Niki C Maragou

Athina Makri

Eugenia N Lampi

Nikolaos S Thomaidis

Michael A Koupparis

Migration of bisphenol A from polycarbonate baby bottles under real use conditions

Keywords: Chromatographic analysis, Chromatography -HPLC Additives/Contaminants: Bis-phenol A, Food contact materials, Food simulants, Simulants BPA, xenoestrogens, migration, infants, feeding bottles, HPLC-FLD, LC-MS, polymer hydrolysis

Migration of the potential endocrine disrupter bisphenol A (BPA) from 31 polycarbonate (PC) baby bottles into aqueous food simulants was studied under real repetitive use, with a sensitive and fully validated liquid chromatographic method with fluorescence detection.

Confirmation of the presence of BPA was performed by liquid chromatography-mass spectrometry (LC-MS). The effects of cleaning with dishwasher or with a brush, sterilization with boiling water and the temperature of migration were examined. It was shown that temperature was the crucial factor for the migration of BPA from the plastic bottles to water. All the samples released BPA in the concentration range of 2.4-14.3 µg kg - 1 , when they were filled with boiled water and left at ambient temperature for 45 min. The decrease of BPA release in the sterilization water and in the food simulant over twelve cycles of use indicated that the hypothesis of polymer degradation in water is rather doubtful. The estimated infantile dietary exposure, regarding the use of PC baby bottles, ranged between 0.2 and 2.2 µg kg -1 bw d -1 , which is below the Tolerable Daily Intake of 50 µg kg -1 bw, recently established by EFSA.

Introduction

It is widely known that bisphenol A (BPA) is used as a monomer in the synthesis of polycarbonates (PC), a type of polymer that is utilized in the manufacture of plastic food containers, such as infant feeding bottles. Exhibiting high transparency, low weight and high heat and impact resistance, polycarbonate plastics are increasingly replacing glass in such applications (Plastics Europe 2007).

In the early 1990s, it was reported that BPA, was released from polycarbonate flasks during autoclaving, exhibits estrogenic activity in in vitro assays at concentrations of 10-25 nM (2-5 ng ml -1 ) [START_REF] Krishnan | Bisphenol-A: An estrogenic substance is released from polycarbonate flasks during autoclaving[END_REF]. Later studies reported that exposure to low doses of BPA causes reproductive toxic effects, such as increases in murine prostate gland and reduced sperm efficiency [START_REF] Saal | A physiologically based approach to the study of bisphenol A and other estrogenic chemicals on the size of reproductive organs, daily sperm productions, and behavior[END_REF]. Additionally, recent studies state that BPA exposure affects brain development of rats at a very low dose range of 10 -12 -10 -10 M (0.23 -23 pg ml -1 ) (Zsarnovszky et al 2005), and brain development of murine at a higher dose of 3 µg g -1 [START_REF] Tando | Effects of pre-and neonatal exposure to bisphenol A on murine brain development[END_REF].

The presence of unreacted BPA in PC baby bottles has been reported, and the residual amount of the monomer was found to range from 7 to 58 µg g -1 in samples purchased in Washington [START_REF] Biles | Determination of bisphenol A in reusable polycarbonate food-contact plastics and migration to food-simulating liquids[END_REF] and from < 3 to 141 µg g -1 in samples purchased in Singapore [START_REF] Wong | Dietary exposure assessment of infants to bisphenol A from the use of polycarbonate baby milk bottles[END_REF]. The occurrence of BPA in the plastic, the fact that baby bottles are intended to be used repeatedly and the potential adverse effects of BPA at low doses, especially regarding infants, has generated the need for a thorough investigation of BPA migration from this type of food contact material.

Migration studies of BPA from infant feeding bottles have been conducted under diverse treatments and diverse conditions using water (Biles et al 1997, Kawamura et [START_REF] Sun | High-Performance liquid chromatography with peroxyoxalate chemiluminescence detection of bisphenol A migrated from polycarbonate baby bottles using 4-(4,5-diphenyl-1Himidazol-2-yl)benzoyl chloride as a label[END_REF][START_REF] D'antuono | Determination of bisphenol A in food-simulating liquids using LCED with a chemically modified electrode[END_REF][START_REF] Brede | Increased migration levels of bisphenol A from polycarbonate baby bottles after dishwashing, boiling and brushing[END_REF], ethanol/water mixtures [START_REF] Biles | Determination of bisphenol A in reusable polycarbonate food-contact plastics and migration to food-simulating liquids[END_REF][START_REF] Kawamura | Migration of Bisphenol A from polycarbonate products[END_REF][START_REF] D'antuono | Determination of bisphenol A in food-simulating liquids using LCED with a chemically modified electrode[END_REF][START_REF] Wong | Dietary exposure assessment of infants to bisphenol A from the use of polycarbonate baby milk bottles[END_REF], 3-4% acetic acid [START_REF] Kawamura | Migration of Bisphenol A from polycarbonate products[END_REF][START_REF] D'antuono | Determination of bisphenol A in food-simulating liquids using LCED with a chemically modified electrode[END_REF], n-heptane [START_REF] Kawamura | Migration of Bisphenol A from polycarbonate products[END_REF] and oil [START_REF] Biles | Determination of bisphenol A in reusable polycarbonate food-contact plastics and migration to food-simulating liquids[END_REF][START_REF] Kawamura | Migration of Bisphenol A from polycarbonate products[END_REF][START_REF] Wong | Dietary exposure assessment of infants to bisphenol A from the use of polycarbonate baby milk bottles[END_REF] as food simulants.

In particular, [START_REF] Biles | Determination of bisphenol A in reusable polycarbonate food-contact plastics and migration to food-simulating liquids[END_REF] reported that residual BPA observed to migrate from PC bottle to 10% aqueous ethanol at 100 ºC for 30 min over four cycles of use, decreased rapidly after an initial "bloom" and then leveled out. Accordingly, migration tests of PC nursing bottles with water at 95 ºC for 30 min for four or five repeated cycles revealed a decrease since the first elution of BPA [START_REF] Kawamura | Migration of Bisphenol A from polycarbonate products[END_REF][START_REF] Sun | High-Performance liquid chromatography with peroxyoxalate chemiluminescence detection of bisphenol A migrated from polycarbonate baby bottles using 4-(4,5-diphenyl-1Himidazol-2-yl)benzoyl chloride as a label[END_REF]. Moreover it has been shown that the amount of BPA eluted to water at 95 ºC for 30 min was greatly decreased by washing and boiling the bottle [START_REF] Kawamura | Migration of Bisphenol A from polycarbonate products[END_REF]. In contrast, another migration study conducted in twelve PC baby bottles supports that dishwashing, boiling and brushing, significantly increased the migration of BPA during incubation with water at 100 ºC for 1 h [START_REF] Brede | Increased migration levels of bisphenol A from polycarbonate baby bottles after dishwashing, boiling and brushing[END_REF]. The authors attributed these findings at possible polymer degradation. The increase of BPA migration to water due to PC degradation has not been verified experimentally by other researchers. Although decomposition of PC has been reported before, only an alcoholic simulant consisted of 50% ethanol or more in water could provoke this observation [START_REF] Biles | Determination of bisphenol A in reusable polycarbonate food-contact plastics and migration to food-simulating liquids[END_REF]. This obscurity concerning the release of BPA from reusable PC baby bottles, the possible decomposition of polycarbonate material and the conditions that could lead to this phenomenon, reveal the need for further investigation on this subject.

The objective of this study is to contribute to the elucidation of BPA migration from PC baby bottles during realistic and repeated handling. The material of the baby bottles was FLD). After validation of the HPLC-FLD method, the effect of various parameters of simulated use with water and acidic food simulants was investigated. The effects of sterilization in boiling water, the way of cleaning (dishwasher or brush) and the effect of different temperatures to two different brand names, were examined by two experimental procedures. Subsequently, a third experimental procedure of twelve cycles of realistic use of baby bottles was applied to four other brand names, including cleaning of the bottles with brush, sterilization in boiling water for 10 min, filling with hot water and leaving at ambient temperature for 45 min. In addition, the sterilization water of the twelve cycles was also analyzed for BPA in order to monitor the behavior of PC bottles during sterilization, and investigate the possibility of polymer degradation under these exaggerated conditions. The presence of BPA was confirmed by LC-MS analysis.

Materials and methods

Chemicals

Standard BPA (≥97%) was purchased from Fluka (Buchs, Switzerland). Stock solution (375 mg l -1 ) was prepared in methanol and used for further dilutions. The stock solution was stored at -15 º C for six months. Methanol of HPLC grade was obtained from LAB SCAN (Dublin, Ireland) and acetic acid of 99.9% purity from Carlo Erba (Rodano, Italy).

Water, used as HPLC solvent, as simulant for the migration tests and for the preparation of

Baby bottle samples

A total of thirty one plastic baby bottles of six different brand names (A-F) were purchased from the Greek market and were used for the migration trials (Table I). The maximum volume of the bottles, up to which they were filled for the migration tests, ranged from 120 to 250 ml.

Identification of baby bottles material with FT-IR

Three bottles of three different brand names were analysed with a Perkin Elmer FT-IR System 2000 (Bucks, England). A small piece of each sample was finely pulverized and mixed with KBr at a ratio of approximately 1:10. The mixture was compressed into an evacuable pellet die in conjunction with a 15 ton manually operated hydraulic press (Graseby Specac, Kent, UK). The produced KBr pellet was placed in the cell of the spectrometer and scanned within the frequency range of 4000-370 cm -1 . The obtained spectra of transmittance were compared by a matching algorithm (Euclidean search) to the spectra of known polymers available in BIO-RAD Sadtler Library (Philadelphia, Pennsylvania). The best match of the Euclidean search list was polycarbonate resin for all the tested baby bottles. BPA was determined with a Hewlett-Packard 1050 series HPLC system, equipped with an HP 1046 fluorescent detector and an HP 1100 autosampler. Injections of 25 µl were performed into an Inertsil ODS-2 (250 mm × 2.1 mm, 5 µm) C18-bonded reverse phase column. The isocratic elution was carried out with a mixture of methanol/water 70:30 v/v at a flow rate of 1.3 ml min -1 . The solvents were degassed using helium prior to analysis. The fluorescent detector was set at 235 nm for the excitation and at 317 nm for the emission.

The HPLC system was operated by HP Chemstation software.

Standard working solutions were prepared in water and 3% w/v acetic acid. In order to cover a wide range of concentrations including very low levels of BPA expected from the migration tests but also including the European Specific Migration Limit (SML 0.6 µg ml - 1 ), two calibration curves were constructed for each simulant, one for low concentrations and one for high concentrations. Low concentration calibration curves were obtained by measuring standard working solutions at five levels in the range 7.5 -180 ng ml -1 , with two replicates per concentration. Similarly, high concentration calibration curves were obtained by measuring standard working solutions at five levels in the range 0.18 -6.0 µg ml -1 with two replicates per concentration. Linear regression analysis was performed using the analyte peak area versus analyte concentration.

