Tomáš Kovalczuk

Ondřej Lacina

Martin Jech

Jan Poustka

Jana Hajšlová
email: jana.hajslova@vscht.cz

Novel approach to fast determination of pesticides using of ultraperformance liquid chromatography -tandem mass spectrometry (UPLC-MS/MS)

Rapid, high throughput method employing ultra-performance liquid chromatography hyphenated with tandem quadrupole mass spectrometry (UPLC-MS/MS) has been developed and optimized for simultaneous quantification and confirmation of 64 pesticide residues and their toxic metabolites in fruit extracts prepared by buffered QuEChERS procedure. Total time required for UPLC-MS/MS analysis was 8 min plus 2 min for re-equilibration to initial UPLC conditions. Performance characteristics were determined for apple extracts spiked at 10 µg kg -1 . Repeatability of measurements expressed as relative standard deviations was in the range 1.5-13% at this level for most analytes. Thanks to very low limits of quantification (< 10 µg kg -1 for majority of pesticides), optimized method allows reliable control of not only common MRLs set in EU regulation for various pesticides/fruit combinations but also uniform MRL 10 µg kg -1 endorsed for baby food.

Introduction

Pesticides applied at various stages of food crops cultivation and/or during their post-harvest storage, play an important role in intensification of agricultural production. The number of active ingredients intended for control of undesirable pests and weeds currently exceeds 800 (Tomlin 2002, Fernández-Alba 2005, Hertherton et al. 2004). Avoiding occurrence of some pesticide residues in food supply is obviously impossible to achieve and, therefore, not surprisingly, health risk associated with a dietary intake of these chemicals has become a safety issue, both for toxicologists and consumers. To address these concerns, reliable and cost effective analytical methods have to be available to control respective regulation requirements.

With regard to the high number of target analytes concerned, multiresidue methods represent the only practical solution that enables meeting the requirements of current extensive surveillance/compliance programs, both in terms of scope of analysis and the number of samples analyzed. Historically, gas chromatography (GC) used to be the main technique employed for this purpose. As documented in recent reviews (Hertherton et al. 2004, Hernández et al. 2006), GC-based multiresidue methods are still widely used in control laboratories worldwide, nevertheless, it should be noted that their scope becomes more and more insufficient. Many registered pesticides are relatively polar compounds, not amenable to direct GC analysis, and on this account, development/validation of a broad-scope procedures employing high-performance liquid chromatography (HPLC) for sample separation and selective detection strategy, such as tandem mass spectrometry (MS/MS), has become an urgent task.

Most of published LC-MS based methods involve either extensive, cost and labour demanding clean-up procedures for processing of crude extracts, what, unavoidably, results in a loss of some target analytes. Similarly, solvent-exchange step, which is in some cases carried out prior to introduction of extract onto LC column, makes the sample preparation process less effective (Hertherton et al. 2004). Challenges exists both in innovation of sample handling strategies and use of novel instrumentation applicable for determinative step.

Regarding the first aspect, significant increase of sample throughput, reduction of labour demands and improvement of cost-effectiveness can be obviously achieved by implementation of so called QuEChERS method (Quick, Easy, Cheap, Effective, Rugged, Safe) originally developed for GC-based analysis of multiple pesticide residues in [START_REF] Anastassiades | A multi-residue screening method for the determination of 73 pesticides and metabolites in fruit and vegetables using high-performance liquid chromatography/tandem mass spectrometry[END_REF]. Acetonitrile extraction accompanied by simultaneous liquid-liquid partitioning is followed by dispersive SPE clean-up. According to recently modified version, either acetonitrile containing 1% acetic acid and acetate buffer (Lehotay et al. a. 2005) or citrate buffer (www.quechers.com) are used in the first step.

Regarding the measurement tools, current LC-MS/MS instruments enable, thanks to novel designs of ion sources and availability of fast electronics, determination of large range of pesticides in complex matrices at (ultra)trace levels and, in addition, also an on-line confirmation of residues identity (Hertherton et al. 2004[START_REF] Hancock | Comparison of ultra-performance liquid chromatography and high-performance liquid chromatography for the determination of priority pesticides in baby foods by tandem quadrupole mass spectrometry[END_REF], Soler et al. 2006). In older studies there was a tendency to develop LC-MS method only for a single residue or small multiresidue set of chemically related pesticides that were not amenable to GC analysis because of their high polarity or low thermal stability (e.g. [START_REF] Mezcua | Application of ultra performance liquid chromatography-tandem mass spectrometry to the analysis of priority pesticides in groundwater[END_REF]. Some of until now published LC-MS/MS-based methods (Hertherton et al. 2004, Hernández et al. 2006[START_REF] Lehotay | Use of buffering and other means to improve results of problematic pesticides in a fast and easy method for residue analysis of fruits and vegetables[END_REF], Hancock et al. 2004[START_REF] Janson | A new multi-residue method for analysis of pesticide residues in fruit and vegetables using liquid chromatography with tandem mass spectrometric detection[END_REF][START_REF] Klein | Applicability of gradient liquid chromatography with tandem mass spectrometry to simultaneous screening for about 100 pesticides in crops[END_REF][START_REF] Alder | The ECHO technique -the more effective way of data evaluation in liquid chromatography-tandem mass spectrometry analysis[END_REF], Ortelli et al. 2004) enable control of more than 50 pesticides in one chromatographic run, however, only one of them (Hertherton et al. 2004) allows simultaneous acquisition of two MS/MS transitions for all analytes in the method. It should be noted that only one MS/MS transition monitored for each analyte does not provide absolute confirmation of identity and further evidence may be required, especially in cases when particular MRL is exceeded (Commission document No. SANCO/10232/2006).

In general terms, several confirmative strategies could be employed. However, some of them, such as a change of LC separation system intended for confirmation based on retention times conformity and/or switching to a MS ionization mode employing different principle (e.g. ESI vs. APCI) are not convenient or feasible from practical point of view. Medium and/or high resolution MS detectors (Commission document SANCO/10232/2006), e.g. those employing time-of-flight (TOF) mass analyzers, represent another alternative for detection of target analytes. It should be noted, however, that only hybrid instruments, such as quadrupole-timeof-flight (Q-TOF), allow full confirmation of particular analyte. Currently, most laboratories specialized in pesticide residue analysis prefer affordable low resolution MS/MS for analysis of target compounds, as an optimal option enabling both quantification and confirmation of target pesticides at trace levels (Hertherton et al. 2004, Hernández et al. 2006[START_REF] Anastassiades | A multi-residue screening method for the determination of 73 pesticides and metabolites in fruit and vegetables using high-performance liquid chromatography/tandem mass spectrometry[END_REF][START_REF] Lehotay | Use of buffering and other means to improve results of problematic pesticides in a fast and easy method for residue analysis of fruits and vegetables[END_REF], Fernández et al. 2000, Hancock et al. 2004[START_REF] Janson | A new multi-residue method for analysis of pesticide residues in fruit and vegetables using liquid chromatography with tandem mass spectrometric detection[END_REF][START_REF] Klein | Applicability of gradient liquid chromatography with tandem mass spectrometry to simultaneous screening for about 100 pesticides in crops[END_REF][START_REF] Alder | The ECHO technique -the more effective way of data evaluation in liquid chromatography-tandem mass spectrometry analysis[END_REF], Ortelli et al. 2004[START_REF] Hancock | Comparison of ultra-performance liquid chromatography and high-performance liquid chromatography for the determination of priority pesticides in baby foods by tandem quadrupole mass spectrometry[END_REF][START_REF] Kovalczuk | Ultra-performance liquid chromatography-tandem mass spectrometry: A novel challenge in multiresidue pesticide analysis in food[END_REF][START_REF] Mezcua | Application of ultra performance liquid chromatography-tandem mass spectrometry to the analysis of priority pesticides in groundwater[END_REF], Díez et al. 2006, Zrostlíková et al. 2003) (Soler et al. 2006[START_REF] Hancock | Comparison of ultra-performance liquid chromatography and high-performance liquid chromatography for the determination of priority pesticides in baby foods by tandem quadrupole mass spectrometry[END_REF], Hertherton et al. 2004).

Regarding instrumental set-up, most of current LC-MS/MS based multiresidue methods employ conventional HPLC systems for separation of sample components. (Hertherton et al. 2004, Hernández et al. 2006[START_REF] Lehotay | Use of buffering and other means to improve results of problematic pesticides in a fast and easy method for residue analysis of fruits and vegetables[END_REF], Hancock et al. 2004[START_REF] Janson | A new multi-residue method for analysis of pesticide residues in fruit and vegetables using liquid chromatography with tandem mass spectrometric detection[END_REF][START_REF] Klein | Applicability of gradient liquid chromatography with tandem mass spectrometry to simultaneous screening for about 100 pesticides in crops[END_REF][START_REF] Alder | The ECHO technique -the more effective way of data evaluation in liquid chromatography-tandem mass spectrometry analysis[END_REF], Ortelli et al. 2004, Soler 2006). The sufficient separation of multiple residues (tens, even hundreds of analytes) in HPLC-MS/MS methods can be accomplished as soon as within 20-30 min, however, the need to carry out post run column reequilibration may increase the total analysis time up to 30-40 min. On this account, instrumental analysis becomes a limiting step in laboratory throughput. In theory, there are also other strategies that can increase the speed of chromatographic separation such as increase of mobile phase flow rate or the use of shorter columns [START_REF] Kovalczuk | Ultra-performance liquid chromatography-tandem mass spectrometry: A novel challenge in multiresidue pesticide analysis in food[END_REF], however none of them is suitable for trace analysis of multiple pesticide residues in complex matrices such as food. The recent introduction of ultra-performance liquid chromatography (UPLC) system, such as Acquity™, is capable to operate conventional "HPLC size" columns with small (1.7 µm) porous particles at pressures as high as 15000 psi (1025 bar) has offered a new challenge to increase significantly number of analyzed samples per day. Under these conditions, the van Deemter equation indicates that a significant gain in efficiency is not diminished at increased flow rates of mobile phase. [START_REF] Hancock | Comparison of ultra-performance liquid chromatography and high-performance liquid chromatography for the determination of priority pesticides in baby foods by tandem quadrupole mass spectrometry[END_REF]. As demonstrated in recently published studies [START_REF] Hancock | Comparison of ultra-performance liquid chromatography and high-performance liquid chromatography for the determination of priority pesticides in baby foods by tandem quadrupole mass spectrometry[END_REF][START_REF] Kovalczuk | Ultra-performance liquid chromatography-tandem mass spectrometry: A novel challenge in multiresidue pesticide analysis in food[END_REF], the improvement of several methods performance parameters including decrease of limits of detection (LODs) and other characteristics depends on particular experimental set-up.

