Emmanuel Kossi Tangni

Luc Pussemier

Yvan Larondelle

Jean Claude Motte

Tangni

& Larondelle

Development and application of analytical methods for the determination of mycotoxins in organic and conventional wheat

Keywords: Chromatography -HPLC, Chromatographic analysis, Clean-up, Inhouse Mycotoxins, Mycotoxins -trichothecenes, Mycotoxins -ochratoxin A, Mycotoxins -zearalenone Food Types: Cereals and grain, Organic foods, Cereals Mycotoxins, Deoxynivalenol, Ochratoxin A, Zearalenone, Analytical method, wheat, Organic, Conventional, Belgium

come

Introduction

Organic farming does not permit the use of synthetic pesticides and relies on the use of resistant varieties of seeds as well as the use of some agricultural techniques including crop rotation, manure and compost fertilization. In contrast to this mode of cultivation, conventional farms use mineral fertilization and pesticides in the vegetation period. The use of pesticides to reduce microbial contamination during cereal production and storage, as well as the addition of preservatives at different stages of foodstuffs production and distribution, may lead to the presence of residues in the final products. In contrast, the nonuse of pesticides or preservatives may result in increasing fungal contamination and consequently in increased levels of mycotoxins in the final products. So, there is evidence that organic food often contains relatively high amounts of natural toxic compounds produced by fungi or plants, whereas corresponding conventional food tends to contain more synthetic toxins such as pesticide residues [START_REF] Finamore | Novel approach for food evaluation. Results of a pilot experiment to evaluate organic and conventional foods[END_REF]). Moreover, the use of pesticides or preservatives in insufficient amounts could lead to an even worse situation since the stress imposed on the moulds is thought to stimulate mycotoxin production.

In principle, although the foodstuffs resulting from conventional production are not free of mycotoxins, the organic foodstuffs may present in some circumstances a greater risk of contamination [START_REF] Pussemier | Chemical safety of conventionally and organically produced foodstuffs: a tentative comparison under Belgian conditions[END_REF]. Moreover, as there is a greater number of microorganism species able to develop during the organic production (in contrast to the conventional counterpart), a wider spectrum of mycotoxins may potentially be detected in the organic foodstuffs than in the conventional counterparts. So, there is a need for information related to food safety, to inform consumers of the health benefits and/or 4 hazards of food products of both origins, in order to optimize the impact on health and minimize the risks. Identification and quantitative assessment of mycotoxins present in specific raw materials, feed and foodstuffs as well as in manufactured food deserves thus further safety evaluation for public health concerns.

So far, most mycotoxin analyses of foodstuffs have usually been conducted using single toxin methods validated according to the criteria established by the European Union. These methods although sensitive, are time-consuming and thus not applicable for monitoring a large panel of mycotoxins. In fact, it is well known that some fungi species are able to produce several mycotoxins and that the co-existence of two or more toxins in cereals has frequently been documented in the literature, e.g. barley, malt, maize and wheat contaminated with deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEN) [START_REF] Lee | The coexistence of the Fusarium mycotoxins nivalenol, deoxynivalenol and zearalenone in Korean cereals harvested in 1983[END_REF][START_REF] Romer | Use of small charcoal/alumina Cleanup Columns in Determination of Trichothecene Mycotoxins in Food and Feeds[END_REF][START_REF] Park | Natural occurrence of Fusarium mycotoxins of the 1990 barley crop in Korea[END_REF][START_REF] Tanaka | A survey of the natural occurrence of Fusarium mycotoxins, deoxynivalenol, nivalenol and zearalenone in cereals harvested in the Netherlands[END_REF][START_REF] Birzele | Deoxynivalenol and ochratoxin A in German wheat and changes of level in relation to storage parameters[END_REF], maize contaminated with fumonisin B 1 (FB1), DON, T-2 toxin, NIV and ZEN [START_REF] Ali | Natural co-occurrence of aflatoxins and Fusarium mycotoxins (fumonisins, deoxynivalenol, nivalenol and zearalenone) in corn from Indonesia[END_REF]), wheat contaminated with DON and ochratoxin A (OTA) [START_REF] Birzele | Deoxynivalenol and ochratoxin A in German wheat and changes of level in relation to storage parameters[END_REF], barley contaminated with OTA and DON [START_REF] Olsson | Detection and quantification of ochratoxin A and deoxynivalenol in barley grains by GC-MS and electronic nose[END_REF], wheat, rye, barley and several cereal-based commodities contaminated with DON and T-2 toxin [START_REF] Biselli | Development of a multicomponent method for Fusarium toxins using LC-MS/MS and its application during a survey for the content of T-2 toxin and deoxynivalenol in various feed and food samples[END_REF]. Consequently, if contaminated raw materials are used for their production, manufactured products (such as cornflakes, meal flour, beer) can also be contaminated by all key cereal mycotoxins, except perhaps citrinin (CIT), which does not seem to survive the mashing step [START_REF] Galvano | Mycotoxins in the human food chain[END_REF].

The wide extent of mycotoxin co-occurrence requires an analytical method for their simultaneous detection and quantification in food and feed. Most common methods for simultaneous determination of A and B-trichothecenes require GC-ECD, GC-MS or LC [START_REF] Krska | State of the art in the analysis of trichothecenes[END_REF]. Recently, LC/MS/MS has been increasingly applied for such purposes [START_REF] Biselli | Development of a multicomponent method for Fusarium toxins using LC-MS/MS and its application during a survey for the content of T-2 toxin and deoxynivalenol in various feed and food samples[END_REF].

The present contribution describes methods for the simultaneous determination of several mycotoxins in wheat using high performance liquid chromatography (HPLC) coupled with photodiode array-UV and fluorescence detectors. Emphasis is put on the clean-up procedures that combine in series an anion exchange (SAX) column and a MycoSep 226 column for the determination of NIV, DON, 15-and 3-acetyl deoxynivalenol (15-AcDON and 3-AcDON), zearalenol (ZOL), ZEN, CIT and OTA in wheat grains and wheat derived products. The validation of the method to determine the major mycotoxins DON, ZEN and OTA in wheat is first reported whilst further work is in progress in order to improve the analytical performances regarding the other toxins. To date, there is not enough information available regarding possible co-occurrence of these toxins taking into account the various contamination sources. A survey was therefore performed on wheat samples taken from organic and conventional farms in Belgium as well as on organic and conventional whole meal flour samples taken from the retail market.

Materials and methods

Reagents and standards

Pure crystalline form of NIV, DON, 3-AcDON, 15-AcDON, ZEN, ZOL, CIT and OTA were purchased from Sigma Chemical Co. (St Louis, MO, USA). Single standards were prepared in acetonitrile and composite working standard solutions were prepared mixing aliquots of stock solution in methanol:water (50:50). and benzene were provided by Acros (New Jersey, NY, USA). Phosphoric acid was supplied by Sigma (St Louis, MO, USA). HPLC grade water was obtained using a ELIX3-Milli-Q combined system from Millipore (Molsheim, France). SPE-SAX Extract Clean up columns (500 mg/4ml) were obtained from Alltech (Deerfield, IL, USA). Romer-MycoSep ® cartridges 226 were provided by Coring Systems Diagnostix GmbH (Gernsheim, Germany). OTA immunoaffinity columns (OchraTest ® ) were obtained from Vicam (Watertown, MA, USA)

Sample collection and handling

Wheat grains and wholemeal wheat flour samples were tested in this study.

Wheat grains, harvested in August 2002 and in August 2003, were obtained directly from the organic and conventional producers. Taking into account the geographical (climatic and pedological factors) variability, samples were taken from different parts of Belgium with the minimum target sample size of 10 per mode of production and per region : Wallonia (20 samples) and Flanders (20 samples). Based on this size, volunteer farmers who were available to participate adhered to the study group. Thus, 20 conventional and 25 organic wheat producers were registered in 2002. In 2003, an additional 2 and 1 other farmers were enrolled in the conventional and organic farming groups, respectively. The sampling was performed by the farmers themselves according to our recommendations. They were asked to sample one kilogram of grains in 5 different buckets or at 5 different places in the heap of grains, in order to obtain a 5-kg sample representative of the parcel of land. The organic route of production was assured thanks to these collaborations. In the case of OTA Samples were ground in a Retsch centrifugal mill to pass a 1 mm sieve and stored at room temperature.