For the determination of the LOD, standard solutions of the three lowest concentrations were analyzed ten times. For every concentration level the mean concentration, C and the standard deviation, SD, of the ten replicates were calculated and linear regression analysis was performed using standard deviations against mean values of concentrations (SD = b× C + S o ). The LOD was estimated using the intercept, S o of the obtained equation curve Within run precision was expressed as the relative standard deviation of ten replicate measurements of a standard solution at three different concentrations for water (30, 75 and 180 ng ml -1 ) and at three different levels for 3% w/v acetic acid (15, 30 and 75 ng ml -1 ).

Between run precision was expressed as the relative standard deviation of six independent replicate analyses (preparation and measurement) of a standard solution of 30 ng ml -1 for both simulants.

LC-MS analysis

Identification of BPA was carried out according to a method described previously [START_REF] Maragou | Determination of bisphenol A in milk by solid phase extraction and liquid chromatography-mass spectrometry[END_REF]. A Thermo Finnigan LC-MS system (San Jose, USA) consisted of a Spectra System P 4000 pump, a Spectra System AS 3000 autosampler with the volume injection set to 10 µl was used with a Surveyor MSQ quadrupole mass spectrometer equipped with an electrospray ionization LC-MS interface (ESI). In brief, the chromatographic separation was performed using a LiChrospher 100-RP18 (250 mm × 4 mm, 5 µm) reversed phase LC column and isocratic elution with a mobile phase consisted of methanol-water (70:30 v/v) at a flow rate of 0.9 ml min -1 . ESI was applied in the negative ionization mode and the capillary was held at a potential of 3.5 kV. The cone voltage was 70 V and the ionization source was set at a temperature of 500 ºC. The full scan spectrum (m/z 100-300) and the single ion monitoring (SIM) chromatograms of the deprotonated molecule of BPA (m/z 227) were acquired at every analysis. The study of BPA migration from PC baby bottles was divided into three experimental procedures (I-III), which differ in the sample treatment before the migration test and/or in the migration conditions. In all cases the bottle treatment and the migration conditions were selected so as to be representative of real use. Water and 3% acetic acid (w/v) in aqueous solution were used as food simulants for the migration testing. Water was used as simulant for milk and 3% w/v acetic acid was used as simulant for fruit juices, according to the Directive in force, 85/572/EEC. Regarding the water as milk simulant, it has been noted that milk should not be experimentally modelled by water [START_REF] Franz | Migration modelling from food-contact plastics into foodstuffs as a new tool for consumer exposure estimation[END_REF], but this argument was based on the results of a migration experiment with a very lipophilic migrant (1,4diphenyl butadiene). On the contrary, BPA is a compound with polar characteristics, containing two hydroxyl groups and with a reported water solubility of 120-300 mg l -1 [START_REF] Staples | A review on the environmental fate, effects, and exposures of Bisphenol A[END_REF]. Additionally, a typical preparation of milk consists of pouring boiling water in the baby bottle, which seems to be the critical point for BPA migration, as it appears from the results of this work. After that, addition of the milk powder follows.

Table I summarizes the number of baby bottles of each brand name, the experimental procedure to which each group was subjected and the simulant used. Before the very first use, all the specimens were sterilized in boiling water for 5 min according to the manufacturer's instructions.

[Insert Table I about were subjected to ten cycles of simulated use with water as simulant. In each cycle, the bottles were washed in a dishwasher at 60 ºC using a common detergent and distilled water for the final wash. Subsequently the bottles were sterilized for 10 min in boiling water, and then they were filled up to the mark with the simulant. Finally, they were incubated for 2h at 70 ºC (Table II). Exactly the same procedure, consisted of ten cycles, was applied to three new bottles of brand name A and three new bottles of brand name B using 3% w/v acetic acid as a simulant.

Experimental Procedure II. In the second experimental procedure (II) the impact of brushing during cleaning, the impact of sterilization and the effect of the incubation temperature and time were examined. The eight bottles of labels A and B, which had already been subjected to the experimental procedure I with water, were used to this experimental procedure, as well, with the same simulant. For this reason the numbering of cycles starts from eleven. This migration test was also consisted of ten cycles of simulated use. From the eleventh to the fourteenth cycle the bottles were brushed with detergent under tap water and rinsed with distilled water. After the cleaning step the bottles were sterilized for ten minutes in boiling water and then they were filled up to their maximum volume with the simulant and were incubated for 2h at 70 ºC. The objective of these four cycles was to examine the impact of brushing during cleaning. The results were compared with those obtained from the experimental procedure I where dishwasher was used. In the fifteenth cycle, the bottles were washed only with distilled water, without brushing and II summarizes the steps of simulated treatment and the migration conditions for the three experimental procedures (I, II and III) where water was used as simulant. Acetic acid was not used to experimental procedures II and III as it is not expected to drink fruit juices at such high temperatures.

[Insert Table II about here]

Results and discussion

HPLC-FLD method performance

Table III shows the equations of the low and high concentration calibration curves generated from standards prepared in water and in 3% w/v acetic acid. The standard deviation of the slope and the intercept, and the correlation coefficient of every equation are also given. It is shown that linear calibration curves were obtained with correlation coefficients always exceeding 0.999 for both simulants. The method LOD for water and 3% acetic acid simulant were found to be 2.4 and 1.8 ng ml -1 , respectively, without any preconcentration applied. These values are sufficient for safety control of the materials intended to come to contact with food since they are much lower than the specific migration legislative limit of BPA (0.6 mg kg -1 or µg ml -1 , assuming that the density of aqueous simulants is 1 g ml -1 ) (Directive 2004/19/EEC). In addition, the LOD of the method is at a concentration level that permits the study of BPA migration and at concentration levels at which it has been reported that BPA can exhibit estrogenic activity [START_REF] Krishnan | Bisphenol-A: An estrogenic substance is released from polycarbonate flasks during autoclaving[END_REF][START_REF] Saal | A physiologically based approach to the study of bisphenol A and other estrogenic chemicals on the size of reproductive organs, daily sperm productions, and behavior[END_REF]. The within-run precision, expressed as % RSD 13 (N=10), at 30, 75 and 180 ng ml -1 of water simulant was 2.2, 1.7 and 1.0, respectively. The corresponding values for 3% w/v acetic acid at 15, 30 and 75 ng ml -1 were 4.4, 3.0 and 2.2, respectively. The between run precision at 30 ng ml -1 , expressed as % RSD (N=6), was 2.9 for water and 4.2 for 3% w/v acetic acid. These values indicate the satisfactory performance of the method.

[Insert Table III about here]

BPA release during migration testing and sterilization

Table IV shows the migration of BPA, as amount of substance (ng) per simulant volume (ml), from the eight baby bottles (brand names A and B) that were subjected to experimental procedure I and subsequently to experimental procedure II, where the used simulant was water. Table V illustrates the corresponding migration results from the seventeen new feeding bottles of brand names C-F that were subjected to procedure (III). In Tables IV andV, BPA migration is expressed as the mean value of N identical migration experiments (N bottles of the same brand name) ± the standard error. Standard error (S.E.) is defined as SD / N , where SD stands for the standard deviation of the N values.

Concerning the migration from PC bottles to the acidic simulant, none of the six specimens released detectable amounts of BPA into 3% w/v acetic acid at 70 ºC for 2h, throughout the ten cycles of the experimental procedure I (< 1.8 ng ml -1 ). Our results are in agreement with previous work that reported non detectable BPA (< 0.5 ng ml -1 ) after migration test of nursing bottle with 4% acetic acid at 60 ºC for 30 min [START_REF] Kawamura | Migration of Bisphenol A from polycarbonate products[END_REF]. The temperature applied at our migration test (70 ºC) rather exceeds realistic handling, provided that 3% w/v acetic acid is representative of fruit juices, a food which is 14 not expected to be heated. Therefore subjection of the baby bottles to more exaggerated conditions with this simulant was not considered essential. However, migration of BPA from baby bottles to 3% acetic acid, at a level of 2.2 ng ml -1 , has been reported when a higher temperature of 80 ºC was applied for a shorter time of 2 min (D'Antuono et al

2001). [Insert Table IV about here] [Insert Table V about here]

The results of the migration tests of cycles 1-10 and 11-14 (Table IV) demonstrate that the repetitive cleaning of bottles either with dishwasher or with brushing (Table II), do not lead to detectable BPA migration (< 2.4 ng ml -1 ) when incubation is taking place with water at 70 ºC for 2h. From the comparison of the two sets of cycles it can be concluded that brushing, which is supposed to increase the wear of bottles, is not likely to influence BPA migration remarkably. The objective of the bottle treatment of the fifteenth cycle was to investigate whether the absence of BPA migration in the previous cycles was ascribed to the fact that the available BPA in the plastic was washed away during brushing and sterilization, and therefore there was no substance left to migrate during incubation. The results indicate that this was not the case, since detectable BPA migration was not observed when neither brushing nor sterilization had preceded. Migration was observed only when the bottles were filled with boiling water (100 ºC) in the sixteenth cycle and afterwards.

From the results of the migration tests of experimental procedure I and II (Table IV) it The data from the cycles 16-20 of procedure II (Table IV) and the data from the cycles 1-12 of procedure III (Table V) show that the very first contact of the PC material with boiling water causes leaching of BPA to the aquatic medium at a concentration ranging from 3.9 to 13.8 ng ml -1 . After four to eight subsequent cycles (depending on the brand name), migration decreases to a level below 2.4 ng ml -1 . In experimental procedure III no migration was observed during the last four cycles 9-12, and for this reason the procedure was not continued further. Considering that the treatment of baby bottles during cycles 16-20 (Exp.Pr. II) and cycles 1-12 (Exp.Pr. III) (Table II) is representative of typical preparation of milk, it could be assumed that BPA migration occurs during actual domestic use of PC baby bottles at least at the first four to eight cycles of use.

Moreover, sterilization water from the cycles of procedure (III) was analyzed and measurable BPA was present in all the samples (Figure 1a,b). The amount of BPA released per bottle during sterilization was calculated by multiplying the concentration of the measured subsample with the actual water volume in the kettle (1000 ml), and then dividing with the number of bottles put in the kettle.

[Insert Figure 1 water declines with the extension of handling. The data clearly reveal that no degradation occurs during sterilization by boiling the bottles in water for 10 min. Besides, due to the insolubility of PC in water, the aqueous depolymerization requires severe conditions such as long reaction times, high temperature and pressure, whereas in alcali-catalyzed methanol-toluene medium, complete PC degradation occurs at 60 ºC in 70 min [START_REF] Hu | Alkali-catalyzed methanolysis of polycarbonate. A study on recycling of bisphenol A and dimethyl carbonate[END_REF].

Based on the findings of Table V and those of Fig. 1, it could be assumed that there is a certain amount of residual BPA available on the surface of the PC bottles which is released during sterilization and during incubation with hot water, but it is not likely that additional BPA is yielded throughout the repeated treatment. These findings are in contrast with a previous work [START_REF] Brede | Increased migration levels of bisphenol A from polycarbonate baby bottles after dishwashing, boiling and brushing[END_REF], which reported significant increase of BPA migration from baby bottles to water due to use. Polymer degradation was proposed for the explanation of those results. However, it should be noted that migration of BPA was measured only three times in that work, after the first use, after 51 cycles and after 169 cycles of washing, and that the increase of migration was observed between first use (0.23 ng ml -1 ) and 51st cycle (8.4 ng ml -1 ) but there was no trend between 51st and 169th cycle (6.7 ng ml -1 ).

