 5 minutes. The supernatant 13 was transferred to a 40 ml glass vial containing saturated sodium sulfate solution (10 mL) and the 14 vial placed on the shaker for 20 minutes. The mixture was centrifuged at 700-1000 x g at 20°C 15 for 5 minutes and the supernatant transferred to a 12 ml vial. 16 17

Alkaline hydrolysis to liberate sucrose 18

The solvent was removed under a stream of nitrogen on a dry-block heater set at 70 ± 2°C. 19

The residue was not allowed to remain in the dry state any longer than necessary. A mixed 20 solvent was added (butanol: hexane, 1:1 v/v, 2 mL) and the vial shaken to dissolve the 21 residue. Hydrolysis of the esters was achieved by adding sodium hydroxide solution (4M, 2 22 mL) and shaking for 40 minutes. The lower aqueous layer was transferred to a 4 ml glass vial

Acidic hydrolysis to glucose and fructose and their derivatisation 1

A portion (100 µL) of the hydrolysed extract was placed in a 4 mL glass vial along with 2 fucose internal standard solution (0.1 mg/mL,100 µL) and hydrochloric acid (6M, 125 µL). 3

The solvent was removed at 50°C under a stream of nitrogen until a dry residue was obtained. 4

The addition of a small volume (1-2mL) of acetonitrile to the vial aided this drying. 5

Immediately on drying, Tri-Sil ® Z derivatising agent (250 µL) was added to the vial which 6 was then capped and heated at 70°C for 30 minutes. The derivatised sample was then cooled 7 and transferred to a GC-MS autosampler vial. 8 9

GC-MS analysis 10

The GC-MS was a Hewlett-Packard Model 5890 series II capillary GC with model 7673 11 autosampler and split/splitless injector, model 5971 mass selective detector (Agilent 12 Tecnologies UK, Stockport). The column was an HP-5MS (5%-Phenyl-methylpolysiloxane) 13 fused silica column, of dimensions 30m x 0.25mm i.d. x 0.25µm d.f. (Esslab, Hadleigh, UK). 14

The column temperature was programmed from 80°C (held 1 min) at 5°C/min to 200°C (held 15 3 min). The carrier gas was helium at 1ml/min. The injection volume was 1µl in splitless 16 mode at 280°C. The MS was operated in impact mode and the ions monitored were m/z 204 17 and 217. 18 19

Calculation of sucrose levels in samples 20

Due to isomeric effects, 2 main peaks were observed for each of the substances monitored -21 glucose and fructose as analytes and fucose as the internal standard. The 2 peaks were 22 integrated and the areas summed to give a single area value for each substance. Using sucrose 23 solutions of 10 and 0.1 mg/mL in water, a series of calibration standards was prepared by 24 The sample/internal standard peak area ratio values derived for calibration standards were 4 plotted against the concentration of sucrose. The correlation coefficient was 0.996 or better. 5

The peak area ratios for the samples were calculated and the sucrose concentration 6 interpolated from the calibration graph. The sucrose ester content was calculated using the 7 following equation: Sucrose ester content (mg/kg) = Sucrose content (mg/kg) x 4.5. 8 9

Results and discussion 10

Since E473 (and E474) emulsifiers are not single substances, it was necessary to chemically 11 characterise appropriate reference materials and a range of additive formulations. The key 12 aspects of available analytical methods were taken forward for development whilst ensuring 13 that the developed method would be readily accessible to a wide base of users. The analytical 14 strategy was based on selective solvent extraction of SuE from food followed by a clean up 15 procedure to remove unwanted co-extractives using solvent partition. Candidate end 16 determination methods considered included colorimetry and gas chromatography, as these 17 offered a lower degree of complexity and were considered as relatively easy to roll out to 18 other laboratories. It was recognised that analysis of intact SuE would be complicated by the 19 wide range sucrose esters that may be present, so hydrolysis of SuE and measurement of the 20 released sucrose was investigated, with derivatisation and gas chromatography considered the 21 upper limit of desired complexity for this aspect. 22

23

The most appropriate way to express the results in terms of SuE content based on sucrose 24 measured, is to apply a conversion factor, based on the sucrose contents of available standard 25 SuE. Theoretically, SuE over the entire HLB range [START_REF] Birch | Determination of sucrose polyesters by high performance gel 2 permeation chromatography[END_REF](2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16) could contain between ca. 22.3 and 1 52.2% sucrose by weight. The calculated arithmetic mean conversion factor falls around the 2 centre of the HLB scale and could therefore be used as an 'average' conversion factor. 3 However, given that the main purpose of determining the SuE content of foods is for 4 estimating intakes, the use of a conversion factor for higher esters of sucrose (low HLB) 5 would seem appropriate because it would incur an overestimation of the SuE content. Thus a 6 factor of 4.5 was chosen to convert from sucrose content to SuE content (i.e. a ratio of ca. 7 100/22.3). This give an upper-bound estimate of SuE content in foods. If information was 8 available for a particular type of SuE used then the more relevant conversion factors could be 9 used. 10 11

Preparation and homogeneity testing of an in-house validation material (IHVM) 12

A pack of (6) mini Cornish pasties were finely chopped in a food processor and a portion (500 13 g) was mixed with water (200 mL) to facilitate further homogenisation. A 250 g portion of the 14 homogenate was spiked with a mixture of 3 different commercial sucrose esters dissolved in 15 THF:EtOAc (7:3 v/v). The spiking details are given in Table 2. The total SuE content was 16 6,060 mg/kg. The IHVM was stored at -20°C. 17 range based on a mean recovery of 81 % (Figure 3) for use in subsequent batch analyses of 4 test samples. An example of a chromatogram obtained from the IHVM is shown in Figure 4. 5 6 Analysis of the blank IHVM (before spiking) showed that the saturated sodium sulfate / 7 solvent partition was effective at removing free sucrose present at high level and that co-8 extracted lipid from the reasonably high fat product used to prepare the IHVM (Cornish 9 pasty) did not interfere with the subsequent hydrolysis, derivatisation and GC-MS stages. The 10 results from the repeat analyses show that the IHVM was homogeneous and that the analysis 11 was repeatable. 12 13

Analysis of food samples 14

The application range for sucrose esters was categorized into 5 main food commodity groups 15 and at least one commodity was selected to cover the range of applications stated by the major 16 manufacturer. Since SuE usage in the UK is very sparse, commodities were spiked at levels in 17 line with those appropriate to the maximum permitted. Samples were purchased locally and 18 the results are given in Table 4. Recoveries of SuE spiked into foodstuffs at a concentration of 19 between 0.5 to 1.0 % were in the range 73% (soft mint sweets) to 106% (ice cream). The 20 mean recovery for all commodities was 91%, with a mean RSD of 11%. 21

22

Quality assurance 23

Samples were analysed on a batch basis with at least one IHVM sample. The recovery values for 24 the IHVM shown on the chart in Figure 3 show that the method was essentially under analytical 25 control. The limit of quantitation (LOQ, s:n 10:1) was 12 mg/kg as sucrose, equivalent to 54 1 mg/kg sucrose ester. The GC-MS chromatograms were free of any significant interferences. 2 During method development, some sample extracts exhibited poor recovery for the internal 3 standard, this was in most instances due to insufficient drying of the acidic hydrolysate prior to 4 derivatisation. 5 6

The sample matrices that presented the most difficulties were marshmallow biscuit and 7 margarine. The presence of gelatine and starch in the marshmallow biscuit product caused the 8 sample to congeal during the initial solvent extraction, thereby compromising SuE extraction. 9

This was ameliorated by the addition of 2 mL of water to the 1 g sample and homogenising 10 prior to solvent extraction. Although the mean recovery of 97% for this commodity was very 11 good, the higher RSD value obtained (18%) reflected the difficulty with analysis of this 12 matrix. The high fat content of margarine effectively neutralised the sodium hydroxide and so 13 the hydrolysis of SuE failed. By taking a smaller sample size (0.25 rather than 1 g) the 14 analysis of margarine was successful and the recovery was very good (mean 101%). 15 16The results from this study suggest that E473 and E474 have very limited use in the UK, 17 borne out by the difficulty in obtaining samples. Nevertheless, the developed method provides 18 a means by which the total sucrose ester contents of a limited range of food commodities may 19 be estimated at levels well below the maximum levels specified in the Council Directive. This 20 is particularly relevant to the generation of intake data for E473/474. 21

22

The factor for converting the free sucrose content to SuE content was based on the SuE with 23 the lowest sucrose content (i.e. higher degree of esterification) and was adopted on the 24 grounds that its use effectively gives an over estimation of lower esters but does err on the 25 side of caution with respect to public protection. The alternative was to express the results as a 1 range (based on factors derived from the highest and lowest theoretical sucrose contents 2 across the HLB range). This was considered to be less appropriate for monitoring SuE intake.