As for flour, eighty samples of wholemeal wheat flours were obtained from retail shops in Belgium. Forty conventionally produced samples (representing 10 brands) and forty organically produced flours (representing 10 brands) were assayed. To check the variation in batches and brands of the sampled materials, two batches per brand and two samples per batch were used in this study. All samples were stored at room temperature before analyses.

Sample preparation

A 25 g-portion of flour (ground and homogenized) sample was transferred into a 300 ml PTFE centrifuge vessel and mixed with 125 ml of extraction solvent (acetonitrile:water 84:16, v:v). The mixture was triturated for 3 min with the IKA Ultra-Turrax mixer (Stauffen, Germany) fitted with a T17-V shaft at 13500 rpm to produce slurry and then centrifuged for 10 min at 15300xg. An aliquot (10 ml) of the supernatant was then submitted to the following purification steps.

Because acidic and neutral mycotoxins could not be simultaneously purified on the MycoSep column, an anion exchange column (SAX) was used prior to the MycoSep column in order to fix the acidic mycotoxins on the SAX column. Before loading the extract, the SAX column was conditioned with methanol (5 ml) and water (5 ml). The centrifuged extract (10 ml) were poured on the column. After flowing through the SAX column, the neutral mycotoxins were further purified by filtration on the MycoSep cartridge. The SAX column was successively washed thereafter with water (5 ml), 8 methanol (5 ml) and mixture (4 ml) of methanol:acetic acid (99:1, v:v). OTA and CIT were eluted with mixture (10 ml) of methanol:trifluoroacetic acid (99:1, v:v). Atmospheric air (about 10 ml) was passed through the column to collect all the acidic eluate which was then evaporated to dryness at 45 ± 5°C using a rotavapor. The residue was dissolved with 1 ml of methanol:water (50:50).

The eluate (9 ml) from SAX column were filled into Mycosep tube and then pushed through the Mycosep column. An aliquot of the purified eluate (4 ml) was taken from the flow-through and then evaporated to dryness at 45 ± 5°C using a rotavapor. The dry residue containing the neutral mycotoxins (DON,NIV,ZOL and ZEN) was dissolved with 1ml of methanol:water (50:50, v:v).

HPLC analysis

Identification and quantification of mycotoxins were performed by HPLC analysis. The Neutral mycotoxins were separated by a linear gradient, starting from 5 % to 100 % of phase B in 20 min. UV detector wavelengths were set at 225 and 236 nm whilst the fluorescence detection was performed at λ excitation = 280 nm and λ emission = 465 nm for ZEN.

OTA and CIT were analysed using an isocratic flow of 48 % phase A and 52 % phase B.

The detection was performed with the fluorescence detector set at λ excitation = 332 nm and λ emission = 462 nm). The injection volume was 25 µl of the eluates.

Validation

Analytical method for multi-mycotoxin determination in wheat was validated in-house and within inter-laboratory ring tests.

A set of nine wheat flour was spiked by adding mixture of standards at 4 different levels ranged from 200 to 2000 µg kg -1 for DON, 20 to 200 µg kg -1 for ZEN and 0.5 to 10 µg kg -1 for OTA. The initial concentration of each toxin in the wheat samples before spiking was determined and taken into account in the recovery calculations. Recovery and repeatability experiments were performed with these spiked samples. The detection and quantification limits (limit of detection (LOD) and limit of quantification (LOQ)) of the chromatographic procedure were determined by the signal-to-noise ratio of 3:1 and 10:1, respectively.

Inter-laboratory (FAPAS ® CSL, UK) proficiency tests (series 17 round 32 of May -July 2004 and series 22 round 14 of April -June 2004) were also used to evaluate the method for the determination of OTA (FAPAS 2004a) andDON (FAPAS 2004b). 2001). Whole wheat flour samples (50 g) were mixed with chloroform (200 ml) and 0.1 M H 3 PO 4 (20 ml). This mixture was triturated for 3 min with the Ultra-Turrax CAT x 620 shaft at 13500 rpm to produce a slurry. After centrifugation for 10 min under 820xg at 8 ± 2°C, the chloroform phase was transferred into a beaker. The remaining part was again extracted with chloroform (200 ml) and 0.1 M H 3 PO 4 (20 ml).

The combined chloroform phases (300 ml) were evaporated to dryness using a rotary evaporator at 35 ± 5°C. The residue was dissolved in 0.5 M NaHCO 3 (100 ml). After 10 min centrifugation under 820xg at 8 ± 2°C, 20 ml of the sample were passed through an Ochratest ® immunoaffinity column. This column was fitted with a 20 ml-syringe reservoir and placed on a vacuum manifold. Before loading the extract, the Ochratest ® column was conditioned with Phosphate Buffered Saline (PBS, pH 7.4) solution (20 ml). The OTA extract was passed through the column at about 1-2 ml min -1 . De-ionized water (20 ml) were used to wash the loaded Ochratest ® and OTA was eluted with methanol (2 ml) and deionized water (2 ml). Atmospheric air (ca 20 ml) was passed through the column to collect all the eluate. This solution was stored at -20°C. Prior to HPLC analysis (fluorescence spectrophotometer detector; λ excitation =332 nm, λ emission =462 nm), it was brought to room temperature and then filtered with a 0.45 µm microfilter prior to HPLC analysis. proposed by [START_REF] Romer | Use of small charcoal/alumina Cleanup Columns in Determination of Trichothecene Mycotoxins in Food and Feeds[END_REF]. Twenty five grams of the milled and well mixed sample were transferred into a 300-ml centrifuge beaker and added with 125 ml of extraction solvent (acetonitrile:water, 84:16). The mixture was triturated for 3 min with the Ultra-Turrax device CAT x 620 shaft at 13500 rpm to produce a slurry. The solvent phase was isolated by centrifugation at 1835xg during 10 min at 8 ± 2°C. The resulting supernatant (10 ml) was placed in the MycoSep ® test tube (Romer Labs, Coring system Diagnostix GmbH, Gernsheim, Germany) for purification. Four ml of the cleaned extract were evaporated to dryness, using a rotary evaporator at 45 ± 5°C. The residue was dissolved in 2 ml of methanol:water (10:90) and was stored at -20°C. Prior to HPLC analysis (UV detection at 218 nm), it was brought to room temperature and then filtered with a 0.45 µm microfilter.

Data handling and statistical analyses

The SAS Enterprise Guide software (SAS Institute, Inc., Cary, USA, version 3.0.0.369) was used for statistical studies. Differences in contamination frequency at level over the limit of quantification between conventional and organic samples were evaluated using Chi-square test performed by the procedure "PROC FREQ" of SAS. Variance analyses were performed on quantifiable concentrations using the GLM procedure of SAS.

Statements of significance were based on probability of p < 0.10.

Results

Analytical method performance

Importance of SAX column. Extract clarification with SAX column (figure 1B) allows to eliminate the impurities or undesirable substances which may interfere with mycotoxin I.

[Insert table I about here]

For DON, OTA and ZEN, the current multi-mycotoxin method fulfills the guidelines of the European Committee of standardization (CEN, CR 13505, 1999).

Comparison with reference method. Figure 2 shows the agreement between the DON concentrations determined on wheat using the multi-mycotoxin and the reference methods.

[Insert figure 2 about here] The fit between both methods is rather satisfying taking into account the quite drastic differences in the clean-up of the samples with both methods and the non-significant deviation between the regression line (r = +0.978) and the identity one (r = +1).

Inter-laboratory performances. The accuracy of the present multicomponent method was checked against the analysis of DON and OTA in test materials within the frame of the inter-laboratory ring-tests organized by Food Analysis Performance Assessment Scheme (FAPAS ® CSL, UK). Proficiency testing gave the respective Z-scores of -0.3 and 0.8 for DON (assigned value = 898.5 µg. kg -1 ) and OTA (assigned value = 5.6 µg. kg -1 ) determination in naturally contaminated wheat samples.. One must note that the results show that the developed method is reliable and exact in quantifying DON and OTA in wheat samples since Z-scores are ranged between -2 and 2.

Applications of the analytical methods to Belgian wheat

Wheat grains and whole meal wheat flour samples were tested in this study taking into account their organic route of production. OTA determination was carried out in the 2002harvests whereas DON and ZEN were studied in wheat collected in 2002 and 2003.

OTA in wheat grains and wholemeal wheat flour. The results of the analysis of 40 samples of wheat harvested in 2002 are summarized in Table II.