On the other hand, it has been reported that migration up to 368% of the available BPA occurred when 50% ethanol in water was used as simulant at 65 ºC for 240 h, indicating hydrolysis of the polymer [START_REF] Biles | Determination of bisphenol A in reusable polycarbonate food-contact plastics and migration to food-simulating liquids[END_REF]. It should be mentioned that in the 4th amendment of the European Directive 2002/72/EC for plastics, which is expected to be published soon, 50% ethanol in water replaces water in simulation tests for milk contact. In 17 that case BPA migration may be higher and further investigation on this issue may be required.

The presence of BPA in sterilization water does not cause any concern regarding the dietary exposure of infants, since the residual water is rejected at domestic use. However, this data can give us an indication for the domestic contribution to environmental pollution, as this water would normally result in an urban sewerage.

Confirmation of the presence of BPA with LC-MS

All the BPA peaks detected by HPLC-FLD were confirmed by LC-MS. The presence of BPA was confirmed when the most abundant ion of the eluted peak corresponded to the ratio m/z 227, which has been assigned as the deprotonated molecule [M-H] -of BPA (MW 228). Figure 2 [Insert Figure 2 about here]

Infantile dietary exposure and safety

Our findings demonstrate a measurable migration of BPA to water simulant in the range of 2.4 -14.3 µg kg -1 (Tables IV andV, assuming that the density of aqueous simulants is 1 g ml -1 ). Considering this migration level and that an infant of 0 to 4 months old, weighs 4.5 18 kg and consumes 0.7 l of formula each day, which is the highest ratio of food intake to body weight for infants, according to the opinion of the Scientific Committee on Food [START_REF] Scf | Opinion of the Scientific Committee on Food, on Bisphenol A[END_REF], the estimated intake is 0.4-2.2 µg kg -1 bw d -1 . In the same way the estimated intake for an infant of 6 to 12 months old, that weighs 8.8 kg and consumes 0.7 l, ranges between 0.2 and 1.1 µg kg -1 bw d -1 . This assessment on infant dietary exposure to BPA from polycarbonate baby bottles is slightly lower than the dietary estimates presented in the latest opinion of [START_REF] Efsa | Opinion of the Scientific Panel on food additives, flavourings, processing aids and materials in contact with food on a request from the commission related to 2,2-bis(4-21 hydroxyphenyl)propane (Bisphenol A). Question number EFSA-Q-2005-100[END_REF]. EFSA proposed that a conservative estimate of total dietary exposure to BPA at 3 month old infant, who is fed formula with PC bottle, is 11 µg kg -1 bw d -1 based on the upper value of 50 µg BPA l -1 of infant formula, and 4 µg kg -1 bw d -1 based on the typical value 10 µg BPA l -1 of infant formula.

Our results suggest that BPA migration from polycarbonate baby bottles are unlikely to be of concern, according to the recently established full TDI (Tolerable Daily Intake) of 0.05 mg kg -1 bw by [START_REF] Efsa | Opinion of the Scientific Panel on food additives, flavourings, processing aids and materials in contact with food on a request from the commission related to 2,2-bis(4-21 hydroxyphenyl)propane (Bisphenol A). Question number EFSA-Q-2005-100[END_REF], derived by applying a 100-fold uncertainty factor to the overall NOAEL (No Observed Adverse Effect Level) of 5 mg kg -1 bw d -1 .

On the other hand, the up-to-date findings of scientific investigations on the adverse effects of BPA in vitro and in vivo have led to an intense controversy regarding the levels of BPA that can be hazardous to humans. In a recent review, concerning low-dose effects of BPA, it is reported that in 31 publications with vertebrate and invertebrate animals, significant effects occurred below the reference dose of 50 µg kg -1 bw d -1 (Vom Saal and Hughes 2005), while in another recent review the updated weight of evidence does not support the hypothesis that low-doses of ≤ 5 mg kg -1 bw d -1 adversely affects human reproductive and developmental health [START_REF] Goodman | An updated weight of the evidence evaluation of reproductive and developmental effects of low doses of bisphenol A[END_REF]. However, it should be mentioned that harmful effects on fertility have been reported after dietary exposure of 19 BPA to 10 member groups of male mice at doses of 0.025 and 0.1 µg kg -1 bw d -1 (Al-Hiyasat et al 2002), and to 6 member groups of male rats at doses of 0.2, 2 and 20 µg kg -1 bw d -1 [START_REF] Chitra | Induction of oxidative stress by bisphenol A in the epididymal sperm of rats[END_REF]. These levels of BPA doses enclose the estimated infant intake of the present work (0.2 -2.2 µg kg -1 bw d -1 ). Additionally, it should be taken into consideration that infants are a susceptible group, possibly exposed to other xenoestrogens except for BPA [START_REF] Kuo | Trace determination of bisphenol A and phytoestrogens in infant formula powders by gas chromatography-mass spectrometry[END_REF].

Taken together, further research on the low dose effect of bisphenol A and on the combined impact of different endocrine disrupters, potentially present in baby feeding bottles or baby food, would be essential for a clearer and more realistic estimation of hazard.

Conclusions

The present study indicates that temperature is the critical factor that favours BPA migration from PC bottles to water. It was shown that migration of residual BPA takes place at a concentration range of 2.4 -14.3 µg kg -1 , when boiling water is poured into PC baby bottles, mainly during the first eight cycles of such use. Moreover, it was demonstrated that BPA release during sterilization decreases throughout twelve cycles of use, indicating that polymer degradation does not occur during boiling in water for 10 min.

The estimated dietary exposure for infants aged up to one year old ranges between 0.2 and 2.2 µg kg -1 bw d -1 . Even though these values are far below the recently established TDI [START_REF] Efsa | Opinion of the Scientific Panel on food additives, flavourings, processing aids and materials in contact with food on a request from the commission related to 2,2-bis(4-21 hydroxyphenyl)propane (Bisphenol A). Question number EFSA-Q-2005-100[END_REF], it should not be ignored that there are indications for hazardous effects at these low doses [START_REF] Darmani | Effects of bisphenol A on adult male mouse fertility[END_REF][START_REF] Chitra | Induction of oxidative stress by bisphenol A in the epididymal sperm of rats[END_REF].

  -IR analysis and the determination of BPA was performed with a sensitive method based on liquid chromatography with fluorescence detection (HPLC-

  solutions, was purified with a Milli-Q water system (Millipore, Bedford, MA, USA).

  defined as 3 × S o . The same methodology was applied for the estimation of LOD for both simulants.

  . In the first experimental procedure (I) the impact of the repetitive use of the dishwasher and the kind of the simulant on the migration of BPA were examined. Four new bottles from brand name A and four new bottles from brand name B

  afterwards they were filled with the simulant and incubated for 2h at 70 ºC. The purpose of the fifteenth cycle was to investigate whether brushing and sterilization remove residual BPA from the plastic surface. During the last five cycles, the bottles were brushed and sterilized as it has already been described for cycles 11-14. Afterward the baby bottles were filled with boiling water and left at room temperature for 45 min (TableII).The conditions of these last five cycles of use were applied as the most representative of actual preparation of milk.Experimental Procedure III. Based on the findings of the experimental procedures I and II and with the intention of expanding this study to a larger number of brand names we proceeded to experimental procedure III. Seventeen more new bottles of the brand names C, D, E and F were subjected to twelve repeated cycles of simulated use. Each cycle consisted of brushing, sterilization, filling with boiling water and incubation at room temperature for 45 min, as it has already been described for cycles 16-20 of experimental procedure II (TableII). In this procedure, each group of bottles of the same brand name was sterilized in one kettle and different kettles were used for the different brand names. The sterilization water of each group was collected and measured in order to investigate the release of BPA during this step and the possible degradation of the polymer.After the expiration of the incubation time of every migration experiment, each simulant was transferred in an Erlenmeyer flask and homogenized by manual agitation. Then, a subsample of the simulant was measured with the validated method of HPLC-FLD. For the quantification of BPA in the solutions of the migration experiments and the sterilization water, external standard calibration was applied.

  temperature is the only critical factor for the increase of BPA migration from PC baby bottles to water. In particular the contact of the surface of polycarbonate bottles with boiling water (100 ºC) seems to affect significantly the migration of BPA.

  observed that in all cases (Figure1a,b) the release of BPA into sterilization

  presents the SIM chromatograms (m/z 227) and the full mass spectra at the retention time of the elution of BPA peak, of a standard solution containing 80 ng ml -1 of BPA (Fig 2a) and of a water simulant containing approximately 2 ng ml -1 after migration testing (Fig 2b).

Figure 1 .Figure 2 .

 12 Figure 1.

Table I .

 I Number of baby bottles of different brand names used for each experimental procedure

	Brand name	Number of samples	Experimental Procedure	Simulant
	A	3	I	3% w/v acetic acid
	B	3	I	3% w/v acetic acid
	A	4	I + II	water
	B	4	I + II	water
	C	4	III	water
	D	4	III	water
	E F F o r	6 3	III III	water water
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Table II .

 II Sample treatment and migration conditions of the three experimental procedures using water as simulant

	Experimental Procedure	Cycle	Cleaning	Sterilization*	Migration conditions
	I		1-10	Dishwasher, detergent, 60 ºC	Yes	70 ºC, 120 min
			11-14	Brushing with detergent	Yes	70 ºC, 120 min
	II		15	Wash only with water	No	70 ºC, 120 min
	III	16-20 1-12 F o r Brushing with detergent Brushing with detergent	Yes Yes	Filling with boiling water and left at ambient temperature for 45 min Filling with boiling water and left at ambient temperature for 45 min
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Table III .

 III Calibration curves of BPA standard solutions in water and in 3% aqueous acetic acid for low and high concentrations. C represents the concentration of the measured solution (ng ml -1 or µg ml -1 ). Calibration points: 5

	Simulant	Concentration range	Calibration Curve	r
	water	7.5 -180 ng ml -1	y = (-0.18 ± 0.05) + (36.8 ± 0.5)×10 -3 C	0.9994
		0.18 -6.0 µg ml -1	y = (-2.1 ± 1.5) + (44.3 ± 0.4) C	0.9998
	3% acetic acid	7.5 -180 ng ml -1	y = (-0.01 ± 0.04) + (38.5 ± 0.4)×10 -3 C	0.9998
	0.18 -6.0 µg ml -1 F o r	y = (2.6 ± 4.3) + (46.1 ± 1.2) C	0.9991
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Table IV .

 IV Migration of BPA to water during experimental procedure I (cycles 1-10) and experimental procedure II (cycles 11-20) for brand names A and B. BPA

	Food Additives and Contaminants				Page 30 of 31
	C	: mean concentration,	. S : standard error .E
	No of cycle	C BPA	±	S	.	E	.	( ng	ml	-	1	)
		A (N=4)							B (N=4)
	1-10	< LOD								< LOD
	11-14	< LOD								< LOD
	15	< LOD								< LOD
	16	13.3 ± 0.2							13.8 ± 0.5
	17 18 19 20 r F o	13.3 ± 0.2 10.3 ± 2.6 13.3 ± 0.5 4.4 ± 2.8							10.6 ± 2.7 14.3 ± 0.4 4.2 ± 2.6 < LOD
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http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Table V .