23 (

 23 SuE and sucroglycerides 15 are permitted in a wide range of foodstuffs under Annex IV of Directive 95/2/EC. SuE are 16 also permitted under Annex V as a carrier for food colours and fat-soluble antioxidants, and 17 under Annex VI Parts 1 and 2. 18 19 These additives have been considered several times by the Joint FAO/WHO Expert 20Committee on Food Additives (JECFA) and have an ADI of 0-30 mg/kg bw, expressed as 21 sucrose ester content(JECFA, 2004). The European Food Safety Authority has also 22 evaluated E473 and E474 recently and a Group ADI of 0-30 mg/kg bw was established

  [START_REF] Chester | Separation of sucrose polyesters by capillary 12 supercritical fluid chromatography / flame ionisation detection with robot-pulled capillary 13 restrictors[END_REF] or high performance gel permeation chromatography[START_REF] Birch | Determination of sucrose polyesters by high performance gel 2 permeation chromatography[END_REF] 1

18

  More recently,[START_REF] Uematsu | Determination of 8 sucrose esters of fatty acids in food additive premixes by gas chromatography and 9 confirmation of identity by gas chromatography-mass spectrometry[END_REF] have reported a GC/ GC-MS method for sucrose 19 monoesters of fatty acids and sucrose acetate isobutyrate (SAIB) in food additive premixes 20 and some foods. A reverse-phase SPE cartridge was used to extract mono-SuE and SAIB 21 from aqueous samples. A silica-gel SPE cartridge removed diglycerides from mono-SuE in 22 THF extracts of solid / fatty samples. Mono-SuEs based on fatty acid residues of C 12 , C 14 , C 16 , 23 C 18 and C 18:1 were acetylated and determined using wide-bore capillary GC with splitless 24 injection and flame-ionization detection. Peak identities were confirmed using GC-MS where 25

  to 0, 1, 5, 10, 25, 50 and 125 µg of sucrose into separate 4 mL 1 vials. Internal standard and HCl were added and the standards then treated as for the samples. 2 3

18 Sucrose

 18 was spiked into Cornish pasty homogenate (i.e. blank IHVM) at 1 and 20 mg/g and 19 this was analysed to determine the efficacy of free sucrose removal from the organic extract 20 prior to hydrolysis. No sucrose was detected in either sample and no peaks due to sucrose 21 were observed in the chromatogram from a reagent blank. An example chromatogram from 22 blank IHVM spiked with 20 mg/g sucrose is shown in Figure2. Two batches of ten replicate 23 sub-samples of the IHVM were extracted on two separate days. The results are given in 24 the mean recovery figures from each batch showed that they were not 2 significantly different (t-test at p=0.05). A control chart was established over the +/-2 s.d. 3

Figure 1 .

 1 Figure 1. Chemical structure of sucrose esters. R 1 -R 3 are usually mixed residues of C 14:0 ,

Figure 2 .

 2 Figure 2. GC-MS analysis of blank IHVM spiked with sucrose at 20 mg/g. The absence of analyte peaks demonstrates that the clean-up was effective. The peaks between 17 and 19 minutes are fucose internal standard.

Figure 3 .

 3 Figure 3. Control chart for batch analysis of sucrose esters based on the IHVM.

Figure 4 .

 4 Figure 4. GC-MS analysis of IHVM extract. Peak identification: Fructose (20.9 and 21.1 min) and glucose (21.8, 22.7 and 24.6 min).

3 4 Page 13 of 24 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants

  Council of the European Union, Report from the Commission on dietary food additive intake 1

		Page 19 of 24
	2	in the European Union, document DENLEG 47, 2001.
	3
	4	Drake, M., Nagel, C., and Swanson, B., 1994, Sucrose polyester content in foods by a
	5	colorimetric method. Journal of Food Science, 59, 655-656.
	6
	7 8	F o r F o r F o r European Parliament and Council Directive 95/2/EC (OJ No. L61, 18.3.95) on food additives other than colours and sweeteners (as amended). F o r F o r F o r
	9 10	P e P e P e Karrer, R., and Herberg, H., 1992, Analysis of sucrose fatty acid esters by high-temperature P P P e e e
	11 12	e r e r e r gas chromatography. Journal of High Resolution Chromatography, 15, 585-589. e r e r e r
	13	R R R Koh, T., Yada, T., Semma, M., Ito, Y., Kato, Y., and Nakamura, M., 1997, A determination R R R
	14	e e e of sucrose ester of fatty acids in foods by IC. Japanese Journal of Food Chemistry, 4 (2). e e e
	15 16 17	v i e w v i e w v i e w Moh, M., Tang, T., and Tan, G., 2000, Improved separation of sucrose ester isomers using v i e v i e v i e gradient high performance liquid chromatography with evaporative scattering detection. Food w w w
	18 19 20	O n l y O n l y O n l y Murakami, C., Maruyama, T., and Niiya, I., 1989, Determination of sucrose esters of fatty Chemistry, 69, 105-110. O n l O n l O n l y y y
	21	acids by high performance liquid chromatography. Shokuhin Eisegaku Zasshi, 30, 306-313.
	22
	23	Opinion of the European Food Safety Authority (EFSA) Scientific Panel on Food Additives,
	24	Flavourings, processing Aids and materials in Contact With Food on Sucrose esters of fatty

Table 2 .

 2 IHVM spiking details

	Sucrose	HLB	Amount added	Spike level in IHVM
	ester	value	(mg)	(mg/kg)
	L1695	16	504	2016
	S570	5	502	2008
	S170	1	509	2036

Table 3 .

 3 Homogeneity and recovery of SuE in IHVM (n=10)

	Batch No.	Mean	Standard	RSD (%)
		recovery (%)*	deviation
	1 (week 1)	89	5.8	6
	2 (week 2)	73	5.8	6
	[*Based on a mean theoretical sucrose content of 45%]

Table 4 .

 4 Recovery of SuE from spiked food commodities.

	Category	Sample	SuE Spike	n	Mean	RSD (%)
			level (%)		recovery
					(%)
	1. Fine bakery	(1) Fruit scone	0.5	6	88	6
	wares	(2) Marshmallow biscuit (i)	0.75	3	97	18
		Marshmallow biscuit (ii)	0.75	6	93	15
	2. Sugar	Soft mint sweets	0.5	6	73	12
	confectionery
	3. Dairy	Ice-cream	0.75	3	106	8
	products
	4. Processed	Margarine	1.0	6	101	12
	fats & oils
	5. Other	(1) Sausage roll	1.0	5	80	10
		(2) Cornish pastie	0.6	20	81	6
		(3) Stir-fry sauce	0.5	6	96	11

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Acknowledgements 1

The authors would like to acknowledge the kind help of Mitsubishi Chemical (U.K.) Plc and 2

support for this work was 3 provided by the UK Food Standards Agency (FSA).


Michael G Kontominas

Antonios E Goulas

Anastasia V Badeka

A Nerantzaki

Migration and sensory properties of plastics based nets used as food contacting materials under ambient and high temperature heating conditions

Migration and sensory properties of plastics based nets used as food contacting materials under ambient and high temperature heating conditions

determination of the volatile compounds' profile in table water exposed to the netting materials using SPME-GC/MS. Test conditions for packaging material/food simulant contact and method of overall migration analysis were according to the EU directives 90/128 and 2002/72. The results showed that for both PET and polyethylene based netting materials overall migration into distilled water ranged between 11.5 and 48.5 mg/L, well below the upper limit (60 mg/L) for overall migration from plastics packaging materials set by the EU.

The overall migration from netting materials into isooctane ranged between 38.0 and 624.0 mg/L, both below and above the EU upper limit for migration. Sensory tests involving contact of representative samples with table water under refluxing (100 o C/4h) conditions showed a number of the netting materials produced both off-odor and/or taste as well as discoloration of the food simulant rendering such materials unfit for the packaging of foodstuffs in applications involving heating at elevated temperatures. GC/MS analysis showed the presence of numerous volatile compounds being produced after netting materials/water contact under refluxing conditions. Even though it is extremely difficult to establish a clear correlation between sensory off-odor development and GC/MS volatile compounds' profile, it may be postulated that plastics oxidation products such as hexanal, heptanal, octanal and 2,6 di-tert-butylquinone may contribute to off-odor development using (PE) are being used for the production of nets or sacks for the packaging of a number of foodstuffs such as processed meats (smoked and/or baked meats, bacon, ham, sausages) and vegetables such as potatoes. While most of vegetable products are peeled before they are consumed (others such as potatoes may be consumed without peeling). National and international authorities such as the US FDA and the EU have set restrictions with regard to the migration behavior of such packaging materials in order to protect consumers from possible migration of low molecular weight compounds from the container into the foodstuff.

The situation becomes critical in cases where such plastics nets are used as wrapping materials for meat cuts which are subsequently cooked in conventional ovens for approximately 1 h at temperatures often exceeding 100 o C. Meat depending on its fat content (pork, lamb, beef etc.) in contact with such plastics netting materials may result in considerable migration especially under extreme conditions of temperature [START_REF] Rossi | European Community controls for high temperature testing of food contact materials[END_REF]. The European Union (EU) has published a number of Directives setting the basic rules for migration testing of packaging materials intended to come into contact with foodstuffs, laying down the list of simulants, test conditions, and test methods to be used for testing overall migration of constituents from packaging materials (EEC 1982, EEC 1985, EEC 1990).