The aim of presented study was to develop a fast multiresidue UPLC-MS/MS method with performance characteristics fully complying with EU legislation requirements (Commission document No SANCO/10232/2006), both for common fruit commodities and fruit-based baby food. The potential of Acquity UPLC system (Waters) hyphenated with Quattro Premier XE tandem quadrupole mass spectrometer (Waters) was demonstrated in determination of multiple pesticide residues in apple extract prepared by QuEChERS procedure.

Experimental

Chemicals and material

Certified pesticide standards obtained from Dr. Ehrenstorfer GmbH (Germany) and/or Riedel de Haen (Germany) were used for preparation of individual stock standard solutions (concentrations in the range 0.3-3 mg mL -1 ) in either methanol, acetonitrile or acetone:acetonitrile mixture (1:9, v/v) depending on the solubility of particular pesticide. These solutions were used for preparation of: (i) individual stock standard solutions in methanol (1-5 µg mL -1 ) for electrospray (ESI) source tuning and MS/MS transitions settings and (ii) preparation of mixed standard solution in acetonitrile (1 µg mL -1 ). The working standard solutions (0.25-5000 ng mL -1 ) used for calibration were then prepared from this solution by dilution with acetonitrile.

Deionised water for preparation of a mobile phase was produced by Milli-Q apparatus (Millipore, Germany). Ammonium acetate 99.99% was obtained from Sigma-Aldrich (Germany), acetonitrile (Sigma-Aldrich, Germany) and methanol (Merck, Germany) were HPLC gradient grade solvents for pesticide residue analysis. The glacial acetic acid 99.99+ was purchased from Sigma-Aldrich (Germany). Anhydrous magnesium sulfate, sodium acetate trihydrate and acetone were obtained from Penta (Czech Republic), and Bondesil-Primary-secondary amine (PSA, 40 µm) sorbent was purchased from Varian (USA). The magnesium sulfate was heated for 8 h at 520°C in a muffle furnace to remove any residual water. Apple samples free of pesticide residues were obtained from an ecological farm.

Apple-based baby food was obtained from retail market.

Sample preparation

The buffered QuEChERS procedure was employed within the pre-analytical step. Blank apples (obtained from organic farm) were used for preparation of blank extracts.

Representative apple sample (3 kg) was thoroughly homogenized using 2094 Homogenizer (Foss Tecator, Sweden). 10 g of homogenate were weighted into PTFE centrifugation tube (50 mL) and shaken vigorously for 1 min with 1 % (v/v) acetic acid in acetonitrile. Anhydrous magnesium sulfate (4 g) and sodium acetate trihydrate (1.6 g) were added and the sample was then immediately vortexed (Ika-Werke, Germany) to prevent formation of coagulated magnesium sulfate. After shaking for 30 s, the samples were centrifuged (Hettich, Germany) at 11 000 RPM for 5 min. 2 mL of supernatant were transferred into another PTFE tube (14 mL) containing anhydrous magnesium sulfate (300 mg) and primary-secondary amine (PSA) sorbent (100 mg). After shaking by Vortex and centrifugation (11 000 RPM, 5 min), the supernatant was filtered through 0.2 µm PTFE filter (National Scientific, USA). All purified blank extracts were combined. 950 µL of this extract were then mixed with 50 µL of appropriate mixed working standard solution in acetonitrile and aliquot (5 µL) of this spiked extract was then analyzed by UPLC-MS/MS. The step following UPLC-MS/MS method optimization was its application to apple-based baby food analysis.

UPLC-MS/MS Analysis

UPLC analyses were performed using an Acquity™ UPLC system (Waters, USA) equipped with Acquity™ UPLC BEH C 18 separation column (100 x 2.1 mm, 1.7 µm) (Waters, USA).

The sample temperature as well as column temperature was maintained at 25 °C. The mobile phase contained 0.005 M ammonium acetate in deionised water (A) and methanol (B), the flow rate was 0.3 mL min -1 .

For UPLC separation the gradient elution was employed, starting composition of 20% B, rising linearly to 100% B over 6 min, and then held for 2 min at 100% B. 2 min reequilibration to initial mobile phase composition followed. Sample injection volume 5 µL was used in all experiments.

Identification/quantitation of target analytes was performed using the Quattro Premier XE tandem mass spectrometer (Waters, USA). The detector was operated in a positive electrospray (ESI+) ionization mode. Multiple reaction monitoring (MRM) conditions (collision energy and cone voltage) were optimized for each pesticide during infusion (5 µL min -1 ) of individual pesticide solution (1-5 µg mL -1 ) into the mobile phase flow (A:B 50:50, (v/v); 0.3 ml mL -1 ). All MS experiments were realized employing following parameters: capillary voltage 3.5 kV, extractor voltage 4 V, source temperature 120°C, desolvation temperature 250°C, cone gas flow 100 L h -1 and desolvation gas flow 700 L h -1 (both gases were nitrogen). Argon was used as a collision gas (3.3 10 -3 mbar). Tuned and optimized MS/MS transitions as well as specific cone voltages and collision energies are summarized in Table 1. Analytes were divided into MRM segments based on their elution characteristics (Table 1). In each of these segments selected MS/MS transitions were monitored at the same dwell time 5 ms, inter-channel and inter-scan delays of 10 ms for all transitions. Generated experimental data were processed using MassLynx software version 4.0 Service Pack 4, Software Change Note #462 (Waters, USA).

[Insert Table 1 about here]

Method performance

The analytes mixture in pure solvent was repeatedly analyzed within the optimization/development of UPLC-MS/MS to tune chromatographic and MS/MS parameters. Repeatability and LODs of UPLC-MS/MS method were obtained on the basis of data generated through six replicate sequences comprising set of matrix-matched standards consisting of 10 purified apple extracts spiked by target analytes mixture at levels: 250; 150; 50; 20; 10; 5; 2; 1; 0.5 and 0.25 µg kg -1 , respectively. Prior to running the sequences, the eletrospray (ESI) source chamber was cleaned and then twelve repeated injections of blank sample extract were performed to minimize the changes of analytes responses within the initial injections of test samples. The LOD for each pesticide was estimated as an analyte concentration, at which signal to noise ratio value for primary MS/MS at least 3 (S/N ≥ 3) was obtained within all six sequences (described above). For S/N estimation Peak-to-peak (PtP) strategy integrated in the MassLynx software was employed. The limit of quantitation (LOQ), (fixed as lowest calibration level -LCL) was defined as an analyte concentration at which following requirements were met: (i) primary MS/MS transition -S/N ≥ 5 and (ii) secondary MS/MS transition -S/N ≥ 3 obtained in all six sequences. Within the study also the matrix effect for each analyte was estimated. The calculation resulted from replicated analyses of six sequences comprising: (i) set of spiked purified apple extracts M 1 -M 3 (prepared as described in chapter 3.3) at levels 10, 20 and 50 µg kg -1 (ii) standards in pure solvent (S 1 -S 3 ) at levels: 10, 20 and 50 ng mL -1 (prepared as described in section "Chemicals and material") and (iii) blank apple extracts (B); (prepared as described in section " Sample preparation"). The injection order within the sequence for ME investigation is shown in Table 2. The blank apple extract was analyzed to check the cross-contamination within the sample injections. The value of matrix effect was calculated as a ratio of average area of matrix-matched standards and average area of solvent standards for each analyte.

[Insert Table 2 about here]

Results and discussion

Sample preparation

Depending on a sample preparation strategy, various co-extracts are contained in analyzed samples (Díez et al. 2006, Hercegová et al. 2004, Zrostlíková et al. 2003, Alder et al.2004).