[Insert table II about here] OTA was quantified in 4 conventional and 10 organic samples. Moreover, trace level of OTA was found in one conventional and one organic sample, leading thus to 25 and 55 % as the overall incidence rates of OTA occurrence in conventional and organic wheat samples. Chi-square test revealed a significant difference between these two ratios (p = 0.047). In contrast, the mean OTA concentrations in conventional (0.067 µg kg -1 ) and organic (0.063 µg kg -1 ) samples were comparable. The highest OTA contamination (1.097 µg kg -1 ) was recorded in a conventional sample. One must note that all contaminated samples had an OTA concentration of < 5 µg kg -1 .

As for the OTA occurrence in wholemeal wheat flour sampled in the Belgian retail market, data are summarized in Table III.

[Insert table III about here]

All organic wheat flour samples were contaminated against 95 % of the conventional ones, with concentrations ranging from 0.141 to 2.199 µg kg -1 and from 0.064 to 1.035 µg kg -1 ,

[Insert table IV about here]

In 2002, DON contamination was most frequently (p = 0.078) quantified in conventional wheat (90 % of the samples), compared to their organic counterparts (68 %). Analysis of variance indicated that the mean concentration (675 µg kg -1 ) in conventional samples was significantly higher (p = 0.074) than the one observed in organic wheat (285 µg kg -1 ).

Moreover, none of the organic samples was found to be contaminated above 1250 µg kg -1 while 3 conventional samples had DON contamination above this limit. One must note that two conventional samples were severely contaminated (1991 and 2842 µg DON kg -1 ). In 2003, DON was quantified in 73 % of organic samples and in 86 % of conventional wheat.

Proportions of contaminated wheat (>LOQ) of conventional and organic origin are statistically equivalent (p = 0.259). Among samples contaminated at a level over LOQ, conventional wheat were statistically more contaminated compared to organic wheat (p = 0.008). Note that only one of the tested wheat samples was contaminated at a concentration above 750 µg kg -1 .

[Insert table V about here]

In contrast to the OTA occurrence in the wheat flour samples, the frequency of DON contamination (>LOQ) was significantly (p < 0.0001) higher in conventional flours (87.5%) than in the organic ones (30.0 %). Among the positive samples (concentrations > LOQ), conventional flours were more DON contaminated than the organic samples, but the magnitude of the difference was not statistically significant (p = 0.232). None of the organic samples exceeded 250 µg kg -1 while four conventional samples (10 % total conventional samples) were contaminated at a concentration above 500 µg kg -1 but remained below 750 µg kg -1 , the future maximum level in wheat flour to be set by European regulations.

ZEN in wheat grains. Occurrence of ZEN at levels exceeding the LOQ was statistically higher (p = 0.020). in conventional samples (85 %) than in organic samples (52 %). A statistical comparison between quantified ZEN concentrations in conventional and organic samples indicated that the organic mode of production influenced the level of contamination (p = 0.013). None of the organic samples showed a contamination level above the proposed limit of 100 µg kg -1 to be set by the European Commission for unprocessed cereals. In contrast, 6 out of 20 conventional samples had a ZEN value above that limit. In addition, nine samples (either organic or conventional) had a ZEN value between 50 and 100 µg kg -1 and have therefore 16 to be considered as not acceptable as bread and breakfast cereals (proposed limit set at 50 µg kg -1 ). As for the 2003 harvest, ZEN concentrations were found to be mostly below the detection limit (LOD = 1.5 µg kg -1 ) or the quantification limit (LOQ = 4 µg kg -1 ). Only 3 samples were found to contain trace levels of ZEN. Because of the high number of nondetected samples, the mean, SD and median calculations are not representative. This finding is in deep contrast with the results obtained in 2002. In fact, the incidence of contamination was found as low as of 4 and 9 % for organic and conventional samples, respectively. The dry weather during June 2003 (flowering period for wheat) may likely explain these differences since a dry period at flowering is not favourable to the growth of Fusarium.

Co-occurrence of DON and ZEN in wheat grains. A significant correlation coefficient (r = +0.8239) was found between DON and ZEN contents in wheat grains harvested in 2002 (Figure 3), showing that a linear relationship exists between the concentrations of these two toxins. ZEN and DON were thus simultaneously found in the wheat samples harvested in 2002.

[Insert figure 3 about here]

Discussion

Mycotoxins in harvested grains

Incidence and levels of OTA contamination in the 2002 harvest were quite low. So far, contamination at levels above the LOQ was frequently found in organic samples than in conventional ones (10 versus 4 samples). The highest OTA level (1.097 µg kg -1 ) was recorded in a conventional sample. One must note that all contaminated samples had an OTA concentration < 5 µg kg -1 , the maximum contamination level fixed by the European (EC N° 123/2005). Within the framework of this study, OTA analysis was not performed in wheat samples harvested in 2003, since it was observed in 2002 samples that low OTA levels can be associated with fungal (pre-harvest) contamination, while extremely high (but not often) occurring levels of OTA are thought to be associated with post-harvest (storage) contamination.

A study carried out in 1997 on wheat samples collected in Poland after harvest detected OTA in none of the 32 samples from conventional farms (LOD = 0.2 µg kg -1 ) while wheat samples from ecological farming showed contamination of almost 8% within the range 0.48-1.2 µg kg -1 (mean = 0.83 µg kg -1 ) (Czerwiecki et al. 2002a). These results are in accordance with the present study as far as the frequencies of contamination are concerned but not in terms of the concentration levels. Conversely, results of the same study performed one year later contrast with our results in terms of frequency. In wheat from conventional farms, OTA concentrations reached extremely high levels since the range stretched from 0.6 to 1024.0 µg kg -1 (mean = 267 µg kg -1 ) and the frequency of contamination was of about 48 %. In contrast, in wheat from ecological farming, OTA was detected in only 23 % of samples (range: 0.8 to 1.6 µg kg -1 and mean = 1.2 µg kg -1 ). This finding which is different from the previous one may be due to the change in weather conditions observed within these 2 years (Czerwiecki et al. 2002b). [START_REF] Jorgensen | Occurrence of ochratoxin A in Danish wheat and rye 1992-1999[END_REF] analysed conventionally (n = 405) and organically (n=14) grown wheat kernels from 1992 to 1999 and found that there was no difference in multiyear means between these two modes of production. OTA mean concentration (3.0 µg kg -1 ) was much higher than the level obtained in Belgian wheat grains harvested in 2002.

As far as DON contamination is concerned, this toxin was detected in almost all the conventional and organic samples harvested in 2002. The incidence at a level above LOQ was of 90 and 68 %, respectively. Statistical analysis indicates a tendency to more frequent quantifiable amounts of DON in conventional samples than in organic ones. When contamination over LOQ occurred, it tended to be higher in conventionally produced wheat (p = 0.077). According to the last European proposal concerning mycotoxins in unprocessed wheat cereals, most of the samples had an acceptable level of contamination (< 1250 µg kg -1 ). Concentrations were below this proposed limit in organically produced wheat while 3 conventional samples were contaminated at levels above 1250 µg kg -1 . High levels of DON (up to 2842 µg kg -1 ) in grains collected at harvest in 2002 were observed because of the rainy weather recorded in June 2002. The climatic conditions were indeed favourable to the growth of Fusarium species. At harvest in 2003, DON concentration was found to be very low and may be explained by the dry weather observed in June 2003 over the flowering period. The mean (median) contamination levels in conventional and organic samples were of 311 ( 248) and of 123 (129) µg kg -1 , respectively. Similar variations in DON contamination in samples from two consecutive years of harvests carried out in 1997 and 1998 were previously attributed to different weather conditions during and after the flowering time [START_REF] Birzele | Deoxynivalenol and ochratoxin A in German wheat and changes of level in relation to storage parameters[END_REF].

ZEN Contamination at a level above LOQ was frequently observed in conventional than in organic samples. Comparison of contaminated samples showed that the conventional ones contained higher levels of ZEN (p = 0.013). Wheat grains collected in 2003 were less affected by DON and ZEN than those harvested in 2002, due to a less favourable weather to contamination. In 2003, DON contamination occurred as frequently as in 2002, but at much lower levels (210 against 460. µg kg -1 ). DON contamination higher than the LOQ occurred nearly as frequently in organic as in conventional wheat but the levels were higher in conventionally produced samples (p = 0.008). In 2003, none of the samples contained

Mycotoxins in wheat flour from the retail market

Using the developed multi-mycotoxin method, the tested wholemeal wheat flours for bread making showed that both OTA and DON contents were very low. OTA was quantified in 42.5 and 87.5 % of the conventionally and organically produced flours, respectively. The organic route of production did not influence the level of contamination. An accidental high contamination may be observed since some indication of contamination differences between batches of a same brand was noticed.