According to Directive 90/128/EEC the overall migration limit is 10 mg for all transferred substances per dm 2 of the food contact material surface. Alternatively when measuring the surface area of the packaging material is not possible the migration limit is expressed as 60mg/kg of food or mg/L of food stimulant. Testing with aqueous simulants involves evaporating the simulant to dryness and determining the residue gravimetrically. Testing with fatty food simulants (olive oil or approved alternatives: sun flower oil or synthetic triglyceride (EEC 1990, CEN 1991).

The use of vegetable oils as food simulants presents a number of difficulties and is a timeconsuming procedure (Figge 1996[START_REF] Van Battum | Draft EEC method for the determination of the global migration of plastics constituents into fatty-food simulants -applicability to lacquers, plastics and laminates[END_REF]. Some alternative fatty food simulants have been proposed -mainly solvents which can be evaporated such as isooctane or heptane and ethanol-water mixtures. Many of the problems encountered in applying fat migration testing can be overcome by using the above alternative solvents because overall migration can been determined directly be weighing a residue after evaporation of the solvent [START_REF] Baner | Alternative fatty food simulants for migration testing of polymeric food contact materials[END_REF][START_REF] Baner | Alternative fatty food simulants for polymer migration testing[END_REF], De Kruijf and Rijk 1996[START_REF] Hamdani | Migration from plasticized poly(vinyl chloride) into fatty media: Importance of simulant selectivity for the choice of volatile fatty simulants[END_REF]. Practical Guide N.1 of the Commission of EU (EEC 1993) and Directive 97/48/EC (EC 1997) refer to isooctane as one of the alternative fatty food simulants. De Kruijf and Rijk (1996) reported that isooctane is a suitable alternative food simulant for overall migration testing under various time and temperature conditions. [START_REF] Hamdani | Migration from plasticized poly(vinyl chloride) into fatty media: Importance of simulant selectivity for the choice of volatile fatty simulants[END_REF] found that the affinities of isooctane and sunflower oil to all migrants were similar and isooctane can be considered as an alternative fatty simulant for plasticized PVC.

Migration of low molecular weight compounds (plasticizers, thermal and light stabilizers, antioxidants, residual monomers, solvents etc.) from plastics packaging materials into foodstuffs and food simulants has been the subject of a large number of studies [START_REF] Badeka | Effect of microwave heating on the migration of dioctyladipate and acetyltributylcitrate plasticizers from food-grade PVC and PVDC/PVC films into olive oil and water[END_REF][START_REF] Castle | Analysis of the plasticizer acetyltributyl citrate in foods by stable isotope-dilution gas-chromatography mass-spectrometry[END_REF][START_REF] Castle | Analysis of the plasticizer acetyltributyl citrate in foods by stable isotope-dilution gas-chromatography mass-spectrometry[END_REF], Dilettato et al. 1991, Gilbert and Startin 1981, Kondyli et al. 1990[START_REF] Nerin | Parts-per-trillion determination of styrene in yoghurt by purge-and-trap gas chromatography with mass spectrometry detection[END_REF][START_REF] Perlstein | Determination of polymeric hindered-amine light stabilizers in plastics by pyrolysis-gas chromatography[END_REF][START_REF] Roberts | Enforcement of regulations governing plastics, materials and articles in contact with food in the UK[END_REF][START_REF] Zygoura | Determination of di-(2-ethylhexyl)adipate and acetyltributyl citrate plasticizers from food-grade PVC film into isooctane: Effect of gamma radiation[END_REF]. The migration of such additives or contaminants from polymeric food Diffusion within the polymer, dissolution at the polymer-food interface and dispersing into bulk food (Lau and Wong 2000).

Apart from the obvious problem of potential toxicity of such low molecular weight migrating species, another possibility is the development of off-odors/off-tastes as well as discoloration as a result of such migration. In both cases the foodstuff becomes unfit for consumption (Figge 1996[START_REF] Hodges | Sensory directed analytical concentration techniques for aroma-flavor characterization and quantitation[END_REF].

Thus the objectives of the present work were (1) to determine the overall migration from a series of commercial plastics-based netting materials into the food simulants: distilled water and isooctane, (2) to run sensory tests in an effort to evaluate possible deterioration of sensory properties of the food stimulant: bottled table water (odor, taste, color) used as a result of migration and (3) to possibly relate sensory attributes (mainly odor) to GC profile of this food simulant using SPME/GC-MS.

Materials and Methods

Materials

Netting and threat samples were based on PET or PE and were supplied by a local commercial company representing several foreign manufacturing companies. The identity of the materials is listed in Table 1. Analytical grade isooctane was supplied by Merck (Darmstadt, Germany). Water used was double distilled or commercially bottled water. Either rectangular strips of net samples (6 g in weight) or plastics threads comprising the raw material for the manufacture of commercial nets (6 g in weight) were immersed in 100 ml of food simulant (distilled water or isooctane) in glass beakers. Beakers were covered with Parafilm ® so as to avoid evaporation of simulant during contact period and kept in a thermostatically controlled chamber at 40±0.5 o C for ten days (EU directive 2002/72). For isooctane the time/temperature contact conditions were chosen on the basis of EU Directive 97/48 to be 4h/60 o C. Following the time/temperature predetermined contact, samples were removed and the simulant was placed in a 250 ml pre-weighed Erlenmeyer flask and evaporated to dryness in a rotary evaporator. The Erlenmeyer flask containing the residue of evaporation was kept in a thermostatically controlled chamber at 105±1.0 o C for 1h followed by 1h in a dessicator and then weighed. A Sartorius analytical balance, model BF221S, capable of weighing to 0.1 mg was used. The overall migration was calculated in mg/L of food simulant. For each net or thread sample three determinations were performed and final migration values were the mean of the three determinations.

Migration experiments

Sensory testing

Sensory testing was carried out using commercially bottled table water packaged in PET as the food simulant. Representative net or thread samples were brought into contact with table water and heated for 4 h at 100 o C under refluxing conditions. Heated, under the same conditions, table water (not in contact with plastics nets) was used as the control sample.

Evaluation was carried out by a 5-member panel of judges experienced in sensory attributes of water. The judges evaluated taste, odor and color of the aqueous simulant on an intensity scale between 0 and 4 where 0 represented no difference between experimental and control sample, 1 represented a slight difference, 2 represented a moderate difference, 3 represented a

Determination of volatile compounds' profile of food simulant using SPME-GC/MS

Heated as described above net samples were placed (10mL) along with a micro-stirring bar in a 20mL glass vial (20×72mm) and 10µL of internal standard solution (20µg/ml of 4-methyl-2-pentanone) which was capped with a PTFE/rubber septum and sealed with an aluminum crimp cap. Solid phase micro-extraction (SPME) was performed with a 50/30 µm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber mounted to a SPME manual holder assembly (Supelco, Bellefonte, CA, USA). The sample vial was placed in a 40 o C water bath and stirred at high speed. After allowing 5 min for the sample to equilibrate at 40 o C, the septum piercing needle of the SPME apparatus was pushed down to expose the DVB/CAR/PDMS fiber to the vial headspace. After 30 min of exposure time with constant stirring, the fiber was retracted into the needle assembly, removed from the vial and inserted in the injector of GC/MS instrument.

GC/MS analysis conditions

Gas chromatographic (GC) analysis of volatile compounds adsorbed onto the SPME fiber was carried out on a HP-5MS (5% phenyl methyl siloxane) 30m×0.25mm×0.25µm fused-silica capillary column (J&W Scientific, Folsom, CA, USA). Flow rate of the helium carrier gas

Results and Discussion

Overall migration

Overall migration values in contact with aqueous or fatty simulant are given in Table 1.

Samples #1 and #8 were polyester based materials also containing rubber and cotton threads destined to be used for wrapping boiled meat and salami products respectively, while sample #4 also containing rubber and cotton threads was a polyethylene based material destined to be used for wrapping smoked meat products. Sample #2, 3 and #5-7 were PET based nets also containing rubber destined to be used for wrapping meat cuts to be baked in a conventional oven or boiled. Thus according to EU Directives 85/572 and 97/48 two sets of test contact conditions were chosen for migration testing: (1) 4h/60 o C and (2) 4h/100 o C under refluxing using isooctane as the fatty food simulant. As shown in Table 1 differences in overall migration values for these samples were not statistically different (p>0.05) between the two sets of conditions. Thus all subsequent testing with regard to isooctane was carried out under the former conditions (4h/60 o C). All of these samples gave migration values higher than the upper limit of 60mg/L set by the EU. At first, one may attribute such high migration values to constituents such as rubber and cotton threads in plastics based nets in which case Directives 90/128 and 2002/72 do not cover such composite materials. In order to test this hypothesis, manufacturers were asked to provide PET threads used to produce PET based nets (i.e. net respectively which are below the above reported upper limit for overall migration. The same rationale holds for samples #4 versus #14 the first giving a very high migration value as compared to the second, consisting solely of PE thread resulting in a migration value within the limit for overall migration (60mg/L). In other cases such as those of samples #1 and 8 the migration value is such that if divided by a factor of 4 falls within the above mentioned migration limit.