Their amount and character may influence, in a different extent, the overall performance of respective analytical method. From a wide range of isolation/purification approaches conceivable in pesticide residue analysis (e.g. described by Fernández-Alba 2005, Lehotay et al. a. 2005[START_REF] Janson | A new multi-residue method for analysis of pesticide residues in fruit and vegetables using liquid chromatography with tandem mass spectrometric detection[END_REF][START_REF] Hancock | Comparison of ultra-performance liquid chromatography and high-performance liquid chromatography for the determination of priority pesticides in baby foods by tandem quadrupole mass spectrometry[END_REF] (Lehotay at al. a. 2005, Díez et al. 2006) for low fat matrices such as fruits/vegetables, QuEChERS currently represents the most challenging preanalytical option for analysis of a wide range of pesticides representing various polarity classes. Based on the list of target analytes involved in these studies, representatives of various pesticide classes were selected for our experiments. Another option we had to decide was the choice of optimal solvent strength for introduction of QuEChERS extract (acetonitrile solution) onto UPLC column. Theoretically, dilution of sample by water should be carried out to obtain a solvent strength similar to initial mobile phase composition (in particular case A:B 8:2, v/v). Such approach was employed in most of LC-MS based studies employing various "classic" sample preparation techniques (e.g. Hertherton 2004, Hernández et al. 2006, Hancock at al. 2004[START_REF] Klein | Applicability of gradient liquid chromatography with tandem mass spectrometry to simultaneous screening for about 100 pesticides in crops[END_REF][START_REF] Alder | The ECHO technique -the more effective way of data evaluation in liquid chromatography-tandem mass spectrometry analysis[END_REF]). However, addition of water to QuEChERS acetonitrile extract may result in the matrix precipitation and, consequently, in some loss of target analytes. With regards to these problems, undiluted sample was injected.

Thanks to its low volume (5 µl), a distortion of early eluting peaks was negligible. Although some sensitivity was sacrificed by reduction of sample equivalent to 5 mg, the LODs were still low enough, to enable reliable control of "baby food limit" (10 µg kg -1 ) for most of analytes. Moreover, decreasing of sample load also increases column life-time and reduces the demands for MS source maintenance.

UPLC-MS/MS

MS/MS parameters

With regards to the principles of confirmation defined in recent Commission document No. SANCO/10232/2006, overwhelming evidence that sample actually contains particular pesticide, i.e. proof of its identity, has to be provided by respective analytical procedure. To meet this requirement and avoid the need of positive samples re-analysis, we aimed to incorporate two MS/MS transitions to get a required specificity of detection process for all analytes. Contrary to the majority of existing multiresidue LC-MS/MS methods in which (as a compromise enabling to achieve low enough LODs for a wide range of analytes) only one MS/MS transitions was monitored, two selective transitions with the highest abundance were (2002/657/EC). This strategy has been demonstrated as suitable for analysis of residues in plant materials by other authors [START_REF] Hancock | Comparison of ultra-performance liquid chromatography and high-performance liquid chromatography for the determination of priority pesticides in baby foods by tandem quadrupole mass spectrometry[END_REF], Hertherton et al. 2004, Soler 2006).

Analytes detection and fragmentation parameters (cone voltages and collision energies) were optimized, under ESI+ conditions by infusing a standard solution by a syringe pump into the mobile phase. Alike in similar studies (Hertherton et al. 2004, Hernández et al. 2006[START_REF] Hancock | Comparison of ultra-performance liquid chromatography and high-performance liquid chromatography for the determination of priority pesticides in baby foods by tandem quadrupole mass spectrometry[END_REF], maximum permitted tolerances for relative ion ratios (Commission document SANCO/10232/2006) were considered for confirmation in presented procedure employing MRM mode. Phorate was only pesticide for which secondary (qualifying) MS/MS transition was not established. Confirmation of this compound, based on ion ratio, was therefore impossible.

MRM optimization

In theory, the replacement of conventional HPLC column (particles in the range 3-5 µm) with novel UPLC columns with sorbent consisting of small particles (2 µm and less) results in reduced heights of theoretical plate (HETP) and increased peak capacity [START_REF] Hancock | Comparison of ultra-performance liquid chromatography and high-performance liquid chromatography for the determination of priority pesticides in baby foods by tandem quadrupole mass spectrometry[END_REF][START_REF] Kovalczuk | Ultra-performance liquid chromatography-tandem mass spectrometry: A novel challenge in multiresidue pesticide analysis in food[END_REF]. As far as narrow peaks such as those generated in UPLC analysis are to be accurately integrated, MS scanning frequency has to be adjusted properly. For meeting the sufficient data density, i.e. at least 10 data points (Hill et al. 2002) across the peak ,even for analytes at the lowest calibration levels, the dwell time period was set down to 5 ms, what is a minimal setting attainable by Quattro Premier XE instrument, inter-scan and inter-channel delays were then 10 ms (under this setting the density for peaks at 150 µg kg -1 was rather excessive, 20-30). With regard to high number of analytes eluted within a short time, and considering strong requirement for low LODs in pesticide residue analysis, several time windows, within which only a limited number of MS/MS transitions is monitored, have to be established across the chromatogram. Unfortunately, under real-life conditions, slight fluctuation of analytes retention times may occur, namely during analysis of large series of samples. Therefore, to avoid running of analytes out of their elution slot, some overlap of these time windows has to be established for reliable acquisition of respective data. Increased number of MS/MS transitions is then monitored in the area of overlap and, consequently, acquisition frequency may significantly decrease. As far as acquisition of data points for particular analyte occurs under largely differing scanning speeds, as shown for aldicarb in Fig. 1A, poor repeatability of generated data (relative standard deviation (RSD) 23% in particular case) is typically encountered. One of conceivable strategies to minimize the above unfavourable effects is illustrated in Fig. 1B. In this case, some peaks in chromatogram are eluted within the overlap of adjacent windows, in which, both the scanning speed and dwell time are equal or very similar.

Repeatability of areas of peaks eluted at the segment "borderline" is fairly improved due to only small differences in acquisition set-up across the analyte elution band. Using this approach repeatability of aldicarb measurement (expressed as RSD) was as low as 6%. It should be noted, however, that optimization of windows setting is a very demanding task and any the expanding method scope (adding of a new analyte), is rather complicated.

In Fig. 1C, further improved, obviously simpler approach is presented. Contrary to the previous strategy, elimination of problems associated with the overlap of time windows characterized by different scanning conditions is achieved by separating analytes into many time segments within witch only a small number of MS/MS transitions is monitored. This setup allows a large flexibility in the method optimization, since modification of windows overlap (when needed), does not result in significant changes in scanning frequencies. In addition, for some peaks a special MRM segment including respective two characteristics MS/MS transitions can be easily added without a significant change of the scanning frequency. Example in Fig. 1C shows flexible settings of "tailor-made" windows for aldicarb.

The later strategy was employed in comprehensive optimization of the current UPLC-MS/MS procedure.

Method performance characteristics

Considering a large number of registered pesticide/fruit combinations involved in various surveillance/compliance studies, it was obviously impossible to carry out the validation of UPLC-MS/MS procedure for all of them. On this account, generic approach was adopted for set-up of this study. The choice of test matrice was based on assumptions on comparable nature of matrix effects occurring in LC-MS/MS analysis of commodities characterized by For most of residues, optimized method allowed to obtain LODs and LOQs (lower than 10 µg kg -1 , see Table 3) what enables to use it also for control of uniform baby food MRL set at this level. For two "priority" pesticides specified in EU Commission Directive 2003/13/EC included into our study (oxydemethon methyl and demethon-S-methyl sulfone) LODs 0.5 µg kg -1 were obtained, what allows their reliable control of even lower level. (MRL for demethon-S-methyl is 6 µg kg -1 , expressed as sum of demethon-S-methyl, oxydemethonmethyl and demethon -S-methyl sulfone). The calibration curves were realized by six calibration sets of spiked QuEChERS apple extracts over a wide concentration range 0.25-250 ng mL -1 (equivalent to contamination level 0.25-250 µg kg -1 , prepared as described in 3.4).

The correlation coefficient (R 2 ) was calculated by MassLynx software for the concentration range LOQ-250 µg kg -1 , (corresponds to LOQ-250 ng mL -1. ). For the majority of tested pesticides the calibration curves were linear (correlation coefficient R 2 > 0.98) over the tested range. As shown in Table 3, for acetamiprid, carbendazim, carbofuran, pirimicarb and thiacloprid, excellent linearity was achieved over the whole calibration range (0.25-250 µg kg - 1 ).

It should be noted that the applicability of QuEChERS pre-analytical procedure for isolation of a wide range of pesticides from apples and similar matrices was reported in earlier published studies [START_REF] Anastassiades | A multi-residue screening method for the determination of 73 pesticides and metabolites in fruit and vegetables using high-performance liquid chromatography/tandem mass spectrometry[END_REF][START_REF] Lehotay | Use of buffering and other means to improve results of problematic pesticides in a fast and easy method for residue analysis of fruits and vegetables[END_REF], Díez et al. 2006) and, therefore, recoveries of target analytes were not examined in our experiments. The current study was mainly focused on critical assessment of performance characteristics of UPLC-MS/MS determinative step, and on this account, only spiked extracts prepared from blank apples were analyzed. Regarding random errors of six repeated injections (as described in 3.4), relatively low RSDs 6 % in average were found for all the tested pesticides at concentration level 10 µg kg -1 . For only few analytes with LOQ > 10 µg kg -1 the repeatability was calculated at their LOQ (see Table 3). Dodine was the only pesticide for which the RSD exceeded 20% (see Table 3). The uncertainty of measurement of this troublesome analyte (see Fig. 2), as documented in our long-term records, increases with growing number of samples analyzed on Acquity BEH C18 separation column. Although column with different stationary phase (Discovery C 18 ) was used in our routine multiresidue HPLC-MS/MS method, similar problems were encountered.

[Insert Table 3 about The suppression/enhancement of individual analytes signal measured in the presence of matrix components, i.e. matrix effect, was estimated on the basis of comparison with pure solvent standard. Within this study, matrix effects ranging from 55 to 160% were observed, with the highest enhancement of response obtained for azoxystrobin, and the most intensive signal suppression found for spiroxamine. Although for most of analytes matrix effects were in acceptable range 80-120%, the matrix-matched standards were used for quantification thorough the study to achieve good accuracy of generated data.