 V Migration of BPA to water during the twelve cycles of procedure III for brand names C, D E and F

	No of cycle		C BPA	±	S	.	E	.	( ng	ml	-	1	)
		C (N=4)	D (N=4)					E (N=6)	F (N=3)
	1	3.9 ± 2.3	10.6 ± 2.7					5.8 ± 2.7	5.5 ± 3.5
	2	4.7 ± 1.0	2.6 ± 1.2					3.7 ± 1.0	3.2 ± 1.6
	3	3.3 ± 1.0	5.2 ± 3.5						< LOD	2.6 ± 1.2
	4	< LOD	2.7 ± 1.3					4.8 ± 1.5	2.8 ± 1.3
	5	2.4 ± 1.0	3.6 ± 1.2					2.8 ± 0.9	2.8 ± 1.3
	6	3.9 ± 1.3	5.3 ± 1.2					3.8 ± 1.1	3.1 ± 1.6
	7	3.9 ± 1.3	< LOD						2.4 ± 0.7	< LOD
	8	2.7 ± 1.2	< LOD						4.8 ± 2.2	< LOD
	9 -12	< LOD	< LOD							< LOD	< LOD

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


Michael Sulyok

Rudolf Krska
email: rudolf.krska@boku.ac.at

Rainer Schuhmacher

Application of a liquid-chromatography -tandem mass 1 spectrometric multi-mycotoxin method to different raw cereals 2 and evaluation of matrix effects 3 4

Keywords: 30 mycotoxins, multi-target analysis, liquid chromatography, tandem mass spectrometry, matrix 31 effects

Application of a

Introduction 34

In the past few years, a trend towards the use of liquid chromatography-tandem mass 35 spectrometry (LC-MS/MS) in mycotoxin analysis has been observed (Sforza et al. 2006, 36 Krska and[START_REF] Krska | Mycotoxins: State-of-the-art and future trends[END_REF]. In contrast to most screening methods for mycotoxins that are 37 based on immunoassays, unambiguous analyte confirmation can be obtained by mass 38 spectrometry (Zöllner and Mayer-Helm 2006). The broad applicability of this analytical 39 approach enables the development of multi-analyte methods and thus the simultaneous 40 screening of different classes of mycotoxins, which is essential to assess the health risks Closely related compounds that are not occurring in naturally contaminated samples and show 69 similar chemical structures as the target toxins, such as verrucarol, deepoxy-deoxynivalenol 70 and zearalanone, are frequently applied as internal standards [START_REF] Zöllner | Determination of zearalenone and its 501 metabolites αand β-zearalenol in beer samples by high performance liquid chromatography-502 tandem mass spectrometry[END_REF], van 71 Bennekom et al. 2002[START_REF] Royer | Quantitative analysis of Fusarium mycotoxins in maize 460 using accelerated solvent extraction before liquid chromatography/atmospheric pressure 461 chemical ionization tandem mass spectrometry[END_REF], Berthiller et al. 2005[START_REF] Klötzel | Determination of 12 type A and B 429 trichothecenes in cereals by liquid chromatography-electrospray ionization tandem mass 430 spectrometry[END_REF], Delmulle 72 et al. 2006[START_REF] Klötzel | A new solid phase extraction clean-up method for 433 the determination of 12 type A and B trichothecenes in cereals and cereal based food by LC-434 MS/MS[END_REF]). However, it has been shown by post-column addition 73 experiments that signal suppression deriving from co-eluting matrix compounds may affect 74 only very small retention time periods [START_REF] Klötzel | Determination of 12 type A and B 429 trichothecenes in cereals by liquid chromatography-electrospray ionization tandem mass 430 spectrometry[END_REF]. In addition, it is known that the 75 extent of these effects also depends on the properties of the analyte (Rundberget and Wilkins 76 2002[START_REF] Vidal | Selection of a representative matrix for calibration in 449 multianalyte determination of pesticides in vegetables by liquid chromatography-electrospray 450 tandem mass spectrometry[END_REF][START_REF] Niessen | Matrix effects in quantitative pesticide analysis 457 using liquid chromatography-mass spectrometry[END_REF]. Therefore, some authors expressed 77 doubts about the applicability of a single non-coeluting analogue compound as internal 78 standard (Royer et al. 2004, Zöllner andMayer-Helm 2006). Studies investigating 2 D-or 13 C-79 labelled internal standards [START_REF] Lindenmeier | Quantification of ochratoxin A in foods by a 444 stable isotope dilution assay using high-performance liquid chromatography-tandem mass 445 spectrometry[END_REF][START_REF] Asam | Synthesis of four carbon-13-labeled type A trichothecene 368 mycotoxins and their application as internal standards in stable isotope dilution assays[END_REF], Bretz et al. 80 2006, Hartmann et al. 2006, Häubl et al. 2006aand 2006b) have shown that stable isotope 81 labelled mycotoxins are the best choice as internal standards as they completely compensate 82 matrix effects even in crude extracts due to their identical chemical and chromatographic 83 properties compared to the target toxins. However, this approach covers only a limited 84 number of analytes of a multi-mycotoxin method as the related standards are available only 85 for a few toxins. 86 87 Another option that is widely used in mycotoxin analysis is the use of matrix-matched 88 calibration, i.e. the fortification of a blank sample extract (representing a typical matrix) with 89 a standard solution containing the analytes. This may be regarded as a simplified version of a 90 standard addition approach in which each individual sample has to be fortified with a series of 91 standard solutions and which is clearly too laborious for routine analysis. Ideally, a single 92 representative matrix can be used to compensate for matrix effects of both, samples that are allocated to other matrices, as has been investigated for the determination of twenty pesticides 94 in different commodities [START_REF] Vidal | Selection of a representative matrix for calibration in 449 multianalyte determination of pesticides in vegetables by liquid chromatography-electrospray 450 tandem mass spectrometry[END_REF] as well as of different varieties 95 (different brands, origins, ..) of the same matrix. Differences in the extent of matrix effects 96 between individual samples of the same type of matrix may have a large influence on the 97 precision and the trueness of a method, as has been shown for the analysis of pharmaceutical 98 compounds in different lots of human plasma [START_REF] Matuszewski | Strategies for the assessment of 453 matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS[END_REF]. 99 100 It can be concluded from the data published so far that the existing LC-MS(/MS) methods for 101 mycotoxin analysis still suffer from large matrix effects (in spite of application of clean-up 102 procedures) whereas differences between individual samples of the same matrix seem to be 103 manageable. In a multi-toxin method developed for milk including protein precipitation and 104 subsequent SPE on Oasis HLB, matrix effects ranged from 75 to 110%, depending on the 105 analyte, while differences in matrix effects within 20 investigated milk brands did not 106 influence the precision of the method, only zearalenone showed slightly higher signal 107 intensities in low-fat milk [START_REF] Sorensen | Determination of mycotoxins in bovine milk by liquid 473 chromatography tandem mass spectrometry[END_REF]. Similarly, fluctuations of matrix 108 effects within 6 corn samples in a multi-Fusarium mycotoxin method including a two-step 109 clean-up occurred only in case of nivalenol (Cavaliere et al 2005a and b). Almost no matrix 110 effect for deoxynivalenol was observed in grain-based commodities such as wheat flour, oats 111 and rye, whereas strong signal suppression was observed in soybeans and in matrices with a 112 high sugar content, although the extracts had been cleaned with Mycosep  columns (Biselli 113 and Hummert 2005). [START_REF] Uhlig | Determination of beauvericin and four other enniatins in grain by 485 liquid chromatography-mass spectrometry[END_REF] analysed enniatins and beauvericin in 114 different types of grain without any clean-up and obtained relative standard deviations of 115 <30% for the recoveries of spiked samples. Signal suppression of trichothecenes and 116 zearalenone in extracts of durum wheat, corn and bread was reduced from >30% to <20% by 117 applying deepoxy-deoxynivalenol as internal standard, and was quite comparable between the 118 three matrices [START_REF] Klötzel | Determination of 12 type A and B 429 trichothecenes in cereals by liquid chromatography-electrospray ionization tandem mass 430 spectrometry[END_REF]. Zearalanone was used as internal standard for the [START_REF] Zöllner | Determination of zearalenone and its 501 metabolites αand β-zearalenol in beer samples by high performance liquid chromatography-502 tandem mass spectrometry[END_REF]. The authors concluded that the elimination of the matrix 121 effect is only possible within each beer brand, but not between the different brands. 122 123 Most of these methods include a clean-up step, as a reduction of sample preparation was 124 found to result in unacceptably large matrix effects and in extreme cases to complete 125 suppression of the detector signal (Biselli and Hummert 2005). In contrast to that, it was 126 shown in our group that the latest generation of MS interfaces tolerates the injection of crude 127 sample extracts of wheat and maize [START_REF] Sulyok | Development and validation of a 476 liquid chromatography/tandem mass spectrometric method for the determination of 39 477 mycotoxins in wheat and maize[END_REF]. In spiked wheat extract, significant 128 matrix effects were observed for only few of the 39 analytes, whereas the maize matrix 129 showed a more pronounced influence on the detector signal intensities, especially in case of 130 aflatoxins and ergot alkaloids. The excellent values obtained for the coefficients of variation 131 of the overall process indicated that these matrix effects were stable within the investigated 132 matrices and caused purely proportional errors, which may in principle be compensated by 133 matrix-matched standards. The present work includes the extension of our recently developed 134 method to three further matrices as well as an investigation of the variability of the matrix 135 effects between three individual samples of maize and rice, respectively. Based on these 136 additional data on matrix effects, the applicability of the concept of matrix matched 137 calibration for the developed method is evaluated. 138 139

Materials and methods 140

Chemicals and reagents 141

Methanol and acetonitrile (both LC gradient grade) were purchased from J.T. Baker 142 (Deventer, The Netherlands), ammonium acetate (MS grade) and glacial acetic acid (p.a.) 143 were obtained from Sigma-Aldrich (Vienna, Austria). Water was purified successively by 144 reverse osmosis and a Milli-Q plus system from Millipore (Molsheim, France). The coefficients of variation of the end determination (CVs) were calculated from linear, 1/x 224 weighted calibration curves obtained from the analysis of spiked samples using Validata  , a 225 Microsoft Excel macro developed by [START_REF] Wegscheider | Validata (Excel-Macro for method validation[END_REF].