Of PET thread samples #16-18 destined for wrapping salami products and samples #19-22 destined for wrapping baked and boiled meat products, samples #18, 21 and 22 meet the above migration criterion, while samples #16, 17, 19 and 20 do not. It is unclear why these four PET samples gave such high migration values given that migrating species from PET packaging materials include mostly PET oligomers responsible for low migration values (Castle et al. 1989).

The rest of the net samples namely #9-11 were made solely of polyethylene and were intended for the packaging of unprocessed vegetables such as potatoes, onions and garlic and were thus tested against distilled water as the food simulant. It is clear that overall migration values for all these samples are well below the upper limit of 60mg/L for migration. It should be stressed, at this point that with the exception of potatoes which might be consumed with their skin intact, most vegetables are peeled before consumption and thus even high overall migration values will most probably not pose any kind of health risk for the consumer. In general migration values in isooctane were significantly higher than corresponding migration values in distilled water. This can be explained by the capability of isooctane to penetrate into the polymer matrix causing swelling of the polymer and thus changes in its structure. The consequence is an increase in the diffusivity of the potential migrants and a resulting increase in both rate and amount of migrating species [START_REF] Shepherd | Trace contamination of foods by migration from plastics packaging -A review[END_REF]). et al. (2003) reported overall migration values for a series of multilayer flexible food packaging materials based on PE containing a layer of recycled PE in distilled water and isooctane in the range of 3.6-10.5 mg/L (0.17-0.52 mg/dm 2 ) for distilled water and 70.2-102 mg/L (3.5-5.3 mg/dm 2 ) for isooctane.

To the best of the authors' knowledge these are no reports in the literature involving overall migration values from plastics based netting materials into either food simulants or foodstuffs.

Sensory evaluation

Results of the sensory evaluation are shown in Table 2. Representative samples made of PET and PE were chosen with overall migration values both above and below 60mg/L (samples #1 through 15). Of all the samples tested only seven (#2, 9-12, 14 and 15) received a score lower or equal to 1 (sensory acceptability limit). Usual undesirable sensory attributes recorded, included development of yellowish discoloration, opaque appearance, slight to moderate plastic off-odor and slight to moderate plastic off-taste. At this point it should be mentioned that the migration behavior of present netting materials does not necessarily correlate well with sensory characteristics of the contacting food stimulant, i.e material #1 exhibits an acceptable level of migration while at the same time it significantly deteriorates sensory characteristics of the contacting aqueous simulant. On the other hand material #2 exhibits a high migration value while it does not substantially deteriorate sensory characteristics of the contacting aqueous simulant. Finally a set of netting materials (#9-12, 14 and 15) exhibits migration values below the upper limit for migration and at the same time substantially retains desirable sensory characteristics of the contacting aqueous food simulant. A general trend shown in Table 2 is that netting materials containing rubber or rubber+cotton result in more objectionable sensory attributes as compared to materials consisting solely of plastics (i.e. #1-8 versus # 12-15). An exception to this trend is material #13 consisting of PET thread producing objectionable taste and color. The reason for this is unclear.

Determination of volatile compounds

Volatile compounds' profiles of representative netting materials (i.e. #2, 8 and 12) including control samples are given in Table 3. Samples were chosen on one hand to represent composite materials made of PET + rubber (#2), of PET + rubber + cotton (#8), of PE + rubber + cotton (#4) and of PET thread (i.e. #12) and on the other hand on the basis of water after refluxing (100 o C/4h). Semi-quantitative determination of volatiles was achieved using 4-methyl-2pentanone as internal standard. Comparison for example between control sample and samples #2 and #8 leads to the suggestion that either a number of compounds show up in the aqueous simulant after contact with the netting material which do not exist in the control sample or the concentration of other compounds significantly increases in the aqueous simulant after contact with the netting material. Examples of the former case are: hexanal, heptanal, octanal, benzaldehyde, butylated hydroxytoluene and 2,6 di(t-butyl)-2,5 cyclohexadien-1-one while an example of the latter case is: 1,2 benzenedicarboxylic acid, bis (2-methyl propyl) ester and butylated hydroxytoluene (BHT).

What should be stressed at this point is that numerous volatile compounds show up in the control sample which was table water packaged in PET. Thus their presence is largely justified. What we were interested in seeing were differences in volatiles produced as a result of heating either plastics based nets or plastics threads. It is also apparent that numerous compounds identified in composite netting materials absent from both the PET thread (sample #12) and the control such as carbon disulfide; [1,1' biphenyl]-2-ol; propanoic acid-2-methyl 1-(1,1 dimethyl)-2-methyl-1,3-propanediyl ester etc. are most probably related to specific treatments applied to the rubber and/or cotton component of the netting materials.

Even though it is extremely difficult to establish a clear correlation between volatile compounds' profile and sensory (odor) evaluation it may be postulated that oxidation products of plastics materials such as 2-ethyl hexanal, heptanal, octanal and 2,6-di-tert butyl quinone probably contribute to off-odor development in heated plastics based netting materials. [START_REF] Riganakos | Effects of ionizing radiation on properties of monolayer and multilayer flexible food packaging materials[END_REF] determined the volatile compounds' profile of a series of based on polyethylene using HS-SPME and reported a series of compounds among which hexanal, heptanal, decanal, nonanal, decane, toluene and 2-ethyl hexanal were also found in the present study. Similar compounds were also identified by [START_REF] Bravo | Identification of odour-active compounds resulting from thermal oxidation of polyethylene[END_REF] as odoractive species resulting from thermal oxidation of polyethylene. [START_REF] Rose | Determining volatile extractives from microwave susceptor food packaging[END_REF] determined volatile extractives from microwave susceptor PET based materials (conditions: Temp=125 o C, time=3-4 min) and reported the production of 72 compounds including many of the compounds determined in the present study.

Conclusions

Based on data of the present study it is concluded that PE netting materials used for the packaging of vegetable products (i.e. potatoes) are absolutely safe as packaging materials.

Netting materials based on PET used for packaging of meat products should be carefully selected especially for applications involving heat treatment at high temperatures (i.e. baked and boiled products) in which case they can produce off-odor/taste as well as discoloration problems.

  water as a food simulant. Likewise compounds such as carbon disulfide, [1,1' biphenyl]-2-ol and propanoic acid, 2 methyl 1-(1,1 dimethyl)-2-methyl-1,3propanediyl ester probably originating from cotton and rubber components of netting materials may also contribute to off-odor/taste development.Key words: plastics based nets, migration, sensory properties threads mainly polyester (polyethylene terephthalate, PET) and polyethylene

  involves weighing of the plastic before and after simulant contact. The amount absorbed into the plastic is determined by extraction and GC analysis

  food may be separated into three different but interrelated stages.

  4 represented a very large difference. Samples receiving a score larger than 1 were considered to be unacceptable.

  was 0.7mL/min. A Hewlett-Packard 6890 series gas chromatograph equipped with a Hewlett-Packard 5973 mass selective detector was used. During the injection phase, the injector was operated in the split mode (2:1 split ration) at a temperature of 260 o C. For thermal desorption, the SPME fiber remained in the injector for 5 min. The initial column temperature was 40 o C and was maintained at this temperature for 4 min. The column was heated to 280 o C at a rate C/min and held at 280 o C for 5 min. Mass spectrometric (MS) conditions were as follows: source temperature 230 o C; quadropole temperature 150 o C; acquisition performed in electron impact (E.I.) mode (70eV) by 2.48 scans/s; the mass range m/z: 30-330. The temperature of the transfer line was held constant at 290 o C. Peak identification was performed by comparison of mass spectra of eluting peaks to those of the Willey library (Wiley, version 275). Spectral match was based on target and top ten ions as well as relative mass intensity signals.