The possibility to obtain significant reduction of analysis time by the use of UPLC-MS/MS system, even in case of such complex sample as multiple residues in plant matrices, was documented. The UPLC-MS/MS examination of apple extract was completed within 8 min (see Fig. 3). Compared to similar procedures employing conventional HPLC-MS/MS systems sample throughput was approx. 3-4 times higher, as summarized in Fig. 4.

The time necessary for re-equilibration the separation column was set to 2 min, and as documented by negligible RSDs of retention times (0.11-0.36%), this time was sufficient enough for their good repeatability.

[Insert Figure 3 and 4 about here]

Conclusions

The UPLC Acquity separation system (Waters) coupled with Premier Quattro XE (Waters) tandem quadrupole mass spectrometric detector, used for analysis of multiple pesticide residues, enabled an overall improvement of method performance characteristics when QUEChERS apple extract was injected:

• Due to the reduced band broadening on a high resolution UPLC column, narrower analyte peaks, hence increased signal to noise ratios, were obtained. This resulted in achieving limits of quantification (LOQs) for most pesticides fairly below 10 µg kg -1 . Under these conditions, not only MRLs established by EU regulation for common fruit commodities, but also uniform baby food limit (10 µg kg -1 ) can be reliably controlled.

• Thanks to the high detection sensitivity, simultaneous acquisition of two characteristic MS/MS transitions was possible for all the target analytes. In this way, contrary to most of currently existing LC-MS based methods, on-line confirmation of residues • Distinct improvement of respectabilities of peak areas measurement was obtained by increasing the number of (partly overlapping) time windows adjusted throughout the chromatographic run, and, at the same time, decreasing the number of MS/MS transitions set within each of them. Large changes in acquisition frequencies across the eluting analyte band that may cause unacceptable variations in recorded signals (too high standard deviations, RSDs); can be eliminated by this data acquisition strategy.

• Matrix effects (peak suppression/enhancement) were not too intensive (only small sample equivalent inject), generally in the range 80 -120%. In spite of that the use of matrix-matched standards for accurate quantification is recommended.

• Distinctly reduced analysis time (10 min in particular case) attainable thanks to possibility to operate UPLC column at high mobile phase flow rates without any loss of resolution enabled significantly increased sample throughput.

In conclusion, the newly developed procedure fully meets the analytical quality control Rather poor repeatability of dodine over the whole calibration range is compared with calibration curve of malathion. In case of malathion as well as most pesticides calibration curves were linear (R 2 >0.98). Spiked apple extracts were used for calibration within one sequence containing six calibration sets (each had 10 points ranging from 0.25-250 µg kg -1 ).

* -calibration range 5-250 µg kg -1 (equivalent to 5-250 ng mL -1 )

+ -calibration range 1-250 µg kg -1 (equivalent to 1-250 ng mL -1 )

0

  selected for detection in our study. The confirmation of identity was based on the ion ratio statistics for two MS/MS transitions monitored, as recommended by Commission document No. SANCO/10232/2006 and as usually applied in the analysis of veterinary drugs residues (Commission Decision of 12 August 2002 implementing Council Directive 96/23/EC of analytical methods and the interpretation of results

  similar composition(Commission document No. SANCO/10232/2006, Hercegová et al. 2004), i.e. similar nature of co-extracts interfering with ionization process. In particular case, apples were selected for our study as a representative of high moisture low fat commodity category. The applicability of a new method in the final phase of study was demonstrated on analyses of apple-based fruit baby foods.

  here], [Insert Figure2about here]

  for any positive signal. Confirmation was also supported by highly stable retention times, even within large series of injected samples.

(

  AQC) requirements to support the validity of data used for checking compliance with maximum residue limits (MRLs), enforcement actions, or assessment of consumer exposure to pesticides as laid down in Commission Document N° SANCO/10476/2003.

Figure 1 :

 1 Figure 1: The impact of data acquisition setting in UPLC-MS/MS (changes of scanning frequency) on recording of analyte elution profile; aldicarb was shown as an example: In each segment a certain number of MS/MS transitions was monitored according to particular MRM method. The total number of MS/MS transitions monitored in each moment, expressed as the sum of all overlapped segments is shown below each chromatogram. The phenomenon of poor repeatability based on largely changed scanning frequencies is demonstrated on peak of aldicarb. For details see paragraph "MRM optimization".

  250 µg kg -1 ). Within one sequence six calibration sets (each had 10 points ranging from 0.25-250 ng mL -1 ).Deleted: ng mL -1 Deleted: µg kg -1 Deleted: .

Figure 3 :

 3 Figure 3: Combined UPLC-MS/MS MRM chromatogram based on the quantifying MS/MS transitions (Table1) of spiked apple extracts at 0.05 mg kg -1 .

  recently introduced QuEChERS strategy[START_REF] Anastassiades | A multi-residue screening method for the determination of 73 pesticides and metabolites in fruit and vegetables using high-performance liquid chromatography/tandem mass spectrometry[END_REF][START_REF] Lehotay | Use of buffering and other means to improve results of problematic pesticides in a fast and easy method for residue analysis of fruits and vegetables[END_REF] was chosen for our study. As demonstrated

	within international collaborative studies
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Table 1 :

 1 Table 1) of spiked apple extracts at 0.05 mg kg -1 . Selected chromatographic and MS/MS (in ESI, positive mode) parameters for

	0
	0.50	1.00	1.50	2.00	2.50	3.00	3.50	4.00	4.50	5.00	5.50	6.00	6.50	7.00	7.50	8.00	8.50	9.00	9.50

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Table 2 :

 2 Sequence used for estimation of matrix effect values

	Injection order	1 2 3 4 5 6 7 8 9 10 11 12
	Sample code	S 3 M 3 S 3 B S 2 M 2 S 2 B S 1 M 1 S 1 B	Deleted: µg
	Concentration levels (ng mL -1 )	50	-	20	-	10	-
	For apple extracts (sample B and M) the concentration correspond to µg kg -1 in the sample
	M1-M3 -spiked purified apple extracts; S1-S3 -standards in pure solvent; B -blank apple
	extracts. For more details see Experimental.

Table 3

 3 Performance characteristics obtained by repeated UPLC-MS/MS analyses (n=6) of spiked apple extracts (concentrations of target analytes corresponded to contamination level 10 µg kg -1 )

	Acceptable limit ║

Table 3 (continued) Acceptable limit ║

 3 -repeatability was calculated on LOQ ‡ -Matrix effect calculated as described in 3.4. § -Correlation coefficient calculated for calibration range LOQ-250 µg kg -1x -Qualifying MS/MS transitions was not established + -µg kg -1 correspond to ng mL -1 ║ Specified in Commission document No.SANCO/10232/2006

	Pesticide	RSD (%)	ME ‡ (%)	LOQ (µg kg -1 )+	(R 2 ) †	(quan./qual.) Ion ratio	RSD (%) Ion ratio	(%)	criteria Met	Formatted Table
	29 Imazalil	3.4	79	5	0.99	1.4	10	20	Yes
	30 Imidacloprid	3.7	83	2	0.99	1.5	5	20	Yes
	31 Kresoxim methyl	9.7	90	5	0.98	1.2	15	20	Yes
	32 Linuron	4.3	85	1	0.98	1.8	3	20	Yes
	33 Malaoxon	4.5	116	1	0.99	4.4	14	25	Yes
	34 Malathion	5.5	106	1	0.98	3.2	12	25	Yes
	35 Metalaxyl	5.9	95	1	0.98	2.3	8	25	Yes
	36 Methamidophos	4.4	80	2	0.99	2.5	19	25	Yes
	37 Methiocarb	3.4	119	1	0.99	1.5	14	20	Yes
	38 Methiocarb sulfone	8.7	92	5	0.99	179	28	50	Yes
	39 Methiocarb sulfoxide	9.2	148	5	0.98	2.8	19	25	Yes
	40 Methomyl	5.5	77	1	0.99	1.1	17	20	Yes
	41 Myclobutanil	2.2	100	1	0.99	2.8	20	25	Yes
	42 Omethoate	3.8	96	1	0.99	6.5	15	30	Yes
	43 Oxydemeton methyl	4.6	97	1	0.99	3.3	4	25	Yes
	44 Penconazole	4.6	85	2	0.99	1.3	10	20	Yes
	45 Phorate	6.1	92	5	0.98	x		x	No
	46 Phorate oxon*	4.5	103	20	0.98	33.8	20	50	Yes
	47 Phorate sulfon	9.9	92	1	0.99	1.9	12	20	Yes
	48 Phorate sulfoxide	7.8	93	1	0.99	1.2	15	20	Yes
	49 Pirimicarb	4.0	86	0.5	0.99	3.7	17	25	Yes
	50 Prochloraz	2.8	85	5	0.98	4.6	5	20	Yes
	51 Propiconazole	3.6	93	5	0.98	1.4	14	20	Yes
	52 Pyrimethanil	5.3	100	2	0.99	1.1	13	20	Yes
	53 Spiroxamin	9.2	55	2	0.98	2.5	5	25	Yes
	54 Tebuconazole	4.1	95	2	0.98	5.6	22	30	Yes
	55 Teflubenzuron	6.8	87	5	0.98	6.6	12	30	Yes
	56 Thiabendazole	3.3	78	1	0.99	6.6	5	20	Yes
	57 Thiacloprid	4.2	94	0.5	0.99	1.4	16	50	Yes

*

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Food Additives and Contaminants

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Acknowledgement

This study was a part of research project MSM 6046137505 granted by Ministry of Education, Youth and Sports of the Czech Republic. UPLC system Acquity loan, before its purchase is acknowledged to WATERS Gesellschaft m.b.H, Prague, Czech Republic.