Of 156 samples of conventional wheat flour and 120 samples of organic wheat flour purchased in Denmark between 1993[START_REF] Cen | Food analysis-Biotoxins-Criteria of analytical methods of mycotoxins[END_REF][START_REF] Jorgensen | Occurrence of ochratoxin A in Danish wheat and rye 1992-1999[END_REF] found little difference in multiyear means of OTA between conventional samples (mean concentration of about 0.3 µg kg -1 ) and organic ones (mean concentration of 0.5 µg kg -1 ). These findings are in accordance with the current study. [START_REF] Biffi | Ochratoxin A in conventional and organic cereal derivatives: a survey of the Italian market, 2001-02[END_REF] found that conventional wheat flours were more OTA contaminated (0.134 µg kg -1 , n = 14) than organic flours (0.053 µg kg -1 , n = 11), but the magnitude of the difference was not significant. OTA analyses were performed on cereal-based baby food from Italy by [START_REF] Beretta | Ochratoxin A in cereal-based baby foods: occurrence and safety evaluation[END_REF]. OTA occurred in 6 out of 10 batches of semolina from conventional agriculture with concentrations ranging from 0.14 to 0.65 µg kg -1 . Only one out of ten semolina batches from organically produced cereals contained a detectable amount of OTA (0.18 µg kg -1 ; LOD = 0.060 µg kg -1 ). In terms of frequency of contamination, these conclusions are opposed to those obtained in this study. As for DON contamination, the 80 samples analysed in the present study showed that concentration above LOQ occurred more frequently in conventional flours than in organically produced ones, but levels of contamination were equivalent in the two types of samples. [START_REF] Schollenberger | Fusarium toxins in wheat flour collected in an area in southwest Germany[END_REF] analyzed Fusariotoxins in wheat flour collected in Germany in 1999. The 36 conventional samples were DON contaminated at a mean level of 394 µg kg -1 (incidence = 100 %) whereas the organic samples (n = 24) contained on average 131 µg DON kg -1 (incidence = 96 %). DON content in conventional samples was statistically higher than that in organic products. Globally, these results match with our findings. The same authors analysed the DON content in conventionally and organically produced wheat bread and showed some indication of a significant influence of the organic mode of production. Median DON levels in bread were higher for the conventional production mode. b : traces means that the concentration of OTA is between the limit of detection (LOD = 0.01 µg kg -1 ) and the limit of quantification (LOQ = 0.035 µg kg -1 ). ) b : traces means that the concentration of OTA is between the limit of detection (LOD = 0.050 µg kg -1 ) and the limit of quantification (LOQ = 0.150 µg kg -1 ) c : mean and median are computed assuming that non-detected samples contain half the LOD while samples with traces levels of OTA contain half the LOD+LOQ d : range of samples with OTA contents ≥ LOQ b : traces means that the concentration of DON is between the limit of detection (LOD = 50 µg kg -1 ) and the limit of quantification (LOQ = 150 µg kg -1 ). c : mean and median are computed assuming that non-detected samples contain half the LOD while samples with traces levels of DON contain half the LOD+LOQ. ) b : traces means that the concentration of ZEN is between the limit of detection (LOD = 1.5 µg kg -1

) and the limit of quantification (LOQ = 4 µg kg

  column derivatization and fluorescence detection

  (AcN), methanol (MeOH) and toluene were obtained from Biosolve (Valkenswaard, The Netherlands). Acetic acid (AcOH), trifluoroacetic acid (TFA)

  20 conventional and 20 organic wheat samples were chosen at random from the 2002 sample set.

  chromatographic system consisted of a system of two Waters 510 pumps (Milford, MA, USA) equipped with an automatic WISP model 712 injector (Waters, Milford, MA, USA), a 2996 Diode array UV detector (DAD) and a 2474 fluorescence detector, all from Waters (Milford, MA, USA). Computer using the Millennium Empower software was used to process the signals of the detector and for data management. The reversed-phase analytical column, Alltima RP-C18 (100 x 4.6 mm id, 3 µm particles) obtained from Alltech (Deerfield, IL, USA) was thermostatically maintained at 35°C by a temperature controller Model 7990 (Jones Chromatography, Hengoed, UK) during HPLC analyses.The analytical method used a linear gradient of two mobile phases eluted at 1 ml min -1 . The mobile phase A was a mixture of acetonitrile:water (2:98, v:v) whereas the mobile phase B was a mixture of acetonitrile:water (25:75, v:v). Water of both mobile phases contained 1 . The mobile phases were filtered and degassed on a 0.22 µm Durapore filter (Millipore, Molsheim, France).

  confirmation of DON and OTA was done by comparing the concentrations obtained with the multi-mycotoxin method with those obtained with reference methods. Analytical methods targeting OTA or DON (single mycotoxin) determination OTA was extracted by using an adaptation of the extraction procedure of the Technical Committee for Standardisation (CEN/TC 275 1998). The method was described by Thirumala-Devi et al. (

  for DON by a method adapted from the extraction procedure

  1 A). As for acidic mycotoxins, the clean up of the extract with SAX column allows the specificity of the peaks of OTA and CIT in the chromatograms. [Insert figure 1 A and B about here] Performance characteristics. Taking the whole analytical procedure of sample extraction, clean-up and HPLC analysis into account, the overall recoveries, RSD R , LOD and LOQ determined with spiked wheat samples are shown in Table

  positive samples, analysis of variance revealed homogeneous contamination levels between different brands (p = 0.678) whatever the mode of production (p = 0.375). In contrast, some indication of significant contamination differences between samples of the same batch were observed (p = 0.081), showing thus the heterogeneity (large range of OTA concentrations) in the collected samples. DON in wheat grains. The spreads of DON contents in wheat harvested in 2002 (45 samples) and in 2003 (48 samples) are summarized in Table IV according to their mode of production.

  wheat flour. Occurrence data in wholemeal wheat flour are shown in TableV.

  level above the LOQ value. These results confirmed once more that the contamination by Fusarium mycotoxins is weather dependent.

  Yoneda A, Inoue S, Sugura Y, Ueno Y. 2000. Simultaneous determination of trichothecene mycotoxins and zearalenone in cereals by gas chromatography-mass spectrometry. Journal of Chromatography A 882:23-28. Thirumala-Devi K, Mayo MA, Reddy G, Tangni EK, Larondelle Y, Reddy DV. 2001. Occurrence of ochratoxin A in black pepper, coriander, ginger and turmeric in India. Food additives and contaminants 18:830-835.

Figure 1 :

 1 Figure 1: Representative HPLC separations of neutral mycotoxins (NIV (1),DON (2); 15-acDON (3), 3-acDON (4), ZOL (5), ZEN (6) in a wheat flour purified without (A) and with (B) SAX column.

Figure 2 :

 2 Figure 2: DON concentrations determined by the multi-mycotoxin against the results obtained with the reference method on the same wheat samples

Figure 3 :Figure 1 :Figure 2 :Figure 3 :

 3123 Figure 3: Correlation between DON and ZEN contents in wheat grains.

  and median are computed assuming that non-detected samples contain half the LOD while samples with traces levels of ZEN contain half the LOD+LOQ d : range of samples with ZEN contents ≥ LOQ Page 34 of 34 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants

  Table VI presents the results of ZEN contamination in the organic and conventional samples collected in 2002 and 2003.

[Insert table

VI about here]

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Table I . Overall recoveries, reproducibility, detection limit and quantification limit of the multi-mycotoxin method.

 I

	Mycotoxins (spiking levels)	Recovery (%)	RSD R (%)	b	LOD c (µg kg -1 )	LOQ d (µg kg -1 )
	DON (50-1500 µg kg -1 )	92	12		50	150
	ZEN a (2-500 µg kg -1 )	83	10		1.5	4
	OTA a (0.5-10 µg kg -1 )	73	9		0.050	0.150
	a : Detected by fluorescence b : RSD R , intra-laboratory reproducibility F o c : LOD, limit of detection (signal/noise = 3) d : LOQ, limit of quantification (signal/noise = 10) r
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Table II . Occurrence of OTA in wheat grains harvested in 2002.