  6). Such PET threads (i.e. samples #12, 13 and 15 respectively) gave migration values significantly lower than the former. If recorded migration values are divided by a factor of 4 as suggested in EU Directive 85/572 for meat and meat products then resulting migration values between 19 and 55 mg/L are obtained for samples #12, 13 and 15

Castle

  at al. (1990) studied overall migration of a series of plastics packaging materials including polyolefins (simulant: water, contact conditions: 100 o C/60min) and PET trays (simulant: olive oil, contact conditions: 175 o C/120min) in high temperature food-use applications and reported very low migration values < 2 and 4 mg/dm 2 respectively. Czerniawski and Pogorzelska (1997) investigated overall migration of various plastics materials including PET sheets into a number of food simulants including isooctane and reported very low migration values in the order of 0.3mg/dm 2 . Castle et al. (1989) studied the migration of PET oligomers from PET trays into sausages (contact conditions: 204 o C/60min) from PET roasting bags into roasted beef and pork (contact conditions: 204 o C/90min) and reported low migration values in the range of 0.4 and 0.8mg/kg respectively. Finally Badeka

  odor development in the food simulant: commercially bottled table mineral

  including LDPE and PET/PE laminates and reported a large number of volatile compounds samples, most of which were found in the present study.Ezquerro et al. (2003) quantified volatile organic compounds in a composite flexible packaging material

  , MAYO, A, CREWS, C., and GILBERT, J., 1989, Migration of PET oligomers from PET plastics into foods during microwave and conventional cooking and into bottled beverages. Journal of FoodProtection, 52, 337-342. CASTLE, L., SHARMAN, M., and GILBERT, J., 1988, Gas-chromatographic massspectrometric determination of epoxidized soybean oil contamination of foods by migration from plastic packaging. Journal of the Association of Official Analytical Chemists, 71, 1183-1186. CEN, 1991, Methods of test for materials and articles in contact with foodstuffs. In: CEN Guide to selection of conditions and methods to test. Part I. Cen draft standard TC194 N11, Brussels. CZERNIAWSKI, B., and POGORZELSKA, Z., 1997, Investigations on overall migration of various plastics materials and articles used in contact with foodstuffs. Packaging Food and Science, 10, 261-270. DE KRUIJF, N., and RIJK, M., 1996, The suitability of alternative fatty food simulants of overall migration testing under both low and high-temperature test conditions. In: International Life Science Institute (ILSI) (ed), Food Packaging Ensuring the Quality and Safety of Foods. Proceeding of the International Symposium on Food Packaging, Sept. 11-13, Budapest, Hungary; ILSI, Brussels, Belgium, p. 27. DILETTATO, D., ARPINO, P.J., NGUYEN, K., and BRUCHET, A., 1991, Investigation of low mass oligomers and polymer additives from plastics. Part II: Application to polyolefin soxhlet extracts. Journal of High Resolution Chromatography, 14, 335-342 EC, 1997, Commission Directive No. 97/48/EC of 29 July 1997 amending Council Directive 82/711/EEC laying down the basic rules necessary for testing migration of constituents of plastic materials and articles intended to come into contact with foodstuffs. Official Journal of the European Communities, L 222, 12 August 1997, pp. 10-15.

  FIGGE, K., 1996, Plastics. In: Katan, L.L., editor. Migration from the food contact materials.

F

Table 1 .

 1 Identity and overall migration values (mg/L) of plastics netting materials intoaqueous (distilled water) and fatty (isooctane) food simulants.

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

Table 2 .

 2 Sensory evaluation of plastic nets and threads materials subjected to refluxing (100 o C/4h) in contact with distilled water.

	Sensory attributes

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	21
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y

Table 3 .

 3 Tentative identification and amounts (µg/mL) of volatile compounds of selected plastics netting materials using SPME-GC/MS.

	RT*	Compound	control	sample #2	sample #4	sample #8 sample #12
	1.66	2-Propanone		30.6	48.3	11.2
	1.83	Carbon disulfide		65.5	39.7	31.0
	2.10	Hexane	6.58	5.13	6.59	9.99	9.6
	2.90	Butane, 2,2,3,3-tetramethyl	10.6	12.0	17.2	1.62
	3.18	pentanal				1.48
	3.70	4-methyl-2-pentanone (i.s.)	20	20	20	20	20
	4.65	toluene		9.06	2.00	3.00	3.06
	5.54	3-penten-2-one, 4-methyl-		4.50	5.47
	5.52	hexanal				1.00
	7.15	Benzene, ethyl-	8.37	3.57	3.97	4.93	5.88
	7.38	Benzene, 1,3-dimethyl	15.5	15.6	18.6	19.8	19.6
	7.98	Benzene, 1,4-dimethyl	11.1	6.19	5.76	12.6	7.43
	8.26	n-heptanal				0.76
	9.70	Benzaldehyde		6.16	5.77	12.6
	10.37 6-methyl-5-hepten-2-one					2.22
	10.71 Octanal		3.38		2.69
	11.28 2-ethyl hexanal		1.12	1.29	2.43	8.26
	12.88 Nonanal	1.27	7.73	2.20	10.2	3.10
	14.64 2-decanone				0.95
	14.84 Decanal	2.63	11.2	2.30	10.8	3.56
	19.10 Geranyl acetone	0.84	1.92	1.71	2.77	1.30
	19.39 2,6 di(t-butyl)-2,5-		1.77	3.11	6.75
		cyclohexadien-1-one
	19.81 pentadecane		1.80
	20.01 phenol, 2,4-bis(1,1-			1.16	1.41	2.19
		dimethylethyl) or
		2,6-di-tert-butylquinone
	20.09 Butylated hydroxytoluene	1.51	6.16	4.37	1.76
		(BHT)
	20.14 [1,1'-biphenyl]-2-ol		1.50	1.79	1.38
	21.29 hexadecane		0.87	1.15
	21.33 Propanoic acid, 2 methyl, 1-		1.31	1.39	0.97
		(1,1 dimethyl)-2-methyl-1,3-
		propanediyl ester
	21.46 Phenol, 4(1,1,3,3-tetramethyl	2.12	0.93	0.90	0.74	2.14
		butyl)
	22.71 heptadecane		1.50
	23.15 Benzenemethanamine, N-		8.60	7.72	13.2	7.85
		(phenylmethyl)
	23.46 Benzenemethanamine, N-		2.13	2.16	2.72
		(phenylmethylene)
	24.06 octadecane			1.20
	25.01 1,2-benzenedicarboxylic acid,	2.16	33.5	40.6	42.8	9.30
		bis(2-methyl propyl) ester
	28.70 phenol, 4,4'-(1-methyl-		3.83	2.64
		ethylidene) bis or
		Bisphenol A

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Acknowledgements

The authors would like to thank the Unit of Food Standardization of the University of Ioannina for providing access to the GC/MS instrument.


C Benetti
email: cbenetti@izsvenezie.it

R Piro

G Binato

R Angeletti

G Biancotto

SIMULTANEOUS DETERMINATION OF LINCOMYCIN AND FIVE MACROLIDE ANTIBIOTIC RESIDUES IN HONEY BY LIQUID CHROMATOGRAPHY COUPLED TO ELECTROSPRAY IONISATION MASS SPECTROMETRY

Keywords: Veterinary drug residues, lincomycin, macrolides lincomycin, macrolides, honey, validation, LC/MS/MS

A sensitive and specific method based on liquid chromatography-tandem mass spectrometry (LC/MS/MS), for the simultaneous determination of lincomycin and five macrolide antibiotics in honey, was developed and validated. The analytes were extracted with Tris buffer 0.1 M pH 10.5 and cleaned-up by a single solid phase extraction step on an OASIS HLB column. The chromatographic separation of analytes was performed on a Synergi Hydro-RP reversed -phase column using a gradient program of aqueous 0.01 M ammonium acetate pH 3.5 and acetonitrile as the mobile phase, at a flow rate 0.25 ml min -1 . Detection of analytes was achieved by positive

Introduction

American foulbrood (AFB) is one of the most important and destructive diseases of honey bees (Apis mellifera) and is caused by the ingestion of the spores of a Gram + bacterium Paenibacillus larvae subsp. larvae (Shimauki 1997). The spores are highly resistant to heat, desiccation, and chemical disinfectants (Hansen et al. 1999) and can remain dormant for many years in beehive, beekeeping equipment, honey and wax. This increases the risk that unwitting manipulation by the beekeeper may transfer AFB spores to new bees-colonies. For this reason AFB has traditionally been controlled by burning, scorching or irradiating contaminated beekeeping equipment (Elzen et al. 2002). In order to lessen this financial loss, sulphonamides and antibiotics are used worldwide in apicultural practices to suppress clinical symptoms of AFB, even if these substances have a transient effectiveness and are totally ineffective against the spores of Paenibacillus larvae subsp. larvae (Kochansky et al. 2001, Feldlaufer et al. 2001, Mutinelli 2003). Home-made uncontrolled treatments with these drugs increase the risk that residues can be present in beehive products (mainly honey) and that undesired effects on consumers like allergic reactions or bacterial resistance can potentially occur.

To avoid these consequences, European Community legislation (Council Directive 96/23/EC) on residues in animal-origin food, considers honey like any other food product, but, differently from the others, in this case even minor traces of antibiotics are not tolerated, since no legal limit (MRL) have been established for any antibiotic drug in honey matrix. As a consequence, analytical methods intended for the detection of residues of antibacterial drug in honey at trace level should be developed and applied to routine samples. The use of tetracyclines and sulphonamides in beekeeping is known since 1940 (Eckert 1947, Katznelson et al. 1949, Gochnauer 1951, Katznelson et al. 1952), and many analytical methods have been developed to detect the presence of their residues in honey matrix (Horie et al. 1992, Kaufmann et al. 2002, Vinas et al. 2004, Oka et al. 1994), but very little information are available regarding methods for detecting other antibiotic drugs like lincomycin, and some macrolide antibiotics that seem to be increasingly used in beekeeping for the prevention and treatment of brood diseases (Feldlaufer et al. 2001, Kochansky et al. 2001, Hitchcock et al. 1970, Moffett et al. 1970, Peng et al. 1996). In fact, currently, there are a few analytical methods reported for the determination of lincomycin and macrolides in honey and no one for the simultaneous determination of these drugs (Thompson et al. 2003, Wang 2004, Benetti et al. 2004).