Analysis of rice, spelt and barley 233

In order to further investigate the limits of the universal applicability of the LC-MS/MS based 234 method developed by our group, the method performance was investigated in 3 additional 235 matrices. Figures 1 and2 show the chromatograms of the spiked rice samples, Tables I-III 236 summarize the results for peeled rice, barley and spelt, respectively. The number of 237 concentration levels that were evaluated for a given toxin were not identical in all three 238 investigated matrices, as the lowest levels were near the respective limits of detection and the 239 sensitivity of the detector varied depending on the condition of the mass spectrometer. For 240 spelt, data evaluation had to be restricted to the 4 highest concentration levels due to an 241 non-linear calibration curve was MON that showed a logarithmic response in all three 250 matrices and in the liquid standard. This effect was already observed by [START_REF] Jestoi | Moniliformin in 421 Finnish grains: Analysis with LC-MS/MS[END_REF] in 251 case of the liquid standard (but not in the presence of matrix) and is probably a result of the 252 unfavourable ionisation conditions due to the large water content of the eluent at the retention 253 time of MON. The relative standard deviations of the whole procedure applied to the spiked 254 samples were generally lower than 10% at the highest concentration levels except for AFG2, 255

ERV and DHE in barley and D3G in rice and spelt. The limits of detection have not been 256 calculated for every analyte/matrix combination (as this work focuses on the transferability of 257 the method to other matrices, which is primarily hampered by signal 258 suppression/enhancement effects caused by co-eluting matrix components), but they are 259 comparable to those presented for wheat and maize [START_REF] Sulyok | Development and validation of a 476 liquid chromatography/tandem mass spectrometric method for the determination of 39 477 mycotoxins in wheat and maize[END_REF], as the related 260 concentration ranges during evaluation of method performance characteristics were very 261 similar. The recoveries of the extraction step were comparable between the three matrices for 262 many analytes, slightly larger variations were observed for those analytes showing incomplete 263 extraction (fumonisins and MON and to a lesser extent other polar analytes such as D3G, 264 Z4G, Z4S and NIV) and for several aflatoxins and ergot alkaloids. 265 266 Signal suppression/enhancement due to co-eluting matrix compounds was pronounced in 267 barley (SSE for 14 analytes outside 100±15%) and spelt (9 analytes outside 100±15%), 268 whereas in the case of the rice matrix only AFB1 was outside that range. As concerns the 269 prediction of the susceptibility of different analyte classes to signal variations and/or 270 deviations from 100% SSE, the data presented so far indicate that the most critical 271 compounds are aflatoxins, ergot alkaloids and polar analytes (like MON and D3G). The latter 272 are considered to be generally critical due to insufficient chromatographic separation from 273 polar matrix components [START_REF] Niessen | Matrix effects in quantitative pesticide analysis 457 using liquid chromatography-mass spectrometry[END_REF]. In this aspect, the different SSE values 274 obtained for the co-eluting DON and D3G seem to be contradictory, but it must be kept in mind that matrix effects are both compound-and matrix-dependent. This might also explain 276 the different SSE values observed for aflatoxins and other analytes such as MAS, DAS and 277 HFB1, that are eluting closely (see Figure 1). Concerning the aflatoxins, the hypothesis of 278 hydrolysis under acidic conditions [START_REF] Ventura | Ultra-493 performance-liquid chromatography/tandem mass spectrometry for the simultaneous analysis 494 of aflatoxins B1, G1, B2, G2 and ochratoxin A in beer[END_REF]) was tested by analysing spiked 279 neutral maize extracts. In addition, spiked acidic maize extracts were analysed omitting the 280 dilution step with ACN+H 2 O+HAc (20+79+1, v+v+v; see "extraction" in the experimental 281 section) to investigate whether aflatoxins adsorb on particulate matter emerging from dilution 282 of the raw extracts [START_REF] Sulyok | Development and validation of a 476 liquid chromatography/tandem mass spectrometric method for the determination of 39 477 mycotoxins in wheat and maize[END_REF]. Neither of these two variations led to a decreased signal 283 suppression (data not shown). 284 285In general, these results were comparable to those obtained for wheat and maize (Sulyok et al. 286 2006). The main conclusion is that all effects/losses deriving from co-eluting matrix 287 compounds as well as from incomplete extraction were repeatable on all concentration levels 288 for a given analyte/matrix combination: They caused changes of the slope of the linear 289 calibration function of spiked samples in comparison to liquid standards, but did not result in 290 non-linear calibration curves. Moreover, low CV values have been observed, which indicates 291 that the precision of the method is not significantly decreased by those effects. This suggests 292 that the method transfer to other grain matrices is feasible using calibration with pure standard 293 solutions together with correction factors for each analyte/matrix combination (for example 294 0.67 for ZON in barley, see Table II). However, this demands for low variations of matrix-295 induced signal suppression within a given matrix type. 296 297

Variation of matrix effects between individual samples of the same matrix 298

In order to assess the applicability of the concept of matrix-matched calibration, the variation 299 of SSE between individual samples was investigated for maize and rice by spiking blank 300 extracts obtained from three different individual samples of the same commodity at multiple concentration levels. Although maize seems to be a more critical matrix compared to rice as 302 concerns the extent of signal suppression/enhancement (especially for aflatoxins and ergot 303 alkaloids), the situation was somewhat different regarding the variation of matrix effects 304 between different varieties (Table IV With the exception of D3G, the relative standard deviation of signal/suppression enhancement 309 was < 15% between the three individual maize samples. In contrast to that, larger differences 310 were observed between the three rice samples (RSD of SSE > 15% for 13 analytes), which 311 was caused by dramatically reduced signal intensities in red rice. It should be pointed out that 312 the variation of the texture of the three rice samples was larger compared to the maize 313 samples: brown and red rice is not peeled and still contains its sprout. In addition, red rice 314 grows on clayey soil, which imparts the colour to the peel. It is highly probable that red rice 315 contains particular matrix components, that are responsible for the pronounced signal 316 suppression. This example shows that the general applicability of standards matched to a 317 single sample for the calibration of all other samples of a given matrix can not be taken as 318 granted. 319 320 In view of a routine application of the developed method, we recommend that matrix-matched 321 calibration is effectively carried out even if samples allocated to a single, validated matrix are 322 analysed. The use of a standard prepared in pure solvent (if necessary in combination with 323 some sort of empirical correction factor for the matrix effect -e.g. 0.56 for AFG1 in maize, 324 see Table IV) seems to be insufficient. That approach does not take into account that under 325 routine conditions the interface of the mass spectrometer is contaminated with matrix 326 components, which may have an effect on the analytical signal intensities and also on the extent of matrix effects. Indeed, we observed a slight continuous increase in the MS response 328 (similar to [START_REF] Klötzel | Determination of 12 type A and B 429 trichothecenes in cereals by liquid chromatography-electrospray ionization tandem mass 430 spectrometry[END_REF], who, however cleaned the extract prior to analysis) in the 329 course of some measurement sequences. 330 331 From the data presented here, it is evident that a thorough compensation of the matrix effects 332 by using standards which are matched to a carefully chosen representative matrix is a 333 prerequisite for a reliable quantitative analysis. Relative biases of as large as 40% must be 334 accepted in a worst-case scenario in the case of the red rice for example, if only a single rice 335 sample is used as typical representative for the matrix rice. This is, however, still acceptable 336 for the purpose of a semi-quantitative screening of a large set of samples followed by target 337 analysis of the positive samples including dedicated clean-up procedures and/or standard 338 addition. Based on the results shown for maize in Table IV, it might be assumed that this error 339 may be significantly reduced if model-samples are available for each individual brand or 340 variety. In view of a routine application, we suggest to carry out standard-addition in case of 341 doubt of positive samples exhibiting mycotoxin concentrations near the levels stated in the 342 related regulations. In addition, an adequate number of random blank samples should be 343 fortified and analysed over a longer period of time in order to obtain statistically meaningful 344 data on the variation of matrix effects within a given type of matrix. 345

346

Conclusion 347

The developed LC-MS/MS multitoxin method may be transferred to other grain matrices 348 without any adaptations concerning the instrumental parameters or sample preparation, as the 349 method performance parameters were found to be comparable in all investigated matrices. 350

The main limitation for obtaining accurate quantitative results are differences in the extent of 351 matrix effects between different matrices as well as between variations of a given matrix. 352

Using a single "model" sample which is used to represent all samples of a chosen matrix, the

  , αand β-zearalenol in different beer brands including an SPE-120 based clean-up

  dissolved in acetonitrile (ACN) if not stated otherwise. Stock 147 solutions of nivalenol (NIV), deoxynivalenol (DON), fusarenon X (FUSX), 3-148 acetyldeoxynivaleonol (3ADON), deepoxy-deoxynivalenol (DOM), neosolaniol (NEO), 149 diacetoxyscirpenol (DAS), HT-2 toxin (HT-2), T-2 toxin (T-2), zearalenone (ZON), alpha-150 zearalenol (α-ZOL), beta-zearalenol (β-ZOL), ochratoxins A and B (OTA, OTB), ochratoxin 151 alpha(OTα, in ACN/H 2 O 1+1, v+v), fumonisins B 1 and B 2 (FB 1 , FB 2 , in ACN/H 2 O 1+1), 152 hydrolysed fumonisin B 1 (HFB 1 , in ACN/ H 2 O 1+1), and aflatoxins B 1 , B 2 , G 1 and G 2 (AFB 1 , 153 AFB 2 , AFG 1 , AFG 2 ) were obtained from Biopure Referenzsubstanzen GmbH (Tulln, 154 Austria). 15-monoacetoxyscirpenol (MAS), verrucarol (VOL), beauvericin (BEA), 155 moniliformin (MON, dissolved in MeOH), ergocornine (ERC, dissolved in MeOH/H 2 O 1+1) 156 and ergotamine-D-tartrate (ERA, dissolved in MeOH/H 2 O 1+1) were received from Sigma-157 Aldrich (Vienna, Austria). A stock solution of enniatin A, A 1 , B and B 1 (ENN A, ENN A 1 , 158 ENN B, ENN B 1 ) was provided by Dr. Marika Jestoi (EELA Helsinki, Finland). Agroclavine 159 (AGR, dissolved in MeOH) was received from Dr. Miroslav Flieger (, dissolved in MeOH) was purchased from Dr. 162 Danka Pericic (Ruder Boscovik Institute, Zagreb, Croatia). Ergovaline (ERV, dissolved in 163 MeOH) was purchased from Prof. Forrest Smith (Auburn University, AL, USA). 164Deoxynivalenol-3-glucoside (D3G) was isolated from wheat treated with DON(Berthiller et 165 al. 2005), zearalenone-4-glucoside (Z4G, dissolved in MeOH) was synthesized according to a 166 modified protocol fromGrabley et al. 1992 and zearalenone-4-sulfate (Z4S, dissolved in 167 MeOH/H 2 O 1+1) was extracted in our laboratory from rice inoculated with Fusarium 168 graminearum. Four combined working standard solutions were prepared weekly by dilution 169 of the stock solutions of the analytes in the related solvents, i.e. MeOH (for Z4G, AGR, FB 2 and HFB 1 ) and ACN (for all other analytes), respectively. All solutions were stored 172 at -20°C and were brought to room temperature before use. 173 174 Maize samples deriving from the harvest 2002 (sample A), 2003 (sample B) and 2004 175 (sample C) were collected from fields in Lower Austria. Spelt, barley, Arborio peeled rice, 176 Arborio brown rice and red rice samples were purchased from a local store in Vienna. 177 178 Sample preparation 179 Spiking. 0.5 g of ground sample were spiked by consecutively adding the appropriate amounts 180 of the 4 combined working solutions. The samples were subsequently stored for three days at 181 40°C to allow the evaporation of the solvent and to establish equilibration between the 182 analytes and the matrix. 183 184 Extraction. 2 mL of extraction solvent (CH 3 CN/H 2 O/HAc 79+20+1) were added to 0.5 g of 185 ground sample. The samples were extracted for 90 min using a GFL 3017 rotary shaker (GFL, 186 Burgwedel, Germany) and subsequently centrifuged for 2 min at 3000 rpm (radius: 15 cm) on 187 a GS-6 centrifuge (Beckman Coulter Inc., Fullerton, CA). The extracts were transferred into 188 glass vials using Pasteur pipettes and aliquots of 350 µL were diluted with the same amount 189 of a mixture containing CH 3 CN/H 2 O/HAc 20+79+1. After appropriate mixing, 5 µL of the 190 diluted extract were injected into the LC-MS/MS system without further pre-treatment. 191 192Instrumental conditions 193Parameters of the instrumental set-up have been described in detail inSulyok et al. 2006. 194 Briefly, a 1100 Series HPLC System (Agilent, Waldbronn, Germany) was coupled to a QTrap 195 4000 LC-MS/MS System (Applied Biosystems, Foster City, CA) equipped with a 196 TurboIonSpray electrospray ionization (ESI) source. Chromatographic separation was on a Gemini  C 18 -column, 150 x 4.6 mm i.d., 5 µm particle size, equipped 198 with a C 18 4 x 3 mm i.d. security guard cartridge (all from Phenomenex, Torrance, CA, US). 199 Both eluents contained 5 mM ammonium acetate and were composed of 200 methanol/water/acetic acid 10+89+1 (v+v+v; eluent A) or 97+2+1 (eluent B), respectively. 201 ESI-MS/MS was performed in multiple reaction monitoring (MRM) mode in both positive 202 and negative polarity using two separate chromatographic runs per sample. 203 204 Performance characteristics 205 For the determination of the analytical performance of the method in spelt, rice and barley, the 206 whole spiking and extraction procedure was carried out at 8 different concentration levels 207 (each in triplicate) with relative concentrations of 1:4:7:10:40:70:100:400. The concentration 208 ranges of the spiked samples were chosen to cover the respective limits of detection of each 209 toxin as well as the concentration ranges found in naturally contaminated samples. For 210 external calibration, liquid standards were diluted in mobile phase A as well as in blank 211 extracts (diluted 1+1, as described in "Extraction"). The related analyte concentrations were 212 matched on each level to the expected concentrations in the final diluted extract of the spiked 213 samples. Linear, 1/x weighted calibration curves were constructed from the data obtained 214 from the analysis of each sample type (spiked sample, liquid standard, matrix-matched 215 standard) using the Analyst  software version 1.4.1. To differentiate between extraction 216 efficiency and matrix induced signal suppression/enhancement, the slope ratios of the linear 217 calibration functions were calculated to yield the apparent recovery (R A ) and the signal 218 suppression/enhancement (SSE) due to matrix effects. Eventually, the recovery of the 219 extraction step (R E ) was obtained as follows (modified after Matuszewski et al. 2003): 220 R A (%) = 100 * slope spiked sample / slope liquid standard (1) 221 SSE (%) = 100 * slope matrix-matched standard / slope liquid standard (2) 222 R E (%) = 100 * R A / SSE (