 II

	Occurrence	Conventional	Organic	Total
	Number of samples	20	20	40
	n.d. a	15 (75%)	9 (45%)	24 (60)
	trace b	1 (5%)	1 (5%)	2 (5)
	LOQ-5 µg kg -1	4 (20%)	10 (50%)	14 (35)
	> 5 µg kg -1	0	0	0
	Incidence	25%	55%	40%
	Levels of contamination (µg kg -1 )
	Mean c ± SD	0.067 ± 0.243	0.063 ± 0.082	0.065 ± 0.179
	Median c	0.005	0.030	0.005
	Range d	0.045 -1.097 0.037 -0.0303 0.037 -1.097

a : n.d., not detected (OTA concentration in sample <0.01 µg kg -1 )

Table III . Occurrence of OTA in wholemeal wheat flour.

 III : n.d., not detected (DON concentration in sample < 0.050 µg kg -1

	Occurrence		Conventional	Organic	Total
	Number of samples	40	40	80
	n.d. a		2 (5%)	0	2 (2.5%)
	traces b		21 (52.5%)	5 (12.5%)	26 (32.5%)
	LOQ-1.0 µg kg -1	16 (40.0%	34 (85.0%)	50 (62.5%)
	> 1.0 µg kg	-1	1 (2.5%)	1 (2.5%)	2 (2.5%)
	Incidence		38 (95%)	40 (100%)	78 (97.5 %)
	F Levels of contamination (µg kg -1 ) Mean c ± SD 0.228 ± 0.262 o Median c 0.100 r Range d 0.064-1.035	0.480 ± 0.530 0.378 0.141 -2.199	0.354 ± 0.434 0.214 0.064-2.199
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c : mean and median are computed assuming that non-detected samples contain half the LOD while samples with traces levels of OTA contain half the LOD+LOQ. d : range of samples with OTA contents ≥ LOQ. a

Table V : Occurrence and levels of DON in wholemeal wheat flour.

 V

	Occurrence	Conventional	Organic	Total samples
	Number of samples	40	40	80
	n.d. (< 30 µg kg -1 ) a	0	12 (30%)	12 (15%)
	Traces (< 100 µg kg -1 ) b	5 (12.5%)	16 (40%)	21 (26%)
	LOQ -250 µg kg -1	22 (55%)	12 (30%)	34 (43%)
	250 -500 µg kg -1	9 (22.5%)	0	9 (11%)
	500 -750 µg kg -1	4 (10%)	0	4 (5%)
	> 750 µg kg -1	0	0	0
	Incidence	40 (100%)	28 (70%)	68 (85%)
	Level of contamination (µg kg -1 )
	Mean c ± SD	249 ± 148	92 ± 54	170 ± 136
	Median c	211	100	100
	Range d	153 -661	157 -210	153 -661

a : n.d., not detected (DON concentration in sample <50 µg kg -1 )

Table VI . Occurrence of ZEN in wheat grains collected in Belgium during harvest 2002 and 2003

 VI

	Occurrence	Conventional	Organic	Total samples
		2002	2003	2002	2003	2002	2003
	Number of samples	20	22	25	26	45	48
	n.d. a	3 (15%)	20 (91%)	12 (48%) 25 (96%)	15 (33%)	45 (94%)
	traces b	0	2 (9%)	0	1 (4%)	0	3 (6%)
	LOQ -50 µg kg -1	7 (35%)	0	8 (32%)	0	15 (33%)	0
	50 -75 µg kg	2 (10%)	0	3 (12%)	0	5 (11%)	0
	75 -100 µg kg -1	2 (10%)	0	2 (8%)	0	4 (9%)	0
	> 100 µg kg -1 Incidence F Levels of contamination (µg kg -1 ) 6 (30%) 17 (85%) Mean c ± SD 75 ± 68 o Median c 53 r Range d 4 -86	0 2 (9%) 2 ± 1 1 0.75 -4	0 -13 (52%) 19 ± 27 8 4 -232	0 1 (4%) 1 ± 1 1 0.75 -4	6 (13%) 30 (67%) 44 ± 67 19 4 -232	0 3 (6%) 1 ± 1 1 0.75 -4
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d : range of samples with DON contents ≥ LOQ. a : n.d., not detected (ZEN concentration in sample <1.5 µg kg -1

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Acknowledgments

This research project has been carried out within the framework of the Scientific support plan for a sustainable development policy (SPSD II) Part I "Sustainable production and consumption patterns" (Project CP/30 Mycotoxin contamination of regular and "organic" foodstuffs). The financial support of the Belgian Federal Public Planning Office (Science Policy) is acknowledged here.

b : traces means that the concentration of DON is between the limit of detection (LOD = 30 µg kg -1 ) and the limit of quantification (LOQ = 100 µg kg -1 ). c : mean and median are computed assuming that non-detected samples contain half the LOD while samples with traces levels of DON contain half the LOD+LOQ. d : range of samples with DON contents ≥ LOQ.


J Polzer

R Hackenberg

C Stachel

P Gowik

Determination of Chloramphenicol Residues in Crustaceans: Preparation and Evaluation of a Proficiency Test in Germany

Keywords: Chloramphenicol, proficiency testing, shrimps, validation, GC-NCI-MS, MRPL, residue control

Chloramphenicol (CAP) is banned for use in food producing animals and is thus controlled on the basis of the National Residue Control Plans in the European Union.

Due to the current problems with residues of CAP in shrimps, crayfish and prawns, a sensitive GC/NCI/MS method was optimised and in-house validated. The validation study resulted in a decision limit (CC α ) of 0.07 µg/kg, a recovery of 95 % and a within-laboratory reproducibility of 9 %. The method was used for preparing a proficiency test in order to assess the quality of residue control in Germany. The proficiency test involved analysis of five samples and the results were very satisfactory. The reproducibility standard deviation for five samples ranged from 17 to 24 %, and the median concentrations lay between 0.43 and 0.51 µg/kg CAP. These values are clearly below the corresponding Horwitz standard deviation of about 50%.

From the study it can be concluded that there are -irrespective of the method applied -well established and properly working analytical procedures for the control of CAP around the MRPL of 0.3 µg/kg.

Chloramphenicol (CAP) is banned for use in food producing animals in the European Union (Annex IV of Council Regulation 2377/90/EC) and is thus to be controlled on the basis of the National Residue Control Plans (Council Directive 96/23/EC). Even though it is an effective broad-spectrum antibiotic, its use was forbidden in the European Union in 1994 due to severe side effects, as e.g. aplastic anemia. Since these toxic effects are not dose dependent, a no effect level could not be established and consequently a zero tolerance level was set for CAP in food.

In 2003, with Commission Decision 2003/181/EC, a minimum required performance limit (MRPL) of 0.3 µg/kg for residues of CAP in different matrices, as e.g. muscle and aquacultures, was fixed. The MRPL was introduced with Commission Decision 2002/657 EC and was meant as the "minimum content of an analyte in the sample, which at least has to be detected and confirmed". The aim of the MRPL was a harmonisation of the control of substances for which no MRL could be established.

Since the level of the MRPL is not based on a risk assessment but on an assessment of a residue level which could be controlled by a majority of laboratories in residue control, it is clear that lower control limits are possible and welcome in order to meet scientific progress (as far as the method is validated according to the requirements of Commission Decision 2002/657 EC). This paper describes a sensitive GC/NCI/MS method, which is able to achieve decision limits for CAP below 0.1 µg/kg and which is validated according to this decision using the in-house validation approach [START_REF] Jülicher | Assessment of detection methods in trace analysis by means of a statistically based in-house validation concept[END_REF]) by applying a statistical software ("InterVal™", quo data ltd., Dresden, Germany). This method was developed because of recent problems with residues of CAP in import samples of shrimps and crayfish. According to these recent findings, also the number of publications dealing with the analysis of CAP has increased significantly since 2003. Meanwhile a number of different methods is available, which allows the control of the actual MRPL (e.g. [START_REF] Rupp | Liquid chromatography/tandem mass spectrometry analysis of chloramphenicol in cooked crab meat[END_REF][START_REF] Peng | Simultaneous determination of residues of chloramphenicol, thiamphenicol and florfenicol in shrimp by high performance liquid chromatography-tandem mass spectrometry[END_REF][START_REF] Penney | Determination of chloramphenicol residues in milk, eggs, and tissues by liquid chromatography/mass spectrometry[END_REF][START_REF] Santos | Determination of chloramphenicol residues in rainbow trouts by gas chromatography-mass spectometry and liquid chromatography-tandem mass spectrometry[END_REF][START_REF] Hammack | Multilaboratory validation of a method to confirm chloramphenicol in shrimp and crabmeat by liquid chromatographytandem mass spectrometry[END_REF][START_REF] Ramos | Determination of chloramphenicol residues in shrimps by liquid chromatography-mass spectrometry[END_REF][START_REF] Mottier | Determination of the antibiotic chloramphenicol in meat and seafood products by liquid chromatographyelectrospray ionization tandem mass spectrometry[END_REF].