Therefore, in absence of maximum residue levels in honey, the aim of this work was the development of a multi-residue method for the simultaneous detection and confirmation of lincomycin and five macrolides (tylosin, erythromycin, tilmicosin, spiramycin and josamycin) in honey at trace levels using roxithromycin as internal standard. Due to the complexity of honey matrix the analytical approach was based on a combination of liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) technique. The following analytical parameters of the method were validated according to the guidelines laid down by Commission Decision 2002/657/EC using an in-house validation: linearity of the standard response both in solvent and in matrix extracts,

Materials and methods

Materials and reagents

Ammonium acetate, tris(hydroxymethyl)-aminomethane (TRIS) and 30% aqueous ammonia solution (Sigma, Milan, Italy) were of the highest available purity grade. Acetonitrile, ethanol, methanol (Carlo Erba, Milan, Italy) were all HPLC grade. Distilled water was de-ionised by a Milli-Q apparatus from Millipore. Tylosin (TYLO), spiramycin (SPIRA), erythromycin (ERYTRO), lincomycin (LINCO) and roxithromycin (ROXI) were purchased from Sigma (Milan, Italy), josamycin (JOSA) was purchased from Fluka (Milan, Italy) and tilmicosin (TILMI) was purchased from Eli Lilly (Sesto Fiorentino FI, Italy). A stock solution of 1000 mg kg -1 for each drug was prepared by dissolving the appropriate amount of each substance in ethanol (for

Fortified samples

Negative honey samples (5 g) were fortified at 0.2, 0.4, 0.6 µg kg -1 of TYLO, TILMI, ERYTRO, JOSA, LINCO and 1.6, 3.2, 4.8 µg kg -1 of SPIRA.

Sample extraction and clean -up

Honey samples were processed according to the following procedure: 5 g of honey were weighed in a 50 ml-tube and diluted with 35 ml of TRIS buffer (0.1 M, pH 10.5).

Then the raw extract was purified and concentrated by OASIS HLB columns (3 ml / 60 mg) (Waters, Milan, Italy). The cartridges were conditioned sequentially with methanol (10 ml) and water (10 ml). The sample extracts were quantitatively loaded into the SPE cartridges. The cartridges were washed with 20 ml of a solution of methanol -water ( 5-95, v/v), then the OASIS columns were vacuum -dried for 5 minutes. Finally the analytes were eluted with 10 ml of a solution of methanol -30% ammonia (95 -5, v/v).

A 100 µl volume of a working solution of ROXI (1mg l -1 ) was added to each eluate: this compound is added at this point as internal standard (IS) to compensate the variation in ionisation efficiency of ESI source during HPLC-MS/MS analysis. The eluates were then dried under nitrogen stream at 50°C and redissolved in 1 ml of reconstitution-phase and transferred into vials, 10 µl were injected into the HPLC-MS/MS system. The flow rate of mobile phase was 0.25 ml min -1 . In these conditions the elution profile was in the following order: LINCO (tr = 9.9), SPIRA (tr = 11.4), TILMI (tr = 12.2), ERYTRO (tr = 13.0), TYLO (tr = 13.3), ROXY (tr = 14.2), JOSA (tr = 14.9).

The MS/MS system used was a Quattro Ultima II triple quadrupole mass spectrometer (Micromass, Altrincham, UK), equipped with electrospray ionisation (ESI) ion source.

The analysis was performed in the positive ion mode. and with a small volume (100 µl) of ROXI working standard solution (1 mg l -1 ) used as internal standard (IS). After stirring, these solutions were evaporated to dryness under nitrogen stream at 50°C and were re-dissolved in 1 ml of reconstitution phase to be transferred into vial.

Results and discussion

This paper describes the validation procedure of a LC/MS/MS method according to the

Linearity of response

The linearity of the LC/MS/MS response was demonstrated with solvent standard solutions at 6 calibration levels in the concentration range 0.10 -2.0 µg kg -1 for TYLO, TILMI, ERYTRO, JOSA, LINCO and in the concentration range 0.80 -16.0 µg kg -1 for SPIRA. The correlation coefficients (R 2 ) of the standard curves in solvent were at least 0.999 for all analytes considered. The linearity of the LC/MS/MS response was proved also with standard solutions in matrix extracts to take into account an ion enhancement matrix effect. The correlation coefficients (R 2 ) of these standard curves were at least 0.995. the identification and quantification of TYLO, TILMI, ERYTRO, JOSA, LINCO and SPIRA.

Specificity

Decision limit (CCα) and detection capability (CCβ)

In the 2002/657/EC European decision CCα and CCβ replace the limit of detection and the limit of quantification. These parameters were determined, at first, by the calibration curve procedure according to the ISO 11843-2. As recommended by the ISO the number of reference states I (number of blank honey samples fortified at equidistant steps with the considered analytes) used in the calibration experiment were 4 (including the value zero represented by a blank honey sample); the number of preparations J for each reference state was 2; the number of repeated measurements L performed per honey samples were spiked at these CCα levels and processed according to the described procedure, but the method was not able to detect or properly identify the analytes. This is not unusual and has already been described in other publications (Jülicher et al. 1998, Poltzer et al. 2001). For this reason another approach, recommended by SANCO/2004/2726 was used to estimate them: by parallel extrapolation to x axis at the lowest spiking level (0.2 µg kg -1 for TYLO, TILMI, ERYTRO, JOSA, LINCO and 1.6 µg kg -1 for SPIRA) and by taking into account the method inter-day repeatability, estimated during recovery and repeatability studies.

More in detail the decision limit was calculated according to the following equation:

CCα = C (mrpl) + 2.33 Sc (mrpl)
Where:

mrpl is the minimum required performance level (which is not the Minimum Required Performance Limit) as defined in SANCO/2004/2726 Sc (mrpl) is the standard deviation of whole series of recalculated concentrations at the mrpl over the total number of replicates (n=18);

The minimum required performance level (for each compound) was chosen during the pre-validation studies as the lowest tested concentration at which the method fulfils the recommended identification criteria (4 identification points).

Results are listed in table 3.

[Insert Table 3

Recovery and intra/inter-repeatability

The absolute recovery and intra and inter-day repeatability of the method were assessed by spiking three different series of 18 blank honey at 0.2, 0.4, 0.6 µg kg -1 for TYLO, TILMI, ERYTRO, JOSA, LINCO and 1.6, 3.2, 4.8 µg kg -1 for SPIRA (six replicates for each concentration level). Each series was processed in different days by different operators using different batches of reagents and solutions. The results are summarised in table 3.

Ruggedness

Ruggedness is the characteristic of an analytical method when minor variations in the procedure produce no significant effects in its performance. The As shown in table 4, eight experiments were conducted for the evaluation of seven selected factors by spiking eight blank honey samples with TYLO, TILMI, ERYTRO, JOSA, LINCO at 0.2 µg kg -1 and SPIRA at 1.6 µg kg -1 (first fortification levels used in the recovery and intra/inter-repeatability study).

The effect of a particular variable was estimated by subtracting the mean result obtained with the variable at "high level" indicated by capital letter, from the mean result achieved with it at "low level", marked with the corresponding small letter. For each variable, the calculated difference was indicated as D i . The standard deviation of the differences (S Di ), was calculated by the formula:

∑         × = 7 2 2 i D D S i
When S Di is significantly larger than the standard deviation of the method, it means that the combination of all the chosen factors have an effect on the analytical result. Besides, by means of a t-test it is possible to evaluate the influence of each investigated factor (Vander Heyden et al. 1995, Scortichini et al. 2005). The experimental t is given by: .

. 2 D S D n t i × × =
where n (n = 4) is the number of experiments carried out at each level for each parameter and S.D. is the estimate of the method precision obtained from the analysis of 18 spiked samples at the first spiking level during the validation study.

The obtained t-value, for all seven variables, was compared with the 2-tailed t-critical value (t crit ) for N-1 degrees of freedom, where N (N=18) is the number of determinations used in the estimation of S.D. at 95%confidence level. If t is greater than t crit the investigated variable shows a significant influence and the method is not sufficiently robust against the chosen modification. The results in table 5, obtained following the experimental plan showed in table 4, indicate that the method is robust and minor but still significant fluctuations in the operative parameters that can occur during the routine application of the method have not significantly effect in its performance characteristics. In fact the experimental t values resulted below the 2-tailed t-critical value for all seven factors: t crit = 2,11 for 17 degrees of freedom at 95% confidence level.