Antonia Susca

Gaetano Stea

Giuseppina Mulé

Giancarlo Perrone

Giuseppina Mulè

PCR identification of Aspergillus niger and Aspergillus tubingensis based on calmodulin gene

Aspergillus niger and A. tubingensis, species belonging to section Nigri, are commonly 2 found in plant products and processed food, such as grapes, cereals, coffee and derived 3 products. These two species are very difficult to differentiate by classical morphological 4 criteria and some isolates are known to produce ochratoxin A. The exact identification 5 of these two species is very important to avoid overestimation of toxicological 6 contamination and related risks. A PCR-based identification and detection assay was 7 developed as a tool to identify A. niger and A. tubingensis, using molecular differences 8 obtained by sequencing the calmodulin gene. Two pairs of species-specific primers 9

tubingensis. Species-specific PCR products generated by each primer set were 505 bp 11 (A. tubingensis) and 245 bp (A. niger) in length, which could be potentially useful for a 12 multiplex PCR assay. The sensitivity of this assay was about 10 pg DNA in 25 µl PCR 13 reaction volume, using pure total DNA of the two species. The method described in this 14 study represents a rapid and reliable procedure to assess the presence in food products 15 of two ochratoxigenic species of section Nigri. [START_REF] Magnoli | Mycoflora and 20 ochratoxin-A producing strains of Aspergillus Section Nigri in wine grapes in 21 Argentina[END_REF]. OTA is a potent nephrotoxin diffusely distributed in food [START_REF] Sugita | PCR identification 12 system for the genus Aspergillus and three major pathogenic species: Aspergillus 13 fumigatus, Aspergillus flavus and Aspergillus niger[END_REF] and feed products such as grains, legumes, coffee, dried fruits, beer, wine, and meats. [START_REF] Patino | PCR detection 13 assays for the ochratoxin-producing Aspergillus carbonarius and Aspergillus 14 ochraceus species[END_REF] Since A. tubingensis and A. niger, together with A. carbonarius, are known to produce Nigri, since it contains some species specific diagnostic traits, suitable for diagnostic 8 purposes. Molecular differences were exploited to set up a qualitative PCR assay for A. 9 carbonarius and A. japonicus/A. aculeatus detection [START_REF] Perrone | PCR assay for identification of Aspergillus 19 carbonarius and Aspergillus japonicus[END_REF]. Moreover a 10 quantitative real-time PCR assay using TaqMan chemistry was recently set up for A. The objective of this work was to set up a species-specific PCR assay based on 14 differences in sequences found in the calmodulin gene for an accurate identification of 15 A. niger and A. tubingensis in pure cultures. PCR amplification of ITS rDNA and 5.8S rDNA followed by RsaI digestion. The importance of a molecular tool for species identification is due to the difficulty of 6 morphological recognition of species within this section, and specifically within the A.

7 niger "aggregate" group. These primers could be a useful tool for investigations of a

  designed and empirically evaluated for PCR identification of A. niger and A.

10

 10

Aspergillus

  Aspergillus niger, Aspergillus tubingensis, ochratoxin A, species-species belonging to section Nigri are distributed worldwide. Many species 3 of them cause food spoilage, and several are used in the fermentation industry (Bennett 4 and Klich, 1992), or candidate in the biotechnology industries, such as A. niger which 5 has been granted the GRAS (Generally Regarded As Safe) status by the Food and Drug 6 Administration of the US Government. Recently, the significance of these species, 7 commonly known as "black aspergilli" has completely changed since they were 8 identified as the main fungi responsible for the ochratoxin A (OTA) accumulation in 9 grapes and wine (Cabanes et al. 2002; Da Rocha Rosa et al., 2002; Battilani and Pietri, 10 2002;

20brasiliensis(

  Kuster van Someren et al., 1991; Accensi et al., 1999; Parenicova et al., 21 2000; Abarca et al., 2004). These four species are very difficult to differentiate by 22 classical morphological criteria, such as conidial shape, colour and size (Samson et al., 23 2004). However, effectiveness in the estimation of OTA contamination is dependent on 24 species and OTA production, hence the use of primers 1 identified on genes involved in the metabolic pathway would be more helpful. Although 2 two primers were recently developed to detect OTA producers by Polymerase Chain 3 Reaction (PCR) (Dao et al., 2005), genes involved in steps of OTA biosynthesis have 4 not yet been identified. A PCR-based identification and detection of species could be 5 an useful tool for identifying potential ochratoxigenic Aspergillus spp. The calmodulin 6 gene has proved to be highly useful in discrimination of species belonging to section 7

11 carbonarius

 11 DNA quantification on grapes[START_REF] Mulè | Development of quantitative 3 real-time PCR assay for the detection of Aspergillus carbonarius in grapes[END_REF].

4tubingensis( 11 DNA extraction 12 Fungal

 1112 Perrone et al., 2006b); and 4 strains as outgroup: 2 A. ochraceus,1 Botrytis 5 cinerea (ITEM 3715) and 1 Saccharomyces cerevisae (ITEM 6904). All strains were 6 obtained from the culture collection of the Institute of Sciences of Food Production, 7 with accession number ITEM. Further information about the strains (year of isolation, 8 depositor, toxin production, etc.) are available in the ITEM web site: 9 http://www.ispa.cnr.it/Collection. 10 strains were grown in shaken cultures (150 rpm) in Wikerham's medium (40 g 13 of glucose, 5 g of peptone, 3 g of yeast extract, 3 g of malt extract and water up to 1 L). 14 About 40 mg of filtered, frozen and lyophilized mycelium from each strain were used 15 for total DNA extraction using the EZNA Fungal DNA Miniprep Kit (Omega Bio-tek, 16 Doraville, USA). DNA was recovered and dissolved in sterile water. Concentrations of 17 DNA were determined by gel electrophoresis, and by measuring the ultraviolet-induced 18 fluorescence emitted by ethidium bromide molecules intercalated into DNA, and 19 comparing the fluorescent yield of the samples with a standard (100 bp DNA ladder, 20 New England Biolabs, Beverly, MA, USA).

  of approximately 700 bp fragments of the calmodulin gene with 2 CL1 and CL2A primers (O'Donnell et al., 2000). Amplifications of the partial 3 calmodulin gene were set up in 100 µl reaction mixtures containing 2.5 U of Taq Gold 4 DNA polymerase (Applied Biosystems, Foster City, CA, USA), 30 pmol of each 5 primer, 20 nmol of each deoxynucleoside triphosphate (Applied Biosystems), and 6 approximately 10 ng of fungal template DNA. PCR reactions were performed using the 7 following conditions: denaturation at 94 °C for 10 min; 35 cycles of denaturation at 94 8 °C for 50 s, annealing at 55 °C for 50 s, extension at 72 °C for 1 min; final extension at 9 72 °C for 7 min, followed by cooling at 4 °C until recovery of the samples. After 10 amplification, PCR products were purified by Microcon-PCR Filter Units (Millipore, 11 Bedford, MA, USA).

12 13 21 Primer design and testing 22 Conservative 3 ( 8 4

 12212238 Sequence analysis was performed with the "Big-Dye Terminator Cycle Sequencing 14 Ready Reaction Kit" for both strands, and the sequences were aligned with the "MT15 Navigator" software (Applied Biosystems). All the sequencing reactions were purified 16 by gel filtration through columns containing Sephadex G-50 (Pharmacia, Uppsala, 17 Sweden) equilibrated in double-distilled water and were analyzed on the automatic 18 sequencer "310-Genetic Analyzer" (Applied Biosystems). The calmodulin sequence 19 data of all 150 isolates were aligned by the Clustal-W method. 20 and polymorphic calmodulin gene sequences were sought in a 23 multialignment of 11 Aspergillus species belonging to Section Nigri using Clustal-W 24 on these data, two species-specific primer pairs were designed from the 1 highly conserved region in A. tubingensis and A. niger species, exploiting polymorphic 2 regions of the calmodulin gene in Aspergillus spp. using Primer Express software Applied Biosystems, Foster City, CA, USA). The sequence identity between the 4 designed primers and the calmodulin regions was analyzed with a BLAST search 5 (http://www.ncbi.nlm.nih.gov). 6 7 PCR products were separated in 1.5% Tris-acetate-EDTA-agarose gels and were 8 visualized with ethidium bromide and ultraviolet illumination (Sambrook et al., 1989). 9 As negative controls, strains belonging to 8 different species of Section Nigri (A. 10 aculeatus, A. ibericus, A. brasiliensis, A. carbonarius, A. ellipticus, A. etheromorphus, 11 A. japonicus, A. foetidus) were used. Strains of A. ochraceus, Botrytis cinerea, and 12 Saccharomyces cerevisae, obtained from ITEM Culture Collection were used as 13 outgroup. Positive controls of PCR reaction were prepared replacing the species-14 specific primer designed with rDNA primers ITS1 and ITS4 (White et al., 1990). 15 Images were captured and stored using a Kodak Easy-Share DX3215 Zoom Digital 16 Camera. The PCRs containing species-specific primers were set up in 25 µl reaction 17 mixtures containing 1.25 U of Taq Gold DNA polymerase (Applied Biosystems), 7.5 18 pmol of each primer, 5 nmol of each deoxynucleoside triphosphate (Applied 19 Biosystems, Foster City, CA, USA), and approximately 3 ng of fungal template DNA. 20 Reactions were performed using the following PCR conditions: denaturation at 95 °C 21 for 5 min; 25 cycles of denaturation at 94 °C for 50 s; annealing at 60 °C for 50 s;22extension at 72 °C for 1 min; final extension at 72 °C for 7 min, followed by cooling at 23 °C until recovery of the samples. Amplification products were checked on 1.5% 1 agarose gel stained with ethidium bromide.