One of the tasks a National Reference Laboratory should fulfil according to Council Regulation 882/2004/EC and Commission Decision 96/23/EC is to organise comparative tests between official national laboratories. In order to meet these requirements, proficiency test material was prepared and tested for homogeneity and stability by applying the above mentioned method. Using this material (five different test samples from four different laboratory samples), a proficiency test according to the International Harmonised Protocol for the Proficiency Testing of (Chemical) Analytical Laboratories (Thompson and Wood 1993, IHPPT) was organised and the official residue control laboratories in Germany were offered to participate. The NRL took part in this study as one of the participants. The second part of the paper gives the results of this proficiency test. Additionally the potential influence of parameters on the analytical results is evaluated in detail. This is done on the basis of the information about the various methods applied by the participating laboratories.

Materials and methods

GC-MS (negative chemical ionisation) method

Materials and instrumentation

Sample preparation

Freeze-dried laboratory sample (2 g) was reconstituted with water to the original fresh sample weight (10 g). The internal standard (CAP-D5) was added to the sample followed by 20 ml of an extraction mixture (acetonitrile / 4% NaCl in water solution 1+1 V+V). The mixture was homogenised in a stomacher (Seward, United Kingdom) and transferred into a centrifuge tube. The stomacher bag was rinsed twice with 4 ml of an extraction mixture, the solution was added to the centrifuge tube and centrifuged (4000 rpm for 15 min). The supernatant was separated and de-fatted twice with 10 ml of n-hexane. Water-saturated ethyl acetate (7 mL) was added to the remains, the mixture vortexed and the supernatant was transferred to a TurboVap tube. This extraction procedure with ethyl acetate was repeated. The combined supernatants were evaporated to dryness and re-dissolved in 3 ml of water/acetonitrile (95+5 V+V). The re-dissolved sample was applied onto a C18 cartridge (500 mg, Separtis, Switzerland, preconditioned with 10 ml of methanol and 10 ml of water/acetonitrile (95+5 V+V). The sample was eluted with 3 ml of water/acetonitrile (45+55 V+V). Water-saturated ethyl acetate (4 ml) was added to the eluted sample, the mixture vortexed and the supernatant separated. This extraction procedure was repeated. The combined extracts were evaporated to dryness under a nitrogen stream. The dry residue was re-dissolved in 1 ml of acetone/toluene (20+80 V+V) and then applied onto a Silica cartridge (1 g, Separtis,

Switzerland, preconditioned with 6 ml of acetone/toluene (20+80 V+V).

The cartridge was washed twice with 3 ml of acetone/toluene (20+80 V+V) and the sample was eluted into a derivatisation tube with 6 ml of acetone/toluene (70+30 V+V). The extract was evaporated to dryness. 50 µl of the derivatisation mixture N,Obis (trimethylsilyl)acetamide (BSA)/n-heptane (1+1 V+V) were added to the dry extract. The derivatisation tubes were closed carefully and set aside to react for approx. 45 min at 60°C. The amount of 2 µl of the mixture was directly injected into the GC-MS system.

Validation and validation data

The validation of the method was carried out according to the alternative validation model suggested in Commission Decision 2002/657 ("in-house validation approach"). For each of the 16 factor level combinations, five analysis were carried out (blank material was spiked at levels of 0.05, 0.08, 0.12 and 0.2 ng/g and a blank with only internal standard); i.e. a total of 80 analyses was carried out. The resulting concentrations were calculated by using a standard calibration curve. The results of these analyses are shown in figure 1. The figure gives the 16 "calibration curves" for the different factor level combinations as well as the prediction interval.

[Insert figure 1 about here] From these data, the method validation characteristics are calculated. The data are summarised in table 2.

[Insert table 2 about here] Furthermore, it is possible to estimate the contributions of the selected factors and the uncertainty due to the matrix/run deviations to the total uncertainty of the result.

Details can be found e.g. described by [START_REF] Uhlig | Performance of a matrix comprehensive inhouse validation study by means of an especially designed software[END_REF].

Incurred material

Production of proficiency test samples

Incurred material was provided by a German import control point. This material consisted of non-compliant samples, remainders of import control tests. There were three different types of material, two from shrimps and one from crayfish (see table 3). The samples were frozen and homogenised when they arrived in the laboratory.

The samples were pre-tested for their CAP content, subsequently freeze-dried and the water content was calculated. The lyophilised material was homogenised in a Grindomix mixer (Retsch, Germany) and in an overhead rotary tumbler for two days.

Two of the samples were used directly in this form. The third sample was diluted with blank material (obtained from the grocery, tested for compliance prior to use) in order to achieve more relevant residue levels. The preparation of the proficiency test samples targeted at concentration levels close to the MRPL-level of 0.3 µg/kg.

[Insert table 3 about here]

The material was portioned and filled into dark glass vials, each with a quantity of approximately 4 g. Although the water content was different for the different materials (60 to 88 %), a joint value of 80 % was given in order to avoid the possibility of identifying the samples by their water content. So each portion of lyophilised material referred to a theoretical amount of approximately 20 g of fresh sample. After having Approximately 100 aliquots of each of the samples were available. Five samples each were combined in a set of samples. The sets contained, as shown in table 3, one blank sample (CAP004) and four non-compliant samples (CAP001,-002,-003,-005). One of the non-compliant samples was sent in duplicate, but without indicating this fact to the participants, in order to be able to check the repeatability of the measurements (CAP002/CAP005).

Homogeneity and stability

For testing homogeneity, 10 randomly selected aliquots of each sample (prior to combining it in test sets) were analysed in duplicate using the method described above. The results were evaluated by analysing variance and by comparing the standard deviation occurring between different aliquots S S with the Horwitz standard deviation S H . It turned out that all samples belonged to the same population and had a ratio S S /S H < 0.3 as requested in the IHPPT, see table 4.

[Insert table 4 about here]

For stability testing, the samples were stored under different conditions (-80°C, -30°C,+4°C, +20°C) and analysed after storage periods of one day to one year. The storage of aliquots at +20°C was stopped after one month since this storage condition was primarily chosen to prove the stability of the samples during shipping and sample handling (and due to the limited number of available samples for stability testing). For each of the storage time/storage condition combinations, two aliquots were randomly selected. Each aliquot was analysed in double. The use of an isochronous approach as suggested by [START_REF] Linsinger | Planning and combining of isochronous stability studies of CRMs[END_REF] was not suitable for this study, because in this connection, two significant disadvantages would come into effect: great number of samples to be analysed at one time and the results not being available until the very end of the stability study. Hence, as a compromise, the tests were performed using a "semi-isochronous" approach. Analysis was done after three different periods of time (2 days, 1 month, 1 year), so that, on the one hand, an acceptable number of samples has be analysed at a time and, on the other hand, stability data which were sufficient to start the proficiency test were already available after one month. In the stability study, no degradation of the analyte could be observed as can be seen in figure 3.

[Insert figure 3 about here]

Proficiency test

The proficiency test started in November 2004 with the shipment of the samples. The frozen samples (-20°C) were shipped without dry-ice because even at room temperature the material proved to be stable for some weeks (see figure 3). Each laboratory received a set of five samples. The samples should be analysed with the method routinely applied (a confirmatory method according to Commission Decision 2002/657EC) and results submitted within six weeks.

Evaluation

The proficiency test was organised and evaluated according to the prescriptions of The laboratories were asked to analyse the samples in double each and to submit the recovery corrected results. The mean value of these determinations was used and taken as the laboratory result (x) without any further correction.

The target value X was determined by calculating the median of the results of all laboratories (consensus value). The National Reference Laboratory; Berlin, took part in the study as one of the laboratories. As target value of the standard deviation, the reproducibility standard deviation was calculated using the Q-method as described in DIN 38402-45. The reproducibility standard deviation was taken instead of the Horwitz standard deviation [START_REF] Horwitz | Quality assurance in the analysis of foods for trace constituents[END_REF][START_REF] Boyer | Interlaboratory variability in trace elements analysis[END_REF] because at these low concentrations, the latter would allow significantly higher standard deviations and considerably limit the significance of the quantitative assessment of the proficiency test.

In addition to the individual quantitative assessment of each sample, the NRL uses a point system with the purpose of evaluating the overall proficiency of the laboratories.