[Insert Table 4 about here]

[Insert Table 5 about here]

Conclusions

The test carried out to verify the performances of the method demonstrated that it is extremely sensitive (with a detection capability CCβ < 0.26 µg kg -1 for TYLO, TILMI, ERYTRO, JOSA, LINCO and CCβ < 2.1 µg kg -1 for SPIRA), accurate (mean absolute recoveries > 84% with CV% < 12 % for all analytes) and specific (no endogenous interfering substances or interferences due to other possible antibiotics used in beekeeping) also in within-laboratory reproducibility conditions. The Youden ruggedness test showed that the method is fairly robust: minor fluctuations in some potential critical operative parameters, that can occur during the routine application of the method over a long period of time, do not significantly affect its performance characteristics. Furthermore, since the method requires only a simple extraction and a single clean up step, up to 24 honey samples per day can be processed.

Preparation (J) Measurements (L) J = 1 J = 2 J = 1 J = 2 I = 4 µ µ µ µg kg -1 L = 1 L = 2 L = 1 L = 2 L = 1 L = 2 L = 1 L = 2 1 0 0,
Preparation (J) Measurements (L) J = 1 J = 2 J = 1 J = 2 I = 4 µ µ µ µg kg -1 L = 1 L = 2 L = 1 L = 2 L = 1 L = 2 L = 1 L = 2 1 0 0,

  , repeatability, decision limit (CCα), detection capability (CCβ) and ruggedness.

  guidelines laid down by the Commission Decision 2002/657/EC for the simultaneous identification and quantification of LINCO and five macrolides (TYLO, TILMI, ERYTRO, JOSA, SPIRA) in honey. For each compound, all parameters considered in the validation study were assessed on the basis of the highest intense transition ion, a part from the analytical limits (Ccα and Ccβ) assessed on the basis of the less intense transition ion.The analyte responses were always related to the internal standard response: in the absence of isotope -labelled form of the considered analytes, particularly suited for MS detection, ROXI, a macrolide not used in veterinary medicine, was used as the internal standard added at the end of sample preparation before the LC/MS/MS analysis to avoid response variability due to variation in the ionisation yield. Figure1and figure2shows typical LC/ESI/MS/MS chromatograms of the mixture of standard solution at 0.2 µg kg - 1 of TYLO, TILMI, ERYTRO, JOSA, LINCO and at 1.6 µg kg -1 of SPIRA (in the left side), a blank honey extract (in the medium) and a fortified honey sample containing 0.2 µ g kg -1 of TYLO, TILMI, ERYTRO, JOSA, LINCO and 1.6 µg kg -1 of SPIRA and clean-up procedure (in the right side). Figure1shows chromatograms obtained by monitoring the quantification transitions, figure2shows chromatograms obtained by monitoring the confirmation transitions.[Insert Figure1about here] [Insert Figure2about here]

  Specificity of the LC/MS/MS method was established by processing and analysing 20 different control honey samples (10 wildflower 5 robinia, 5 chestnut). No interference peaks were found around the retention time of analytes in the matrices under investigation. Moreover, blank honey extracts were spiked with a relevant concentration of tetracycline, sulphonamides and amphenicols (other substances that may occur in honey like residues of chemical treatments to prevent AFB) but no one interfere with

  ruggedness test was conducted by the Youden procedure (Youden et al. 1975) cited by Commission Decision 2002/657/EC. Seven variables were chosen and deliberately altered: the volume of dilution buffer (TRIS), the pH and molarity of dilution buffer, the methanol percentage during the washing steps of the SPE purification, the ammonia solution percentage in elution solvent, the SPE elution volume and the evaporation temperature of solvents in the final extract.

  Some recent reports of the mission carried out in the European countries by FVO to evaluate the control of residues in live animals and animal products (DG(SANCO)/3389/2001, DG(SANCO)7504/2005) and some notifications reported by the Rapid Alert System for Food and Feed (RASFF) in 2004(RASFF Annual Report, 2004) indicate the potential use of some of these substances in beekeeping. Therefore a validated method for the detection and identification of these analytes is required and this need is fulfilled by the method reported in this paper.ReferencesBenetti, C.,Dainese, N., Biancotto, G., Piro, R., and Mutinelli, F., 2004, Unauthorised antibiotic treatments in beekeeping. Development and validation of a method to quantify and confirm tylosin residues in honey using liquid chromatography-tandem mass spectrometric detection. Analytica Chimica Acta 520 87-92 Commission Decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results (2002/657/EC), Official Journal L221 (2002) 8-36 Council Directive 96/23/EC of 29 April 1996 on measures to monitor certain substances and residues thereof in live animals and animal products and repealing Directives 85/358/EEC and 86/469/EEC and Decisions 89/187/EEC and 91/664/EEC, )/3389/2001-MR final, European Commission Health & Consumer Protection Directorate-General, Directorate F -Food and Veterinary Office, Final Report of a mission carried out in Turkey from 8 to 12 october 2001 in order to evaluate the control of residues in live animals and animal products DG(SANCO)/7504/2005-MR final, European Commission Health & Consumer Protection Directorate-General, Directorate F -Food and Veterinary Office, Final Report of a mission carried out in Greece from 4 to 8 april 2005 concerning the evaluation of the control of residues and contaminants in live animals and animal products, including controls on veterinary medicinal products Eckert, J. E., 1947, Use of sulfa drugs in the treatment of American foulbrood disease of honeybees. Journal of Economic Entomology 40, 41-44 Elzen, P., Westervelt, D., Causey, D., Rivera, R., Baxter, J., and Feldlaufer, M., 2002, Control of oxytetracycline-resistant American foulbrood with tylosin and its toxicity to honey bees (Apis mellifera). Journal of Apicultural Research 41, 97-100 Feldlaufer, M.F., Pettis, J. S., Kochansky, J. P., and Stiles, G., 2001, Lincomycin hydrochloride for the control of American foulbrood disease of honey bees. Apidologie 32, 547-554 Gochnauer, T. A., 1951, Drugs fight foulbrood diseases in bees. ., and Brødsgaard, C. J., 1999, American foulbrood a review of its biology, diagnosis and control. Bee World, 80, 5-23 Hitchcock, J.D., Moffett, J.O., Lackett, J.J., and Elliott, J.R., 1970,Tylosin for control of American foulbrood disease in honey bees. Journal of Economic Entomology 63, 204-207 Horie, M., Saito, K., and Nose, N., 1992, Simultaneus determination of sulphonamides in honey by liquid chromatography. Journal of AOAC International 75, 786-789 ISO 11843-2: 2000 Capability of detection-Part 2: Methodology in the linear calibration case Julicher, B., Gowik, P. and Uhlig, S., 1998, Assessment of detection methods in trace analysis by means of a statistically based in-house validation concept. The Analyst 123, 173-179 Katznelson, H., and Gooderham, C. B., 1949, Sulfathiazole in relation to American Foulbrood. Scientia Agricula 29, 340-344 Katznelson, H., Arnott, J., and Bland, S. E., 1952, Preliminary report of the treatment of European foulbrood of honey bees with antibiotics. Scientia Agricula 32, 180-184 Kaufmann, A., Roth, S., Ryser, B., and Widmer, M., 2002, Quantitative LC/MS-MS Determination of sulfonamides and some other antibiotics in honey. Journal of AOAC International 85., Knox, D. A., Feldlaufer, M., and Pettis, J. S., 2001, Screening alternative antibiotics against oxytetracycline-susceptible and -resistant Paenibacillus larvae. Apidologie 32, 215-222 Mutinelli, F., 2003, Practical Application of antibacterial drugs for the control of honey bee diseases. Apiacta 38, 149-155 Moffett, J.O., Hitchcock, J.D., Lackett, J.J., and Elliott, J.R., 1970, Evaluation of some new compounds in controlling American foulbrood. Journal of Apicultural Research 9, 39-44 Oka, H., Ikai, Y., Hayakawa, J., Harada K., Asukabe, H., Suzuki, M., Himei, R., Horie, M., Nakazawa, H., and MacNeil, JD., 1994, Improvement of chemical analysis of antibiotics.22. Identification of residual tetracyclines in honey by frit FAB LC-MS using a volatile mobile phase. Journal of Agricultural Food Chemistry 42, (10) 2215-2219 Peng, C.Y.S. C., Mussen, E., Fong, A., Cheng, P., Wong, G., and Montague, M.A., 1996, Laboratory and field studies on the effects of the antibiotic tylosin on honey bee, Apis mellifera L. (Hymenoptera: Apidae). Development and prevention of American foulbrood disease. Journal of Invertebrate Pathology 67, ., Gowik, P., 2001, Validation of a method for the detection and confirmation of nitroimidazoles and corresponding hydroxy metabolites in turkey and swine muscle by means of gas chromatographic-negative ion chemical ionisation mass spectrometry. Journal of Chromatography B, 761, 47-60. Rapid Alert System for Food and Feed (RASFF) Annual Report on functioning of the RASFF 2004, European Commission Health & Consumer Protection Directorate-General, Directorate D -Food Safety: production and distribution chain D5 -Relations with the European Food Safety Autority; Rapid Alert System, 1-38 SANCO/2004/2726: Conclusions of the meeting on the interpretation of the implementation of Decision 2002/657/EC, European Commission Health & Consumer Protection Directorate-General, Directorate D -Food Safety: production and distribution chain, D3 -Chemical and physical risk; surveillance Scortichini, G., Annunziata, L., Haouet, M.N., Benedetti, F., Krusteva, I., Galarini, R., 2005, ELISA qualitative screening of chloramphenicol in muscle, eggs, honey and milk: method validation accordino to the Commission Decision 2002/657/EC criteria. Analytica Chimica Acta 535, 43-48 Shimauki, H. in: R.A. Morse, K. Flottum (Eds.) Honey Bee, Pests, Predators and Diseases, 3 rd ed., A.I. Root Co., Medina, OH, 1997, Chapter 3.S., Noot, D.K., Calvert, J., and Pernal, S.F., 2003, Determination of lincomycin and tylosin residues in honey using solid-phase extraction and liquid chromatography -atmospheric pressure chemical ionisation mass spectrometry. Journal of Chromatography A 1020 241-250 Wang, J., 2004, Determination of five macrolide antibiotic residues in honey by LC-ESI-MS and LC-ESI-MS/MS. Journal of Agricultural and Food Chemistry 52 171-181 Vander Heyden, Y., Luypaert, K., Hartmann, C., Massart, D.L., Hoogmartens, J., De Beer, J., 1995, Ruggedness tests on the high-performance liquid chromatography assay of the United States Pharmacopeia XXII for tetracycline hydrochloride. A comparison of experimental designs and statistical interpretations. Analytica Chimica Acta 312 245-262 Vinas, P., Balsalobre, N., Lopez-Erroz, C., Hernandez-Cordoba, M., 2004, Liquid chromatography with ultraviolet absorbance detection for the analysis of tetracycline residues in honey. Journal of Chromatography A 1022(1) 125-129 Youden, W.J., Steiner, E.H., 1975, Statistical Manual of AOAC, Association of Official Analytical Chemists, Arlington Va