2 348

 2 ResultsWe evaluated the usefulness of two PCR assays, that target a region of calmodulin gene, 5 for identifying Aspergillus niger and Aspergillus tubingensis within Aspergillus niger 6 "aggregate" of species. 7 The partial calmodulin gene sequence has both conserved and polymorphic regions 9 corresponding respectively mainly to exons and introns of the gene in the examined 10 species, useful for designing species-specific primers for A. niger and A. tubingensis. 11 Calmodulin sequences showed 92 % identity (figure 1) between A. niger and A.

12 tubingensis. 13 (

 1213 In this analysis were included the type strains of A. tubingensis ITEM 7040 AJ964876) and A. niger ITEM 4501 (AJ964872) respectively; their sequences were 14 previously deposited in the EMBL nucleotide sequence database(Perrone et al., 2006a).15 The visual inspection of the aligned calmodulin partial gene sequences readily identified 16 unique regions within the fragment. Primers were chosen to target a region displaying 17 high nucleotide divergence at the 3'-end among the species considered in this study.[START_REF] Perrone | PCR assay for identification of Aspergillus 19 carbonarius and Aspergillus japonicus[END_REF] Two pairs of species-specific primers for Aspergillus species belonging to Section Nigri 19 were designed after consideration of product size and melting temperature (table 1). All 20 primers were designed in order to: a) operate at same high annealing temperatures 21 (60°C), b) work simultaneously under the same PCR conditions and c) prevent the co-22 amplification of non-specific target of DNA. Specific primer pair NIG1/ NIG2 23 amplified a 245 bp fragment in A. niger, whereas TUB1/ TUB2 amplified a 505 bp 24 fragment in A. tubingensis and in the type strain of A. phoenicis (CBS 136.52), recently 25 A. tubingensis species according to the revision made by Samson et al. 1 (2004) using extrolites and ß-tubulin sequencing (figure 2).

2 3PCR4

 2 conditions using the two primer pairs were optimized and then tested on 44 Aspergillus strains (table2). The sensitivity of this assay was about 10 pg DNA in 25 µl 5 PCR reaction volume, using pure total DNA of the two species (data not shown). 6 7 Primers NIG1/NIG2 amplify only DNA belonging to A. niger strains and primers 8 TUB1/TUB2 amplify only DNA belonging to A. tubingensis, confirming their 9 specificity (figure 3). PCR amplicons were confirmed as originating from Aspergillus 10 tubingensis and Aspergillus niger by sequencing in both directions.

16 PCR

 16 assays have been introduced successfully for the identification and detection of 17 fungal species based on sequence analysis. PCR technology to detect fungi uses primers 18 which can target two kinds of sequences: 19 (i) fungus-specific regions of conserved proteins such as calmodulin, beta-20 tubulin, elongation factor 1 alfa, and 21 (ii) repeated sequences such as those of rDNA and mitochondrial DNA. Setting 22 up PCR assay requires definition of oligonucleotide primers to amplify a 23

14 15 Specific 18 (

 1518 primers described in this work have been designed on the basis of calmodulin 16 gene sequence comparisons of several strains of Aspergillus species and taking into 17 consideration the phylogenetic and taxonomic analysis made in previous publications Abarca et al., 2004; Samson et al., 2004). In accordance with previous studies (Perrone 19 et al., 2004; Mulè et al., 2006) we demonstrate that the calmodulin-based PCR method 20 has high degree of specificity for the identification of Aspergillus at species level within 21 the black aspergilli. in contrast, ITS regions showed low degree of specificity consisting 22 in variation of only three different nucleotides between these species (Parenicova et al.risks of misidentification. In addition, this method is suitable 2 for investigations that involve a large number of fungal isolates due to rapid DNA 3 extraction and simple PCR amplification.

  Fig.1Sequence alignment between calmodulin gene representative of A. tubingensis and A. niger spp. Dark regions correspond to high conserved regions and light regions correspond to species-specific regions, considered in primer's designing.

  Fig. 2 Species-specific PCR product obtained amplifying A. tubingensis DNA (505 bp) in lane 1 and A. niger DNA (245 bp) in lane 2.

Fig. 3

 3 Fig. 3 PCR products obtained amplifying Aspergillus spp. genomic DNA with NIG F/R (lanes 1-16) and TUB F/R (lanes 17-32): A. niger (lanes 1-3 and 17-19 ), A. tubingensis (lanes 4-6 and 20-22), A. phoenicis/A. tubingensis (lanes 7 and 23), A. awamori/niger (lanes 8 and 24), A. ellipticus (lanes 9 and 25), A. brasiliensis (lanes 10 and 26), A. heteromorphus (lanes 11 and 27), A. japonicus (lanes 12 and 28), A. ochraceus (lanes 13 and 29), A. foetidus (lanes 14 and 30), A. carbonarius (lanes 15 and 31), A. aculeatus (lanes 16 and 32).

Table 1

 1 Details of the two species-specific PCR primer pairs for A. niger and A. tubingensis

16 17 Material and methods 18 Fungal strains

	1	ibericus ITEM 6600 (AJ971806), A. japonicus ITEM 4497 (AJ582717), A. niger
	2	ITEM 4502 (AJ964872), A. tubingensis ITEM 7040 (AJ964876); 136 strains of "black
	3	aspergilli" isolated from grapes grown in different geographical areas: 1 A. aculeatus, 4
		A. ibericus, 4 A. brasiliensis, 31 A. carbonarius, 23 A. japonicus, 44 A. niger, 29 A.
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	20	One hundred and fifty strains were analyzed in this study: 10 type-strains belonging to y
	21	Aspergillus sect. Nigri, A. aculeatus ITEM 7046 (GenBank accession no. for
	22	calmodulin gene sequences, AJ964877), A. brasiliensis ITEM 7048 (AM295175), A.
	23	carbonarius ITEM 4503 (AJ964873), A. ellipticus ITEM 4505 (AM117809), A.
	24	foetidus ITEM 4507 (AM419749), A. heteromorphus ITEM 7045 (AM421461), A.
		5

Table 2 Fungal strains tested by PCR reaction with the species-specific primers for A. niger (NIG1/NIG2) and A. tubingensis (TUB1/TUB2).

 2 = representative strains utilized for PCR reactions showed in figure3

		Page 20 of 20
	Species	ITEM Strains
	A. aculeatus	7046*, 7047
	A. brasiliensis	7048*
	A. carbonarius	4503*, 4504, 4544
	A. ellipticus	4505*
	A. foetidus	4506*
	A. heteromorphus	7045*
	A. ibericus	6600
	A. japonicus	7044*, 7047
	A. niger	4501*, 4502*, 4774, 4775*, 4717, 4730*, 4541,
		4547, 4551*, 4552*, 5219, 5276, 4509, 7042
	A. ochraceus	7043*
	A. phoenicis	4498*
	A. tubingensis	4208*, 4500*, 4542, 4543, 4546*, 4720, 4721*,
		4725, 4728, 4815*, 4816, 4817, 4818, 7040*,
		7041,4510

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Paolo Montuori

Eric Jover

Marcello ; Morgantini

Josep Maria Bayona

Maria Triassi

Assessing human exposure to

Keywords: Bottled water, Polyethylene terephthalate, Phthalic acid, Phthalate esters, SPME, EI GC-MS

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

The bottled water industry in European countries and North America has expanded over the last 30 years, and it is also increasing rapidly in many developing countries (FDA, 2003).

Consumption reached 155 L per capita per year in Italy, 136 L in Mexico, 112 L in France, 123 L in Belgium, 99 L in Germany, 98 L in Spain, 97 L in Switzerland and63 L in the United States in 2000/2001 [START_REF] Potera | The price of bottled water[END_REF]Thurman et al., 2002).

In Italy, as in many developed countries, polyethylene terephthalate (PET) is widely used as a container for commercial bottled water, and its use is also increasing rapidly due to its lower production costs in comparison to glass containers [START_REF] Petrelli | Evoluzione normativa Della disciplina delle acque destinate al consumo umano[END_REF]. PET is synthesized by reacting ethylene glycol (C 2 H 6 O 2 ) with either terephthalic acid or its methyl ester catalysed by antimony oxide. The reaction is carried out under vacuum at high temperatures to achieve high molecular weight polymers. Some studies have shown PET decomposition and phthalate migration in the absence of an accurate temperature and humidity control during PET synthesis [START_REF] Castle | Migration of plasticizers from printing inks into foods[END_REF][START_REF] Calà | Atti della 24a Giornata di Studio di Ingegneria Sanitaria -Ambientale "Acque confezionate per il consumo umano[END_REF]. Other studies have shown that water, PET bottled, can release phthalate additives used in the plastic molding process especially in critical conditions of use (e.g. long storage times) [START_REF] Sauvant | Chemical and in-vitro toxicological evaluations of water packaged in polyvinyl-chloride and polyethylene terephthalate bottles[END_REF][START_REF] Biscardi | Evaluation of the migration of mutagens/carcinogens from PET bottles into mineral water by Tradescantia/micronuclei test, Comet assay on leukocytes and GC/MS[END_REF] as already shown for PVC [START_REF] Hakkarainen | Qualitative and quantitative solid-phase microextraction gas chromatographic-mass spectrometric determination of the low-molecular-mass compounds released from poly(vinyl chloride)/polycaprolactone-polycarbonate during ageing[END_REF].