In order to take into account not only the quantitative but also the qualitative This system is applied per analyte and per sample, i.e. in this case, there are a maximum of six attainable points (4 * 1.5). The minimum score for passing this proficiency test was set at 4.0 points (corresponding to 66.7 % of the maximum score). The score for banned compounds is generally set in such a manner that it is possible to be successful in the overall proficiency by simply identifying the banned compound (which is, in principle, sufficient), if there are no false positive findings in the blank samples.

Results and discussion

A total of 20 participants submitted results. For each sample, the median was calculated as a target value, including all results into the calculation (no outlier elimination). No false negative results and only one false positive result were reported for a total of 100 analyses. For the quantitative evaluation on the basis of zscores, the reproducibility standard deviation was used, the results and the used techniques are summarised in figure 4. Four of the results (from three different labs, [Insert figure 4 about here]

In contrast to the other samples, this matrix was a processed food sample. With the method applied by the NRL, an unusual behaviour during the extraction process (problems in phase separation) was observable. The difficulties with this sample are also reflected in the results of the proficiency test (results out of the z-scores and high reproducibility standard deviations for GC methods)).

[Insert table 5 about here]

The Horrat ratio (see table 5), which gives a certain indication of the overall performance level of the laboratories, should be between 0.5 to 2 for acceptable method performance as suggested by [START_REF] Pocklington | Harmonised protocols for the adaoption of standardised analytical methods and for the presentation of their performance characteristics[END_REF]. The ratio here is even below 0.5, indicating a very good performance of the laboratories.

The evaluation of the overall proficiency based on the point system (see above) added up to a mean of 5.85 points (out of six attainable points, referring to 97.5 %), all laboratories have been successful.

The participants were asked to provide details of their analytical method. The results were also evaluated with respect to this additional information. The very different internal standard. Meta-CAP was used only in three laboratories as the internal standard all of them applied GC-methods. The use of meta-CAP as an internal standard showed a tendency towards a higher scatter of the results but this conclusion is uncertain because of the very small number of measurements.

The only parameter which showed a bias was the comparison of GC/MS(MS) and LC/MSMS results (see table 6). GC/MS results showed a tendency towards higher concentration levels with a worse reproducibility compared to the LCMSMS results.

[Insert table 6 about here]

This effect would be similar, if the results of the two labs, which applied GC/MSMS, would be treated separately. An explanation of this effect is difficult due to the principle difference between the methods, e.g. the different ionisation mechanism, the necessary derivatisation for GC analysis and the different selectivity of the detection systems. Further studies using certified reference material would be necessary in order to verify whether there is a bias between the two techniques or whether it is just a matrix dependent effect.

All in all, the results of the study were very positive. The median concentrations were between 0.43 and 0.51 µg/kg CAP, the reproducibility ranged from 17 to 24 % (clearly below the Horwitz standard deviation of about 50%). None of the laboratories found false negative samples.

A conclusion of the results of the study is that the analysis of CAP residues is well established in many labs and the decision limits between 0.05 and 0.36 ng/g, indicated by the labs, were realistic because all labs were successful in confirming the analytes in the non-compliant samples. Accordingly the methods tested in the 1 5 .5 5 1 5 .6 0 1 5 .6 5 1 5 .7 0 1 5 .7 5 1 5 .8 0 1 5 .8 5 1 5 .9 0 1 5 .9 5 1 6 .0 0 1 6 .0 5 5 0 0 1 0 0 0

T im e --> A b u n d a n ce Io n 4 7 1 .0 0 ( 4 7 0 .7 0 to 4 7 1 .7 0 ) : M 1 2 4 3 P 1 A .D 5 .5 0 1 5 .5 5 1 5 .6 0 1 5 .6 5 1 5 .7 0 1 5 .7 5 1 5 .8 0 1 5 .8 5 1 5 .9 0 1 5 .9 5 1 6 .0 0 1 6 .0 5

0 1 0 1 0 T im e --> A b u n d a n ce
Io n 4 7 0 .0 0 ( 4 6 9 .7 0 to 4 7 0 .7 0 ) : M 1 2 4 3 P 1 A .D 5 .5 0 1 5 .5 5 1 5 .6 0 1 5 .6 5 1 5 .7 0 1 5 .7 5 1 5 .8 0 1 5 .8 5 1 5 .9 0 1 5 .9 5 1 6 .0 0 1 6 .0 5

0 1 0 1 0 2 0 T im e --> A b u n d a n ce
Io n 4 6 6 .0 0 ( 4 6 5 .7 0 to 4 6 6 .7 0 ) : M 1 2 4 3 P 1 A .D 5 .5 0 1 5 .5 5 1 5 .6 0 1 5 .6 5 1 5 .7 0 1 5 .7 5 1 5 .8 0 1 5 .8 5 1 5 .9 0 1 5 .9 5 1 6 .0 0 1 6 .0 5

1 0 2 0 3 0 T im e --> A b u n d a n ce
Io n 4 6 8 .0 0 ( 4 6 7 .7 0 to 4 6 8 .7 0 ) : M 1 2 4 3 P 1 A .D 5 .5 0 1 5 .5 5 1 5 .6 0 1 5 .6 5 1 5 .7 0 1 5 .7 5 1 5 .8 0 1 5 .8 5 1 5 .9 0 1 5 .9 5 1 6 .0 0 1 6 .0 5 5 0

1 0 0 T im e --> A b u n d a n ce
Io n 3 7 6 .0 0 ( 3 7 5 .7 0 to 3 7 6 .7 0 ) : M 1 2 4 3 P 1 A .D 5 .5 0 1 5 .5 5 1 5 .6 0 1 5 .6 5 1 5 .7 0 1 5 .7 5 1 5 .8 0 1 5 .8 5 1 5 .9 0 1 5 .9 5 1 6 .0 0 1 6 .0 5 2 0 3 0 4 0 5 0

T im e --> A b u n d a n ce Io n 3 7 8 .0 0 ( 3 7 7 .7 0 to 3 7 8 .7 0 ) : M 1 2 4 3 P 1 A .D 5 .5 0 1 5 .5 5 1 5 .6 0 1 5 .6 5 1 5 .7 0 1 5 .7 5 1 5 .8 0 1 5 .8 5 1 5 .9 0 1 5 .9 5 1 6 .0 0 1 6 .0 5 2 0 0 4 0 0 6 0 0 8 0 0

T im e --> A b u n d a n ce Io n 4 7 1 .0 0 ( 4 7 0 .7 0 to 4 7 1 .7 0 ) : M 1 2 4 3 P 1 C .D 5 .5 0 1 5 .5 5 1 5 .6 0 1 5 .6 5 1 5 .7 0 1 5 .7 5 1 5 .8 0 1 5 .8 5 1 5 .9 0 1 5 .9 5 1 6 .0 0 1 6 .0 5 Io n 4 7 0 .0 0 ( 4 6 9 .7 0 to 4 7 0 .7 0 ) : M 1 2 4 3 P 1 C .D 5 .5 0 1 5 .5 5 1 5 .6 0 1 5 .6 5 1 5 .7 0 1 5 .7 5 1 5 .8 0 1 5 .8 5 1 5 .9 0 1 5 .9 5 1 6 .0 0 1 6 .0 5 1 0 0 2 0 0 3 0 0

T im e --> A b u n d a n ce Io n 4 6 6 .0 0 ( 4 6 5 .7 0 to 4 6 6 .7 0 ) : M 1 2 4 3 P 1 C .D 5 .5 0 1 5 .5 5 1 5 .6 0 1 5 .6 5 1 5 .7 0 1 5 .7 5 1 5 .8 0 1 5 .8 5 1 5 .9 0 1 5 .9 5 1 6 .0 0 1 6 .0 5 5 0 1 0 0 1 5 0 2 0 0