Figure 1 :Figure 2 :

 12 Figure 1: HPLC-ESI-MS/MS chromatograms of a reference standard solution (left side), a blank honey extract (in the medium) and a fortified honey sample submitted to extraction and clean-up procedure (right side)

  The extrapolated theoretical values for CCα and CCβ, estimated by accurate application of ISO 11843 part 2 annex B.2, corresponded to very low concentration values. Blank

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preparation was 2. Data collected were elaborated following the experimental design, indicated in the ISO 11843-2 Part 2, based on the assumptions that the obtained calibration function is linear and that the standard deviation is linearly dependent on the net state variable. The general equation is:

Y ij = a + bx i +ε ij

where x i is the symbol for the net state variable in state i, Y ij is the response variable ε ij are random variables which describe the random component of sampling, preparation and measurement error.

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	Confirmation	Collision energy
	transition	(eV)

  Intra-day and inter-day repeatibility for the determination of lincomycin and macrolides in fortified honey samples

	Compound	Spike level (µg kg -1 )	Day 1 (n=6) Mean found R.S.D. kg -1 ) (µg (%)	Recovery (%)	Day 2 (n=6) Mean found R.S.D. kg -1 ) (µg (%)	Recovery (%)	Day 3 (n=6) Mean found R.S.D. kg -1 ) (µg (%)	Recovery (%)	Inter-day (n=18) Mean found R.S.D. kg -1 ) (µg (%)	Recovery (%)
	LINCO	0.2	0.19	12.1	93.5		0.19	9.1		96.1	0.20	3.2	101.1	0.19	8.5	96.9
		0.4	0.39	7.1	96.0		0.41	5.5		102.2	0.41	5.3	102.7	0.40	6.3	100.3
		0.6	0.62	3.9	102.2		0.61	7.8		102.2	0.61	3.9	100.9	0.61	5.2	101.8
		Ccα		0.23 µg kg -1
		Ccβ		0.26 µg kg -1
	SPIRA	1.6 3.2 4.8 Ccα Ccβ	1.46 2.99 4.24	6.0 4.3 F 91.2 93.5 4.5 88.3 o 1.9 µg kg -1 r 1.41 2.85 4.29 2.1 µg kg -1	10.8 12.0 3.1	88.1 89.0 89.3	1.58 3.02 4.27	2.9 2.1 6.0	99.0 94.5 88.9	1.48 2.95 4.26	8.4 7.3 4.5	92.8 92.3 88.8
	TILMI	0.2 0.4	0.15 0.34	12.0 9.6	76.7 85.3	0.19 P 6.4 0.39 9.9		93.2 96.5	0.18 0.37	5.7 5.4	91.1 93.0	0.17 0.37	11.5 9.6	87.0 91.6
	ERYTRO	0.6 Ccα Ccβ 0.2	0.51 0.17	6.5 0.23 µg kg -1 84.8 0.25 µg kg -1 10.0 84.4		0.57 e 3.8 e 95.4 r 0.19 3.9 95.7	0.57 0.19	4.8 3.5	95.2 94.8	0.55 0.18	7.3 8.2	91.8 91.6
		0.4 0.6 Ccα Ccβ	0.35 0.54	8.6 3.4 0.22 µg kg -1 87.1 90.7 0.24 µg kg -1		0.36 0.51	1.4 2.9		90.2 85.6 R 0.40 0.55 e	4.1 5.8	99.2 91.5	0.37 0.54	7.6 5.0	92.2 89.3
	TYLO	0.2 0.4 0.6 Ccα Ccβ	0.18 0.37 0.51	6.0 5.1 6.5 0.22 µg kg -1 90.4 91.7 84.7 0.24 µg kg -1		0.17 0.36 0.55	3.5 6.2 5.2	Table 2 86.3 89.0 91.3	0.17 v 5.9 0.34 3.4 i 85.3 86.2 0.53 3.3 87.9 e w	0.17 0.36 0.53	5.6 5.4 5.8	87.3 89.0 88.0
	JOSA	0.2	0.16	6.2	80.2		0.16	11.8	82.4	0.20	3.0	97.8	0.17	11.6	86.8
		0.4 0.6 Ccα Ccβ	0.33 0.49	3.5 3.0 0.24 µg kg -1 82.3 82.3 0.26 µg kg -1		0.33 0.50	8.3 1.6		83.6 83.2	0.38 0.52	1.8 2.4	94.8 87.0 O 0.35 0.51 l 8.2 3.4 n	86.9 84.2
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Table 3

 3

	Selected variables	Units	Abbreviation a	High level	Low level
	Volume TRIS Buffer	ml	A,a	40	30
	Molarity TRIS Buffer	M	B,b	0.11	0.09
	pH TRIS Buffer	pH	C,c	11.5	9.5
	%MeOH in SPE washing solution	%	D,d	5.5	4.5
	%NH 3 in SPE elution solvent	%	E,e	5.5	4.5
	SPE elution volume	ml	F,f	11	9
	Final extract evaporation temperature	°C	G,g	55	45
	a Upper case letter represents high level, lower case letter represents low level value of variable according to the
	r o F experiment design for ruggedness studies.
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Table 5

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Table 1 :

 1 MS/MS conditions for detection, quantification and confirmation ofTYLO, TILMI, ERYTRO, JOSA, LINCO and SPIRA in MRM mode.

Table 2 :

 2 CCα and CCβ of analytes calculated by calibration curve procedure according to the ISO 11843-2. Values reported for each reference state correspond to the ratio between the area of each analyte and the area of the internal standard.

Table 3 :

 3 validation data for honey spiked samples (n = 6 of each fortification level on each day, three series on three different days)

Table 4 :

 4 Variables and their levels in the Youden robustness test experimental design (see table 11 of Commission Decision 2002/657/EC)

Table 5 :

 5 Ruggedness test resultsFigure1: HPLC-ESI-MS/MS chromatograms of a reference standard solution (0.2 µg kg -1 of TYLO, TILMI, ERYTRO, JOSA; LINCO and 1.6 µg kg -1 of SPIRA, left side), a blank honey extract (in the medium) and a fortified honey sample at 0.2 µg kg -1 of TYLO, TILMI, ERYTRO, JOSA; LINCO and 1.6 µg kg -1 of SPIRA (right side). blank honey extract (in the medium) and a fortified honey sample at 0.2 µg kg -1 of TYLO, TILMI, ERYTRO, JOSA; LINCO and 1.6 µg kg -1 of SPIRA (right side).

	Quantification transitions.
	Figure 2: HPLC-ESI-MS/MS chromatograms of a reference standard solution (0.2 µg
	kg -1 of TYLO, TILMI, ERYTRO, JOSA; LINCO and 1.6 µg kg -1 of SPIRA, left side), a

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