  determine the variation of matrix effects between individual samples of the same 228 matrix (rice and maize), blank extracts of three different samples of each matrix were spiked 229 on multiple levels and SSE was calculated according to equation 2

  I-III approximately here, preferably on the same double page] 245 246The CVs obtained from linear calibration data were < 6.0% for all analytes and matrices with 247 the exception of AFB1 and AFB2 in barley and of Z4S in all matrices. The latter was 248 probably a result of the tendency of this compound to hydrolyse. The sole analyte exhibiting a

Figure 1 :Figure 2 :

 12 Figure captions: 508

Table I .

 I Performance characteristics for peeled Arborio rice (n=3): apparent recoveries (R A ), signal suppression/enhancement (SSE), recoveries (R E ), relative standard deviations at the highest concentration levels and coefficients of variation of the overall procedure (CV)

		Conc. range	evaluable	R A (%)	SSE (%)	R E (%)	RSD (%) CV (%)
		[µg/kg]	levels
	NEO	16-1620		7	98	99		99	1.7	1.2
	VOL	531-5310		4	98	99		99	2.8	0.7
	MAS	17-1680		7	105	101		104	10.4	1.6
	DAS	4.2-1660		8	105	105		100	2.5	1.0
	HT-2	16-1570		7	101	105		96	2.0	1.2
	T-2	4.2-1660		8	101	103		97	3.5	1.2
	NIV DON D3G FUSX DOM	40-1600 28-1600 10-400 28-1620 27-1560 F o r	5 6 5 6 6	88 96 93 105 97	99 100 97 105 106		89 96 96 101 91	6.5 4.4 26 2.3 1.8	0.9 1.5 5.9 1.2 0.8
	3ADON AFG 2	28-1600 4.0-160	6 5 P	102 95	104 101		98 93	2.8 4.6	0.7 3.8
	AFG 1 AFB 2 AFB 1 AGR ERV	2.8-160 4.0-160 16-160 0.30-118 8.6-86	6 5 e 4 8 4 e 98 94 96 95 97 r	93 86 82 99 95		106 109 116 96 102	7.0 8.7 5.8 4.4 4.0	5.6 3.7 2.7 3.4 1.5
	DHE ERA	3.4-339 4.2-419		7 7	93 112 R	98 106		94 106	6.2 4.0	2.1 1.7
	ERC BEA ENN B ENN B 1 ENN A 1 ENN A HFB 1 FB 1	14-778 0.80-320 32-320 6.1-346 3.2-224 0.87-345 26-262 41-1630		6 8 4 6 6 8 4 5	147 101 e 104 100 101 105 111 104 100 105 98 99 v i e 88 103 74 100 w	142 101 97 107 96 99 85 74	8.4 0.3 2.1 3.9 2.3 1.6 2.0 1.7	2.4 4.1 2.9 2.6 1.7 1.4 1.4 0.7
	FB 2 OTα OTB OTA Z4G β-ZOL Z4S	40-1620 8.2-330 2.0-800 3.3-330 20-800 2.0-800 0.30-12		5 5 8 7 5 8 5	86 71 98 96 89 102 90	108 114 103 99 100 103 108	80 62 95 97 88 O n l 3.2 5.5 3.4 5.7 3.1 99 2.4 83 3.0 y	0.9 1.9 0.9 2.2 1.3 1.1 8.9
	α-ZOL	2.0-800		8	104	99		105	0.5	0.7
	ZON	4.0-1620		8	110	109		102	0.8	0.7
	MON	71 -4060		6	58	113		51	5.4	-

Table II .

 II Performance characteristics for barley (n=3): apparent recoveries (R A ), signal suppression/enhancement (SSE), recoveries (R E ), relative standard deviations at highest concentration levels and coefficients of variation of the overall procedure (CV); (enniatins and OTA are not included due to contamination of the blank barley)

		conc. range	evaluable	R A (%)	SSE (%)	R E (%)	RSD (%) CV (%)
		[µg/Kg]	levels
	NEO	4.1-1620	8	96	96	101	4.4	1.5
	VOL	531-5310	4	94	95	99	9.4	0.6
	MAS	17-1680	7	93	96	97	8.0	1.2
	DAS	4.2-1660	8	99	94	106	4.3	1.8
	HT-2	4.0-1570 105 F 8 105 o r
			P
			e
			e r
				R
				e
				v i e
					w
						O n l
							y

Table III .

 III Performance characteristics for spelt (n=3): apparent recoveries (R A ), signal suppression/enhancement (SSE), recoveries (R E ), relative standard deviations at the highest concentration levels and coefficients of variation of the overall procedure (CV); (enniatins are not included due to contamination of the blank spelt)

	Conc. range	evaluable	R A (%)	SSE (%)	R E (%)	RSD (%) CV (%)
	[µg/kg]	levels
	F o r
		P
		e
		e r
			R
			e
			v i e
				w
					O n l
						y

Table IV :

 IV Variation of signal suppression/enhancement (in %) within spiked blank extracts of individual samples of maize and rice. Values for maize C are taken from[START_REF] Sulyok | Development and validation of a 476 liquid chromatography/tandem mass spectrometric method for the determination of 39 477 mycotoxins in wheat and maize[END_REF].

	Maize	Maize
	A	B

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Acknowledgements 358

The authors thank the Lower Austrian Government and the Christian Doppler Society for the 359 financial support and Marika Jestoi for providing the enniatin standard. Greatly acknowledged 360 are also Claus Zeppelzauer and the Ecoplus business agency of Lower Austria. 361


B J A Berendsen

J A Van Rhijn

Residue analysis of tetracyclines in poultry muscle; shortcomings revealed by a proficiency test

Keywords: Veterinary drug residues -tetracycline Antibiotics, veterinary residues, tetracyclines, proficiency test

A proficiency test for tetracycline drug residues in poultry muscle was organised according to the guidelines of ILAC-G13:2000. For the proficiency test three test materials were prepared.

Homogeneity and stability of the materials during the study was demonstrated. Sixteen laboratories accepted the invitation to participate in the proficiency test, eleven laboratories reported results within the time frame of the study. Most notably, only four of the participating laboratories complied with the definition of the MRL concerning the inclusion of 4-epimers as stated in Commission Regulation [EC] 281/96. Most participants reported values for CC α and CC ß and hence were already in compliance with Commission Decision [EC] 2002/657/EC for this aspect of method validation. However, some CC α and CC ß values were not in agreement with the actual within-lab reproducibility calculated from the results reported in this proficiency test.

Although most laboratories obtained satisfactory results, it is clear that an effort is needed to include 4-epiOTC, 4-epiTC and 4-epiCTC in the analytical methods. Moreover, reconsideration of values determined for CC α and CC ß with respect to their accuracy may be necessary in some cases.

Oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC) and doxycycline (DC) are the most commonly used tetracycline compounds. Tetracyclines have a broad-spectrum activity against both Gram-positive and Gram-negative bacteria. They interfere with the bacterial protein synthesis in rapidly growing and reproducing bacterial cells and inhibit the metabolism of the bacteria (FIDIN Repertorium Diergeneesmiddelen 2000).

The toxicity of tetracyclines is low, but after prolonged therapy or contact, infections with resistant organisms, allergic reactions and vitamin B deficiencies may occur. The use of tetracyclines during pregnancy and by young children and animals has adverse effects on skeleton formation (Mol 1975). The metabolism of tetracyclines was extensively studied in several species (FAO Food and Nutrition Paper 1990). With the exception of metal chelate formation, no chemical transformation occurred in the body. Small amounts of 4-epimers were detected, however, that was attributed to chemical instability at physiological conditions rather than to metabolic transformation.