Phthalates display a variety of toxic effects in animal studies including decreased fertility in females [START_REF] Biscardi | Evaluation of the migration of mutagens/carcinogens from PET bottles into mineral water by Tradescantia/micronuclei test, Comet assay on leukocytes and GC/MS[END_REF], fetal defect [START_REF] Saillenfait | Effects of mono-n-butyl phthalate on the development of rat embryos : In vivo and in vitro observations[END_REF] and reduced survival of offspring [START_REF] Jr | Perinatal exposure to the phthalates DEHP, BBP, and DINP, but not DEP, DMP, or DOTP, alters sexual differentiation of the male rat[END_REF], altered hormone levels (Thompson et al., 2004), uterine damage [START_REF] Seidlova-Wuttke | Pure estrogenic effect of benzophenone-2 (BP2) but not of bisphenol A (BPA) and dibutylphthalate (DBP) in uterus, vagina and bone[END_REF] and male reproduction abnormalities such as reduced sperm production and motility [START_REF] Sharpe | Gestational and lactational exposure of rats to xenoestrogens results in reduced testicular size and sperm production[END_REF], Sertoli cell damage [START_REF] Heindel | Phthalate ester effects on rat sertoli-cell function-invitroeffects of phthalate side-chain and age of animal[END_REF], Leyding cell tumors [START_REF] Jones | The influence of phthalate-esters on leydig-cell structure and function in-vitro and in-vivo[END_REF], cryptorchidism and hypospadias which may be manifestations of one condition termed as "testicular dysgenesis syndrome" [START_REF] Skakkebaek | Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects[END_REF]. The effects of human exposure to phthalates have not been fully studied [START_REF] Colon | Identification of phthalate esters in the serum of young Puerto Rican girls with premature breast development[END_REF]Health Care Without Harm, 2002;Duty et al., 2003b). Long latency periods between relevant exposures and health impacts, unquantified exposures, and subtle effects that are difficult to detect are added difficulties to the few (Health Care Without Harm, 2002). In one of these few human studies, phthalates were investigated as a cause of precocious puberty in young Puerto Rican girls [START_REF] Colon | Identification of phthalate esters in the serum of young Puerto Rican girls with premature breast development[END_REF]. In this study, the serum levels of phthalates obtained from 41 girls with premature appearance of breast tissue were compared with 35 controls. Phthalate esters were detected in 68% of the cases and in the 17% of the controls and they were found significantly lower levels of phthalates than the cases. For the bis(2-ethylhexyl)phthalate (DEHP), the average concentration was 70 ppb in the controls compared to 450 ppb in the cases. Even if the study conclusions were limited due to small population size and the possibility of contaminated serum samples, the association between phthalates and premature thelarche is biologically plausible. Two studies present the first human data which demonstrate that phthalates are associated with increased DNA damage in sperm (Duty et al., 2003a;Duty et al., 2003b). Finally, a recent study concludes that monobenzyl phthalate (MBzP) exposure was significantly associated with a 10% decrease in follicle stimulating hormones (FSH) concentration in adult men [START_REF] Duty | Phthalate exposure and reproductive hormones in adult men[END_REF]. Furthermore, phthalates are chemicals of concern also due to their large production volume and to a non negligible human intake which has been estimated, for example, ranging from 2 to 10 µg/kg bw/day of dibutyl phthalate (DBP) for the U.S. population (NTP-CERHR, 2006) and from 3 to 30 µg/kg bw/day for DEHP [START_REF] Latini | Monitoring phthalate exposure in humans[END_REF].

In order to assess the safety issues of food containing phthalates their intake can be compared to the reference doses (RfD) defined by the US-EPA. The RfD being an estimate of a daily oral exposure to the human population (including sensitive subgroups) that is likely to be without an appreciable risk of deleterious effects during a lifetime. European Union has included several phthalates as priority substances for evaluation (European Union, 2006) and dibutylphtalate has been proposed by the Committee on the Environment, Public Health and Food Safety to be included as a priority substance in the Water Framework Directive 2000/60/EC (European Union, 2007). Solid-phase microextraction (SPME) is a solvent free pre-concentration technique, which has recently been applied to the extraction of phthalates from aqueous matrices [START_REF] Kelly | Trace determination of diethylphthalate in aqueous media by solid-phase microextraction-liquid chromatography[END_REF]. However, liquid-liquid extraction (LLE) [START_REF] Jobling | A variety of environmentally persistent chemicals, including some phthalate plasticizers, are weakly estrogenic[END_REF][START_REF] Castillo | Analysis of industrial effluents to determine endocrinedisrupting chemicals[END_REF] with dichloromethane or hexane, and solid-phase extraction (SPE) [START_REF] Jobling | A variety of environmentally persistent chemicals, including some phthalate plasticizers, are weakly estrogenic[END_REF][START_REF] Holadova | A comparison of different ways of sample preparation for the determination of phthalic-acid esters in water and plant matrices[END_REF][START_REF] Castillo | Analysis of industrial effluents to determine endocrinedisrupting chemicals[END_REF] , 1998) are usually applied. Nevertheless, a source of error of particular concern for phthalic acid and phthalate esters determination is their high levels in the procedural blanks originating from laboratory plastics, solvents and polymeric sorbents from the preconcentration techniques [START_REF] Durand | Solid-phase extraction using C18 bonded silica disksinterferences and analysis of chlorotriazines in seawater samples[END_REF][START_REF] Castillo | Evaluation of ELISA kits followed by liquid chromatography atmospheric pressure chemical ionization mass spectrometry for the determination of organic pollutants in industrial effluents[END_REF]. In this way, the US Environmental Protection Agency (EPA) reports that DEHP, along with other common phthalate esters, cannot be accurately or precisely measured at concentrations below 2 µg L - 1 , due to high blank levels when the conventional methods LLE or SPE are employed [START_REF] Lawrence | EPA's Sampling and analysis methods database[END_REF], and at the same time the EPA has established a maximum concentration limit (MCL) in drinking water of 6 µg L -1 in its National Primary Drinking Water Regulations (NPDWR) (EPA, 1991). Nowadays, phthalates are not considered in the European Union drinking water regulations even if, dibutylphtalate has been proposed by the Committee on the Environment, Public Health and Food Safety to be included as a priority substance in the Water Framework Directive 2000/60/EC (European Union, 2007).

Recent studies show that detection limits well below this level can be achieved when using SPME coupled to GC-MS [START_REF] Peñalver | Determination of phthalate esters in water samples by solid-phase microextraction and gas chromatography with mass spectrometric detection[END_REF][START_REF] Alzaga | Determination of phthalic monoesters in aqueous and urine simples by solid-phase microextraction-diazomethane on-fibre derivatization-gas chromatography-mass spectrometry[END_REF]. In this way, SPME diminishes the risk of contamination in the extraction of phthalates since it is a solvent free technique and it minimizes the materials use, which can be potentially polluted with phthalates (e.g. SPE cartridges, solvents). At present, there are different published papers in which the suitability of SPME phthalate esters extraction from water is shown [START_REF] Moder | Characterization of water-soluble components of slurries using solid-phase microextraction coupled to liquid chromatography mass spectrometry[END_REF] and even coupled to GC-MS [START_REF] Peñalver | Determination of phthalate esters in water samples by solid-phase microextraction and gas chromatography with mass spectrometric detection[END_REF][START_REF] Peñalver | Comparison of different fibers for the solid-phase microextraction of phthalate esters from water[END_REF]Suzuki et al., 2001;[START_REF] Alzaga | Determination of phthalic monoesters in aqueous and urine simples by solid-phase microextraction-diazomethane on-fibre derivatization-gas chromatography-mass spectrometry[END_REF]) and to LC-UV [START_REF] Kayali | Determination of diethylhexyl phthalate in water by solid phase microextraction coupled to high performance liquid chromatography[END_REF]. However, in these studies only a small number of bottled water samples were analyzed in order to evaluate the developed methodology. Recently, stir bar sorptive extraction (SBSE) technique has been used to analyze phthalates in water obtaining lower limits of detection than with SPME due to its higher volume of polymeric phase compared to SPME but as a drawback, it needs a dedicated analytical instrumentation (Serodio et al., 2003;[START_REF] Serodio | Considerations on ultra-trace analysis of phthalates in drinking water[END_REF].

The aim of this study was to determine the phthalic acid and phthalate esters content in Italian mineral water both bottled in glass or in PET bottles. Moreover, the relevance of bottled water consumption in human exposure to phthalic acid and phthalate esters will be assessed. For this reason, phthalic acid (PhA) as the main degradation product of diesters, bis(2-ethylhexyl) phthalate (DEHP), dimethyl phthalate (DMP), diethyl phthalate (DEP),

Material and methods.

Study area and sampling.

The survey was conducted in Italy and a variety of commercial bottled water samples were collected. Commercial bottled water samples included different water type, such as spring water, mineral water, light water (low mineralization water) and sparkling water from miscellaneous commercial brands. The sampling of each commercial brand was carried out twice, packed in polyethylene and in glass bottles. A total of 71 commercial brands coming from 16 different Italian regions were collected. Therefore, 142 samples, 71 packed in PET and 71 in glass containers were analysed.