Tetracyclines are included in Annex I of Council Regulation [ECC] 2377/ 90 (1990) and Maximum Residue Limits (MRLs) have been established. The MRL for OTC, TC and CTC is defined as the sum of the tetracycline and its 4-epimer and is set at 100 µ g/kg in muscle for all food producing species. For DC the MRL is set at 100 µ g/kg in muscle tissue for DC only (Committee for Veterinary Medical Products 1995 and1997). A proficiency test for tetracyclines in poultry muscle was organised, to allow laboratories to evaluate and demonstrate the reliability of the data that are produced. In addition to validation and accreditation, proficiency testing is an important requirement of the European Union (EU) (Council Directive 93/99/EEC 1999) and is increasingly important in laboratory accreditation according to NEN-EN-ISO/IEC 17025 (2005). This proficiency test was carried out according to the guidelines in ILAC-G13(2000). Statistical analysis was performed according to ISO/IEC Guide 43-1 and ISO/IEC Guide 43-2.

Materials and Methods

Test Materials

Three materials (Table I) were prepared containing different concentrations of OTC, 4-epiOTC and DC by adding solutions of OTC, 4-epiOTC and DC (Sigma-Aldrich, St Louis, MO, USA) in methanol to minced blank poultry muscle. The materials were manually homogenised and subsequently frozen using liquid nitrogen and kept under cryogenic conditions during further grinding and homogenisation. The deep-frozen materials were minced using a Stephan (Stephan Machinery, Hameln, Germany) UMC 5 Electronic food processor and subsequently finely ground using a Retsch (Haan, Germany) Grindomix GM200 mill. The resulting cryogenic powder was seived to remove lumbs of material which were minced again. The seived powder was homogenised by stirring for 10 min.

[Insert table I about here]

Materials A, B and C were weighed in portions (20 g) and stored in polyethylene containers. These containers were randomly numbered and sample sets were prepared consisting of two randomly chosen samples of each material. Because a duplicate analysis for each sample was requested, the laboratories would report four results for each material. From these data the repeatability of the laboratories could be calculated. Until dispatch, the samples were stored at -80 °C.

Sample distribution

Participants were invited to take part in this study based on their involvement in the field of veterinary drug residue analysis. In total seventeen laboratories where invited from both within and outside the EU including several National Reference Laboratories (NRLs). Sixteen laboratories agreed to participate. Sample sets were packed in insulating boxes containing dry ice and were sent to the participants, accompanied by instructions and a result form. Participants were requested to carry out duplicate analysis using the methods they routinely use in the analysis of tetracycline residues. The results were due within seven weeks after shipment of the samples.

Method of analysis

For the purpose of homogeneity and stability testing an in-house validated LC/MS/MS method was used compliant with Commission Decision 2002/657/EC. An aliquot (2 g) of the sample was weighed and transferred to a centrifuge tube. Demeclocycline (demethylchlortetracycline, DMC) was added as the internal standard. The sample was extracted twice with 25 mL of McIlvain buffer (0.1N, pH=4.0). The combined extract was centrifuged (3500 g) and filtered using a glass microfibre filter (Whatman , Brentford, Middlesex, UK). Filtered extract (30 mL) was applied to a preconditioned OASIS® HLB (Waters, Milford, MA, US) extraction column (60 mg, 3CC).

After washing with McIlvain buffer and water, the analytes were eluted using 3mL of methanol. The eluent was evaporated to dryness (N2, 50°C) and the residues were dissolved in HPLC mobile phase. The LC-system consisted of a Waters Alliance 2690 equipped with a Waters SymmetryR C18 (3 x 150 mm, 5 µm) analytical column and a thermostatic oven (Spark, Emmen, NL) maintained at a temperature of 10°C. The tetracyclines were chromatographically separated using a lineair acetonitrile gradient in 1 mM ammonium formate (pH=2.6) starting at t=0 min at 0% acetonitrile, increasing the acetonitrile content to 50% at t=10 min and further to 100% at 14 min. Detection was carried out in multiple reaction monitoring (MRM) mode using a Waters-Micromass (Waters, Milford, MA) Quattro Ultima triple quadrupole mass spectrometer equipped with an electrospray interface and operated in positive ion mode.

Mass spectrometric data acquisition was performed in MRM mode monitoring the following MRM transitions; OTC and 4-epiOTC: m/z 461→337 and 461→201; TC and 4-epiTC: m/z 445→410 and 445→154; CTC and 4-epiCTC: m/z 479→444 and 479→154 and DC: m/z 445→428 and 445→154. For the internal standard DMC the transition m/z 465→154 was monitored. The amount of tetracyclines in the samples was calculated with the internal standard method using the ratio of the peak area of the most abundant product ion of the tetracycline and the peak area of the internal standard. Calibration was carried out using extracted matrix-matched standards fortified at concentration levels ranging from 0.25 -5*MRL. The limit of detection (LoD) for this method was 10µ g/kg (0.1*MRL). The homogeneity study was carried out according to The International Harmonized Protocol for Proficiency Testing of Analytical Laboratories [START_REF] Thompson | The International Harmonized Protocol for Proficiency Testing of (Chemical) Analytical Laboratories[END_REF] and ISO/DIS 13528 (2005), taking into account the insights discussed by Fearn et al. (2001) and [START_REF] Thompson | Recent trends in inter-laboratory precision at ppb and sub-ppb concentrations in relation to fitness for purpose criteria in proficiency testing[END_REF].

Homogeneity of the test materials

Ten randomly chosen containers of materials B and C were each analysed in duplicate for OTC, TC, CTC, their 4-epimers and DC. Statistical analysis of the results was carried out using single factor Analysis of Variance (ANOVA). The homogeneity study demonstrated sufficient homogeneity for both materials for their use in the proficiency test.

Stability of the test materials

From the homogeneity data, the amount of tetracycline residues in the materials, just after preparation, was calculated. Ten randomly chosen samples of each material were stored at -80°C to assure the absence of degradation of the tetracycline residues. After 71 days, three containers each of material B and C were analysed. After this analysis, the containers were stored at -20°C to study the stability at generally applied storage conditions. Fourteen days hereafter (corresponding with the deadline for reporting results), the containers were analysed again. In this way, the procedure includes an extra defrosting and freezing cycle. This is essential because some laboratories carried out a duplicate analyses of each sample on different days (i.e. sreening and quantitative analysis). This most likely involves an extra freezing and defrosting cycle.

For both points in time, the average result was calculated. The results of the initial analyses were compared to the results after 71 and 85 days of storage, using a Students t-test (Fearn et al. 2001).

In this test, the standard deviation of the analyses at the three points in time was considered the same, because the same analytical procedure was applied to obtain the results, and was derived from the validation results. The results of the stability study are presented in table II and III. The Students t-test demonstrated that no significant loss of 4-epiOTC, OTC or DC occurred during the time scale of the proficiency test at the chosen storage conditions.

[Insert table IIa andb around here] [Insert table IIIa and b around here]

Statistical evaluation

Accuracy. The statistical evaluation was carried out according to the International Harmonized Protocol for the Proficiency Testing of Analytical Laboratories [START_REF] Thompson | The International Harmonized Protocol for Proficiency Testing of (Chemical) Analytical Laboratories[END_REF] 1982). The target standard deviation (σH) was calculated from the Horwitz equation, taking into account the insights of [START_REF] Thompson | Recent trends in inter-laboratory precision at ppb and sub-ppb concentrations in relation to fitness for purpose criteria in proficiency testing[END_REF]. According to ISO 13528 (2005), when u ≤ 0.3σH , the uncertainty of the assigned value is negligible and therefore should not be included in the statistical evaluation. When this situation applied, the z a -score for each laboratory was calculated according to equation 1. When the uncertainty of the assigned value did not comply with this criterion, the uncertainty of the assigned value was taken into account and the z a '-score was calculated according to equation 2. A z-score between -2 and 2 is regarded as a satisfactory result. Z-scores between 2 and 3 as well as between -3 and -2, are considered as questionable results. Z-scores below -3 and above 3 are considered unsatisfactory. a p

x -X z = σ

(1)

2 2 p a u + σ X - x = ' z (2)
Precision. In the design of this proficiency test, blind duplicate samples of each material were submitted to the participants. Therefore, every laboratory reported two pairs of results for each material. From the results of the blind pairs of material B and C the repeatability standard deviation (s r ) and the within-lab-reproducibility standard deviation (s RL ) were calculated according to ISO 5725-2 (1994).

To inform participants about their performance for precision, the Horwitz-ratio (HORRAT) (Official Methods of Analysis Program Manual 2002) was used. The HORRAT was calculated from the within-lab reproducibility (Eq. 3). The reproducibility standard deviation (s R ) includes inter-laboratory variation and therefore is likely to be higher than the within-lab reproducibility standard deviation (s RL ). Because the HORRAT value is calculated from s RL instead of s R , this value presents a best case situation and is not suitable for an exact numerical evaluation but is only indicative of the achieved precision. reason that the HORRAT value should be substantially below 1.0. Nonetheless, for the purpose of this study, a HORRAT value is not regarded as a questionable result unless it exceeds 1.0.

Results and Discussion

Participants

Sixteen out of seventeen invited laboratories agreed to participate in the proficiency test. Eleven of those laboratories managed to submit their results for OTC. From those laboratories, two did not include DC in the analysis. Consequently nine laboratories submitted results for DC. None of the participating laboratories reported values for TC and CTC. The majority of participants analysed the samples in duplicate. Only participants 1 and 16 reported one result per sample.

Participants methodologies

The participants applied different sample preparation methods and detection techniques for the analysis of tetracyclines. The majority of the participants extracted the poultry muscle samples 2002) most of the participants carried out a confirmatory analysis using LC/MS/MS. Two participants applied HPLC-DAD for confirmation. In one laboratory a quantitative analysis using HPLC-UV preceded the confirmatory analysis (LC/MS/MS). One participant carried out a semi-quantitative screening analysis using one calibrant followed by a quantitative analysis using a multi level matrix calibration. One participant did not confirm the identity of the analytes but merely carried out a quantitative analysis using LC-UV.

Four participants used an internal standard for their quantitative analyses. In three cases DMC was used. The fourth participant used tetracycline as the internal standard for the quantitative analysis. This quantitative analysis was preceded by a semi-quantative screening analysis to confirm the absence of TC in the test materials.

Although 4-epimers are not metabolically formed in significant amounts (FAO Food and Nutrition Paper 1990), they may be formed during sample storage, preparation and extraction especially under acidic conditions. It is therefore of importance to include the 4-epimers in the method of analysis. Only four of the participating laboratories paid specific attention to the presence of 4-epiOTC, 4-epiTC and 4-epiCTC. Two other laboratories included just one of the 4epimers. When epimer analysis was included, 4-epiOTC often co-eluted with OTC. Those participants quantified the sum of 4-epiOTC and OTC using OTC calibrants. When 4-epiOTC and OTC were chromatographically separated, the participants quantified these compounds using separate calibrants for both 4-epiOTC and OTC or they quantified both compounds using OTC calibrants. Considering the short retention times of OTC reported by some participants that also stated that they did not include 4-epiOTC in their analysis, it is likely that in those cases 4-epiOTC and OTC co-eluted. It could well be possible that those participants included 4-epiOTC in the quantitative analysis without being aware or specifically aiming to do so. Summarizing, it is evident that the analysis of 4-epimers of tetracyclines is not yet generally implemented in the laboratories. Hence, seven out of the eleven participants (i.e. 63%) do not fully comply with Commission Regulation [EC] 281/96 (1996) regarding the definition of the MRL (inclusion of the 4-epimers).

Results of analysis

Quantitative results. Eleven participants reported results for the materials A, B and C. Laboratory 1 and 16 reported only a single value for all samples. Laboratory 3 and 7 did not include DC in their analyses and were therefore not able to report any values for this analyte.

Laboratory 5 detected OTC in both samples of material A with amounts ranging from 40 to 113 µg/kg in contrast to the other participants that unequivocally did not detect any tetracycline residues in this material. Laboratory 5 did not detect any DC in these samples, however, none of the materials sent out to the participants contained OTC as the only analyte. Despite that the identity of OTC was confirmed by participant 5 using LC/MS/MS, this result was considered as a false-positive result which is most likely attributed to contamination of the sample at the laboratory.

Laboratory 6 did not find any tetracycline residues in one of the samples of material C. This was considered as a false-negative result.

[Insert An overview of the participants results, including the calculated assigned value, uncertainty of the assigned value and the target standard deviation is presented in Tables IV-VII. The z a -scores and HORRAT values are graphically presented in figure 1 and 2. In the statistical analysis of OTC in both material B and C, the uncertainty of the assigned value could not be neglected and consequently z a '-scores were calculated. The quantitative results of all participants were satisfactory with respect to accuracy and precision for DC. For OTC, a significant variation in the reported results is observed, ranging from 56 to 176 µ g/kg for material B and 78 to 250 µ g/kg for material C. Surprisingly, no correlation was found between low results and laboratories that did not include 4-epiOTC in their analysis. Other differences in methodology (i.e. LC/MS/MS versus LC-UV and SPE versus no SPE) were examined to clarify divergent results. No correlation between results and methodology was found. Two laboratories (5 and 7) obtained z a '-scores just below 3, indicating questionable results for accuracy. For laboratory 5, a HORRAT was calculated above 1.0, indicating unsatisfactory performance for precision.

[Insert figure 1 VIII. Laboratory 1 reported surprisingly low values for CC α and CC ß compared to the other participants. This suggests very good within-lab reproducibility. The CC α reported by the laboratories were compared to the within-lab reproducibility calculated from the results in the proficiency test. From the reported duplicates of the unknown pairs, the within lab-reproducibility standard deviation was calculated for each laboratory [ISO 5725-2 1994]. With this, the CC α that could be expected from the reported data was calculated for each laboratory using formula 4

(Commission Decision [EC] 2002/657/EC). L R CCα = MRL + 1.64s (4)
For OTC, this calculation resulted in an expected CC α of 152 µ g/kg for laboratory 5, whereas the reported CC α is 115 µg/kg (Table VIII). This indicates that the within-lab reproducibility for OTC for this laboratory is worse than is suggested by the reported CC α even when considering that due to the limited number of data, the uncertainty in the calculated CC α is considerable.

Regarding the analysis of DC of laboratory 9 an expected CCα of 132 µg/kg was calculated, whereas the reported CC α is 119 µ g/kg. This indicates that also for this laboratory the within-lab reproducibility for DC is worse than is suggested by the reported CCα. This comparison could not be made for laboratory 1 and 16 due to the lack of duplicate results.

The comparison showed however, that the suggested good within-lab reproducibility of some laboratories is not supported by the data reported in the framework of this proficiency study.

[Insert table VII about here]

Conclusions

This proficiency study was carried out to enable laboratories to demonstrate or evaluate their performance for the analysis of tetracycline residues in poultry muscle. Seventeen laboratories were invited to participate of which sixteen laboratories agreed to take part. Eleven of those reported results within the time frame of the study. Although most laboratories obtained satisfactory results for accuracy and precision, it is clear that in some laboratories there are difficulties regarding the analysis of tetracyclines residues.

Eight out of eleven participants (i.e. 72%) demonstrated acceptable performance for the analysis of OTC and DC in poultry muscle with respect to accuracy, precision and the occurrence of falsepositive and false-negative results. A significant variation in the results for OTC was observed.

This could not be explained by whether or not participants included 4-epiOTC in their method or any other differences in the applied methods.

F

  important group of antibiotic agents used in human and veterinary medicine.

  , elaborated by the International Organization for Standardization (ISO), International Union of Pure and Applied Chemistry (IUPAC) and Association of Analytical Communities (AOAC), and ISO 13528 (2005). The assigned value (X) and the uncertainty of the assigned value (u) were calculated from the individual results of the laboratories (x) using robust statistics according to the Analytical Methods Committee (1989a and b). According to Commission Decision [EC] 2002/657/EC (2002) the inter-laboratory coefficient of variation for the repeated analysis of a fortified material shall not exceed the level calculated from the Horwitz equation (Horwitz W.

  equal to 1.0 indicates that the within-lab reproducibility is equal to the reproducibility standard deviation calculated from the Horwitz equation. Therefore it is within

  using an aqueous and slightly acidic extraction medium. An EDTA-McIlvain buffer was commonly applied. Other extraction solvents include sodium succinate solution, acetonitrile and heptane, or trichloroacetic acid solution.By far the most common sample clean-up procedure reported was solid phase extraction (SPE) using a polymer-based phase. In one laboratory this step was preceded by clean-up using a chelated sepharose column loaded with copper ions. Two participants used a C18 material for the SPE procedure. Two other participants only filtered their extract before analysis without further purification.In accordance with Commission Decision [EC] 2002/657/EC (

  Figure 1.Graphical presentation of the laboratories z a '-scores (black bars) and HORRAT

Figure 1 .Figure 2 .

 12 Figure 1. Graphical presentation of the laboratories za'-scores (black bars) and HORRAT values (grey bars) for OTC + 4epi-OTC in (a) material B and (b) material C.

table IV

 IV

	[Insert table VI about here]
	[Insert table VII about here]
	F o
	r
	P
	e e r
	R e v i e w
	O n
	l y
	about here]
	[Insert table V about here]

  Laboratory 10, on the other hand, reported relatively high values for CC α (153 µ/kg) and CCß (206 µg/kg) for DC, indicating variable method performance for this compound. The expected CC α calculated from the data in this proficiency study however, is 114 µg/kg. This indicates that, for this laboratory the within-lab reproducibility for DC is better than is suggested by the reported CC α .

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

Table I .

 I It is mandatory, and also relevant from an analytical point of view, to include the 4-epimers of tetracyclines in the method of analysis. At present not all laboratories can meet this requirement.Most participants reported values for CC α and CC ß and hence, the majority already comply with Commission Decision 2002/657/EC(2002). Considering the precision that is apparent from the data reported in the framework of this study, in some cases values reported for CC α and CC ß may be too optimistic and hence may need reconsideration. Target concentrations of tetracyclines in the inter-laboratory test material.

	EC. 2002. Commission Decision 2002/657/EC. Implementing Council Directive 96/23/EC concerning
	the performance of analytical methods and the interpretation of results. Official Journal L221: 67A-Material Amount of OTC Amount of DC
	code	(µg/kg)	(µg/kg)
	76A. A	Blank	Blank
	B	Just above MRL a Just below MRL
	EEC. 1999. Council Directive 93/99/EEC. Official Journal L 290: 0014-0017. C Just above MRL Approximately twice the MRL
	a Sum of 4-epiOTC and OTC (present in the material at about equal
	FAO Food and Nutrition Paper 41/3, Residues of Some Veterinary Drugs in Animals and Foods, 1990. concentrations)
	o r F o r Fearn T, Thompson M. 2001. A new test for 'sufficient homogeneity'. Analyst 126: 1414-1417. FIDIN Repertorium Diergeneesmiddelen. 2000. 8th edition, Den Haag. F o F o Horwitz W. 1982. Evaluation of analytical methods used for regulation Analysis of food products. r r
	P P Journal of the Association of Official Analytical Chemists 65:525-530. P P
	e e r e e r ILAC-G13:2000, ILAC Guidelines for the Requirements for the Competence of Providers of Proficiency Testing Schemes, 2000. e e r e e r
	R e v i e w R e v i e w ISO 5725-2. 1994. Accuracy (trueness and precision) of measurement methods and results -part 2: Basic methods for the determination of repeatability and reproducibility of a standard measurement method. 1st edition. ISO/IEC Guide 43-1. 1997. Proficiency testing by inter-laboratory comparisons -Part 1: Development and operation of proficiency testing schemes. R e v i e w R e v i e w
	O n l y O n l y ISO/IEC Guide 43-2. 1997. Proficiency testing by inter-laboratory comparisons -Part 2: Selection and use of proficiency testing schemes by laboratory accreditation bodies. O n O n ISO 13528. 2005. Statistical methods for use in proficiency testing by inter-laboratory comparison. Mol H. 1975. Antibiotics and Milk. l y l y
	ISO/IEC 17025. 2005. General Requirements for the Competence of Calibration and Testing
	Laboratories.
	Official Methods of Analysis Program Manual. 2002. Appendix D: Guidelines for Collaborative Study
	Procedures To Validate Characteristics of a Method of Analysis. Association Of Analytical

Table IIa .

 IIa Statistical evaluation of the results of OTC + 4-epiOTC obtained from the stability study of material B..

	Time (days)	Result (µg/kg)	std (µg/kg)	t	tcritical
	0	129
	71	132	10.9	0.43	2.08
	85	135	10.9	0.83	2.08

Table IIb .

 IIb Statistical evaluation of the results of DC obtained from the stability study of material B..

	Time (days)	Result (µg/kg)	std (µg/kg)	t	tcritical
	0	55
	71	57	7.5	0.52	2.08
	85	59	7.5	0.92	2.08

Table IIIb .

 IIIb Statistical evaluation of the results of DC obtained from the stability study of material C.

	Time (days)	Result (µg/kg)	std (µg/kg)	t	tcritical
	0	195
	71	202	26.8	0.44	2.08
	85	214	27.0	1.16	2.08

Table IIIa .

 IIIa Statistical evaluation of the results of OTC obtained from the stability study of material C.

		F o
		r
			P
			e e r
	Time (days) 0 71 85	Result (µg/kg) 152 146 138	std (µg/kg) 12.7 12.7	t 0.84 1.77	tcritical 2.08 2.08 R e v i e w
					O n
					l y

Table IV .

 IV Overview of the laboratory results obtained from the analysis of 4-epiOTC + OTC in material B.Assigned value 104.0 µg/kg Uncertainty of assigned value 12.0 µg/kg Target standard deviation (Horwitz, Thompson) 22.9 µg/kg

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

Table VIII .

 VIII Overview of reported CCα and CCß values for 4-epi-OTC, OTC and DC (µg/kg).

	Page 21 of 23
		4-epiOTC	OTC		DC
	Lab	CCα	CCß	CCα	CCß	CCα	CCß
	Lab1			104.3	108.6	103.5	106.9
	Lab2	122.9	147.1	124.2	144.4	118.5	135.4
	Lab5			115	129	129	158
	Lab8			115	130	125	154
	Lab9			112	126	119	146
	Lab10			111	121	153	206
	Lab12	113	137	107	120	112	133

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Acknowledgement

This study was conducted with financial support of the Ministry of Agriculture, Nature and Food Quality of The Netherlands.

Code

Result 1