Sample preparation and analysis

Water samples (5 mL) were placed in a 7 mL glass vial and pH was adjusted at pH 2 by using HCl and were continuously stirred using a magnetic stirrer. Immersion SPME was carried out using the polydimethylsiloxane-divinylbenzene (PDMS/DVB) fibre from Supelco (Bellefonte, PA, USA) adapting an already reported methodology [START_REF] Peñalver | Comparison of different fibers for the solid-phase microextraction of phthalate esters from water[END_REF]. After an extraction of 20 min at 25ºC the fibre was thermally desorbed at 250ºC (3 min splitless time) in the GC-MS standard split/splitless injector. The analysis was carried out using a quadrupole GC-MS QP5050A Shimadzu (Kyoto, Japan) with a GC-MS (version 1.1, data acquisition software ), working in the electron impact mode at 70 eV. A SPB 20 (20% diphenyl 80% dimethylpolysiloxane) (60 m, 0.25 mm ID) coated with a 0.25 µm film thickness column was used. The gas chromatographic conditions were as follow.

The initial oven temperature was 50ºC for 2 min, then programmed from 50ºC to 250ºC at 10ºC min -1 with a final holding time of 30 min. MS transfer line and ion source were kept at 250ºC. Acquisition was carried out in the single ion monitoring mode using two characteristic ions for each target analyte. Compound identification was carried out comparing their retention times with standards, using two characteristic ions and their ratio for each target analyte. Furthermore, for the samples presenting higher concentrations, target analytes identity was confirmed in full-scan mode (m/z range from 60 to 350).

Quantification was done by using the external calibration method showing linear correlations with R 2 >0.98 for all the target analytes from 0.01 to 1 µg L -1 .

Data analysis was performed with the statistical software SPSS (release 13.0, SPSS Inc., Chicago, IL, USA). All data was presented as the mean + SD. The level of significance was set at P < 0.05.

Procedural blanks were carried out and used for the calculation of the detection limits (LOD). They ranged from 0.01 µg L -1 for the DBP to 0.08 µg L -1 for phthalic acid and quantification limits (LOQ) ranged from 0.02 µg L -1 for DEHP to 0.1 µg L -1 for phthalic acid.

Results & Discussion

Despite few data being available, the results reported here are compared in Table 1 with bibliographic phthalate concentrations for tap and bottled water. It is important to point out that the scarcity of data on this topic is mainly due to the analytical difficulties in the phthalate determination. In this way, the large number of non-detected samples in the USA National Resources Defence Council study (NRDC, 2006) (DEHP was not detected in 98.5% of the samples) is explained by the high detection limits of the applied methodologies (e.g. EPA 2 µg L -1 ). However, for the most studied compounds such as DEP, DnBP and DEHP the results found in this study are in agreement with the literature.

The use of SPME GC-MS as analytical technique achieved lower detection limits due to its improved blank level. In this way, lower target analytes concentrations were detected In Table 2 the quartiles are shown of the target analytes concentrations for the glass and PET bottled water. PET bottled water shows, as expected, a higher content for all phthalates, being PhA, degradation product of the phthalates esters, the most abundant individual compound. Box plots of the different target analytes concentrations depending on bottle material are shown in Figure 1. In this figure, it is evident that higher concentrations are found for all the analysed phthalates in the PET bottled water. However, these differences were confirmed using statistical tools and significant differences (Mann-Whitney test p<0.05) were found between glass and PET for all the compounds. It should be pointed out, that also different phthalate patterns were obtained in the two cases (Figure 2). For the PET bottles PhA (69%) and DnBP (10%) are the most abundant compounds and for the glass bottles the most abundant are DiisoBP (25%) and the DnBP (15%). In both cases, the lowest concentrations were found for DEHP, which was always far below the EPA regulation limit of 6 µg L -1 with a maximum of 0.17 µg L -1 for PET bottles and of 0.02 µg L -1 for glass bottles. These results confirm that the use of PET containers is the main cause of higher concentrations of phthalates in bottled water, as the concentration of the sum of the studied compounds is more than twelve times higher in PET than in glass bottled water. The presence of phthalates in glass bottled water could come from the other water processing steps (PVC tubing, storage tanks, filtration steps, cap-sealing). In the case of PET, four different bottle volumes have been sampled (0.75, 1, 1.5 and 2 L) but no correlation was found between the bottle volume and the phthalate content. Moreover, no correlation was found between the phthalates content and several water physicochemical parameters such as conductivity, pH, solid residue, sodium, calcium, potassium, magnesium, chlorine, fluorine, sulphates, carbonates, nitrates and silicates (data taken from the bottle label). Therefore, it could be considered that these parameters are not relevant in controlling the leaching of the phthalates from the bottle to the water. No significant differences were found between sparkling and still waters when there was no segregation of the data between PET and glass containers. However, when considering PET 8 bottles alone, DEP was found to be significantly higher in still water (n=55) than in sparkling water (n=16) (p<0.05). Until now, no reason has been found, to explain this result.

From a toxicological point of view, and considering that a MCL is only available for DEHP (6 µg L -1 EPA limit for drinking water), the concentrations found are lower. Another way to assess the health impact of phthalates is to study the importance of drinking PET bottled water referred to the EPA available phthalates reference doses (RfD) (EPA, 2006) (0.1 mg/kg/day for the DnBP, 0.8 mg/kg/day for the DEP, 2 mg/kg/day for PhA and 0.02 mg/kg/day for DEHP). In this way, we will consider a body weight of 70 kg and a daily water consumption of 2 L. In Table 3, it is shown the proportion of the different RfDs, which can be achieved by drinking PET bottled water. The results show that, due to the low phthalate concentrations, PET bottles do not represent any health risk related to the phthalate intake as they do not contribute significantly to the estimated RfD. The maximum expected contribution will be 0.051% of the RfD for the DnBP and only for the most contaminated sample. However, it is important to point out the lack of data on this subject and the need of further studies involving both food analysis and epidemiological research to estimate the phthalates uptake in a comprehensive way.

Conclusions

Due to analytical difficulties in achieving low detection limits, there is a lack of data on the phthalate occurrence in drinking water. The use of SPME and GC-MS as an analytical technique has proven to be suitable for the analysis of phthalates at low concentrations and for a large number of samples. The use of PET bottles has been clearly correlated with the concentration of phthalates in the bottled water. In this way, phthalates concentrations are significantly higher (nearly twenty times) in this water than in glass bottled water.

However, the concentrations found do not represent any risk for human health as can be seen by comparing the concentrations found to the existing EPA regulation (only available for DEHP) or by referring to the EPA RfDs. The contribution from drinking water is

  of phthalate toxicity in humans

  DiisoBP) and dibutyl phthalate (DBP) have been analyzed in Italian samples of commercial mineral water stored in PET and in glass bottles using SPME in combination with GC-MS. To the best of our knowledge this study represents the first survey in which phthalic acid and phthalate esters, in an extensive sample pool, were investigated by using SPME and GC-MS.

  of a large number of non-detected samples as occurred in the NRDC study(NRDC, 2006).

  of the RfD for all the target analytes. Therefore, the main conclusion of this study is that Italian bottled water does not represent a relevant ingestion source of phthalates esters and phthalic acid for the population who consume bottled water. Due to similarities in the materials being used and in the technologies in water processing industries all around Europe, similar conclusions to the ones presented here should be expected for the whole European Union.

Figure 1 .

 1 Figure legends

Figure 2 .

 2 Figure 2. Normalised phthalates patterns for the PET and the glass bottled water.

  Figure 1

Table I .

 I Comparison of bibliographic data on phthalates content in tap and bottled water. Presented results are the mean of the different analysed samples expressed

	in µg L -1 .
	(µgL -1 )	NRDC, 2006	Casajuana et al.

, 2003 Peñalver et al., 2001 Kayali et al., 2006 Serodio et al., 2006 Serodio et al., 2006 Luks et al., 2001 Luks et al., 2001 Current Study

	Sample		Bottled water	Bottled water	Bottled water	Bottled water	Bottled water	Tap water	Tap water Poland	Tap water Germany	Bottled water
	Samples number		132	20	3	4	1	1	1	1	142
	Phtalic acid (PhA)		-	-	-	-	-	-	-	-	1.28
	Dimethylphthalate (DMP) Diethylphthalate (DEP) Diisobutylphthalate (DiisoBP) F o		---	0.002 0.254 -	ND 0.24 -	---	ND 0.04 -	0.04 0.19 -	-0.16 -	-0.20 -	0.07 0.17 0.20
	Di-n-butyl-phthalate (DBP) r	-	0.047	0.10	-	0.35	0.52	0.64	0.38	0.21
	Bis(2-ethylhexyl)phthalate (DEHP) 1 DEHP was only detected in two samples 8.5 1 P	0.164	1.0	ND	0.17	0.06	0.06	0.05	0.02
	ND not detected -not analysed	e e r
				R e v i e w
					O n
						l y
								18

Table 2 .

 2 Target analytes quartiles in (µg L -1 ) for glass and PET bottled water.

			Food Additives and Contaminants			Page 18 of 21
	(µgL -1 )	Quartiles	PhA	DMP	DEP	DiisoBP	DBP	DEHP	Total
		(%)
		25	<LOD	<LOQ	<LOQ	0.02	0.02	<LOQ	0.13
	GLASS	50	<LOD	0.02	0.02	0.03	0.04	<LOQ	0.19
		75	<LOD	0.04	0.06	0.06	0.09	<LOQ	0.36
		25	1.24	<LOQ	0.14	0.221	0.17	<LOQ	2.11
	PET	50	2.20	0.06	0.22	0.32	0.23	<LOQ	3.52
		75	3.50	0.10	0.35	0.45	0.52	0.02	4.81

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Table 3 .

 3 Minimum, maximum and quartiles values of the water consumption contribution to the RfDs for the PET bottled water. For the PhA the RfD of the phthalic anhydre was considered

	RfD PhA *	RfD DEP	RfD DnBP	RfD DEHP

*

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Acknowledgements