T im e --> A b u n d a n ce Io n 4 6 8 .0 0 ( 4 6 7 .7 0 to 4 6 8 .7 0 ) : M 1 2 4 3 P 1 C .D 5 .5 0 1 5 .5 5 1 5 .6 0 1 5 .6 5 1 5 .7 0 1 5 .7 5 1 5 .8 0 1 5 .8 5 1 5 .9 0 1 5 .9 5 1 6 .0 0 1 6 .0 5 2 0 4 0 6 0 8 0 1 0 0 T im e --> A b u n d a n ce Io n 3 7 6 .0 0 ( 3 7 5 .7 0 to 3 7 6 .7 0 ) : M 1 2 4 3 P 1 C .D 5 .5 0 1 5 .5 5 1 5 .6 0 1 5 .6 5 1 5 .7 0 1 5 .7 5 1 5 .8 0 1 5 .8 5 1 5 .9 0 1 5 .9 5 1 6 .0 0 1 6 .0 5

  from Sigma Aldrich (CAP, >99 %) and from Euroisotope/Cambridge Isotope Laboratories (deuterated CAP, D5-CAP, >98 %). For the evaporation of solvents TurboVap LV™ and TurboVap II™ stations (Zymarck, United States) were used.An Agilent 5973N mass spectrometer coupled with a GC (HP 6890 plus) and equipped with an automatic sampler (HP 7683) was used for the measurements. The mass spectrometer was operated in the negative ion chemical ionisation mode (NCI with methane) with the following ions for confirmatory purposes: m/z 466, 468, 470, 376 (CAP), 466, 468 (meta-CAP), 471 D5-CAP.2 µl of the sample are injected splitless into the GC. The separation is performed on a 30 m * 0.25 mm id column, 0.25 µm ZB5 (Phenomenex) using a flow rate of 1 ml/min (Helium 5.0) and the following temperature programme: 85 o C (0.1 min); 12 o C/min to 240 o C; 5 o C/min to 260 o C/min; 15 o C/min to 290 o C (5 min hold). The injection block is operated at 280 o C, the transfer line at 290 o C and the ion source at 160 o C.

  samples, the dark glass vials are labelled with randomised sample codes (prepared by using the software "Prolab™").

  the IHPPT, ISO/IEC-Guide43-1: 1977 and DIN 38402-45: 2003-09 using the Prolab™" (quo data Ltd. Dresden, Germany). The z-scores for the laboratories´ quantitative results were calculated from the laboratory results x, the target value X (here: consensus value of all submitted results) and the target value of the standard deviation σ.

  matrix, namely CAP002/CAP005) lay outside the range of +/-2, nevertheless three of these four results were close to a z-score of 2.

  sample clean-ups (extraction with different solvents; clean-up with solid phase extraction, liquid-liquid extraction, silica clean-up, or a combination thereof) as well as a hydrolysis step, which was included by some of the laboratories, did not have any significant effect on the results. Most of the laboratories used deuterated CAP as the

  the one used by the NRL, proved to be suitable for the control of residue levels around the MRPL of 0.3 µg/kg.

  Figure 2a and 2b : Matrix blank (2a) and spiked matrix blank (2b) in crustaceans, spike level at 0.08 ng/g (m/z 471 referring to the internal standard, the other ion traces to CAP)

Figure 4 :

 4 Figure 4: Summary of the z-scores of the participating laboratories (dark triangles indicating results out of the accepted tolerance of +/-2)

  The resulting experimental plan is given in table 1. In order to get reliable validation data, it is required that firstly, for each of the runs a different matrix is used, and secondly, the experiments are carried out at different times (not more than one or two runs at a time). This study was carried out within 10 weeks.

	varied during the validation on two levels each: species (as a leading factor),
	operator, cartridge lots, storage times of the extracts, sample amount, extraction and
	centrifugation conditions.
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	This model is based on an experimental plan, which was created by means of the
	validation software InterVal™. In the validation study, 16 different factor-level-
	combinations (using 16 different matrices) were tested. The following factors were

[Insert table 1 about here]

Table captions :

 captions Table 1: Experimental plan for the in-house validation

	Page 15 of 27	Food Additives and Contaminants
	F o r F o r F o r F o r
	P P P P
	e e e e
		e r e r e r e r
		R R R R
		e e e e
		v i e v i e v i e v i e
		w w w w
		O n l O n l O n l O n l
		y y y y
		1

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Table 2 :

 2 Method performance characteristics

Table 3 :

 3 Matrix information of test kit submitted to the participants and sample codes

Table 4 :

 4 Results of homogeneity tests

Table 5 :

 5 Median, Horrat ratio, repeatability and Horwitz standard deviation (S R and S H of the individual samples

Table 6 :

 6 Median and repeatability standard deviation (separate calculation for the

	different systems applied)

Table 1 :

 1 Experimental plan for the in-house validation

				cartridge	test	storage of
	run	species	operator	lot	sample	extracts	centrifugation	extraction	elution
	S_16	pig	B	Y	15 g	2-3 days	with 4000 rpm	with 2 * 8 ml	60/40 ACN/water
	S_06	crustaceans	B	X	15 g	2-3 days	with 3000 rpm	with 2 * 10 ml	50/50 ACN/water
	S_01	crustaceans	A	X	10 g	0-1 day	with 3000 rpm	with 2 * 8 ml	50/50 ACN/water
	S_10	pig	A	X	15 g	2-3 days	with 3000 rpm	with 2 * 10 ml	60/40 ACN/water
	S_12	pig	A	Y	15 g	0-1 day	with 3000 rpm	with 2 * 8 ml	50/50 ACN/water
	S_09	pig	A	X	10 g	2-3 days	with 4000 rpm	with 2 * 8 ml	50/50 ACN/water
	S_14	pig	B	X	15 g	0-1 day	with 4000 rpm	with 2 * 10 ml	50/50 ACN/water
	S_15	pig	B	Y	10 g	2-3 days	with 3000 rpm	with 2 * 10 ml	50/50 ACN/water
	S_04	crustaceans	A	Y	15 g	2-3 days	with 4000 rpm	with 2 * 8 ml	50/50 ACN/water
	S_02	crustaceans	A	X	15 g	0-1 day	with 4000 rpm	with 2 * 10 ml	60/40 ACN/water
	S_11	pig	A	Y	10 g	0-1 day	with 4000 rpm	with 2 * 10 ml	60/40 ACN/water
	S_03	crustaceans	A	Y	10 g	2-3 days	with 3000 rpm	with 2 * 10 ml	60/40 ACN/water
	S_08	crustaceans	B	Y	15 g	0-1 day	with 3000 rpm	with 2 * 8 ml	60/40 ACN/water
	S_13	pig	B	X	10 g	0-1 day	with 3000 rpm	with 2 * 8 ml	60/40 ACN/water
	S_05	crustaceans	B	X	10 g	2-3 days	with 4000 rpm	with 2 * 8 ml	60/40 ACN/water
	S_07	crustaceans	B	Y	10 g	0-1 day	with 4000 rpm	with 2 * 10 ml	50/50 ACN/water

Table 2 .

 2 Method performance characteristics

	Decision limit CC α	0.074 µg/kg
	Detection capability CC β	0.087 µg/kg
	Recovery*	95%
	Repeatability*:	8%
	Within-laboratory reproducibility*:	9 %.
	*) at the concentration of CC β

Table 3 :

 3 Matrix information of test kit submitted to the participants and sample codes

	Sample code	Kind of sample
	CAP 001	CraYfish; non-compliant import sample, Asia
	CAP 002	Shrimps, salted, non-compliant import sample, Asia
	CAP 003	Prawns, non-compliant import sample; diluted with
		blank material, Asia
	CAP 004	Polar sea shrimps, blank
	CAP 005	As CAP 002

Table 4 :

 4 Results of homogeneity tests

	Sample	Test	Critical F-	Mean [µg/kg]	s S	s H	s S / s H
		statistic F	value
	CAP 001	1.876	3.020	0.452	0.011 0.230	0.05
	CAP 002/5	1.227	3.020	0.460	0.015 0.234	0.06
	CAP 003	1.578	3.020	0.356	0.048 0.188	0.26
	S S : Standard deviation between different samples (n = 10)
	S H : Horwitz standard deviation at the respective concentration

Table 5 :

 5 Median, Horrat ratio, repeatability and Horwitz standard deviation (S R and S H of the individual samples

	Sample	Median	Horrat	s R		s H
			ratio
		[µg/kg]	s R / s H	abs.	%	abs.	%
	CAP 001	0.512	0.49	0.124	24.3	0.256	50.0
	CAP 002	0.500	0.37	0.093	18.6	0.251	50.2
	CAP 003	0.427	0.34	0.073	17.0	0.219	51.4
	CAP 005	0.491	0.43	0.107	21.7	0.247	50.4
	Table 6: Median and repeatability standard deviation (separate calculation for the
	different systems applied)
	Sample	GC-

MS (including GC-MS/MS) n=10 (n=2) LC-MS/MS n=10

	Figure captions: Figures:
	Figure 1: Confidence interval of the 16 calibration curves calculated from the 16
							Calibration
	different runs (factor-level-combinations) according to table 1.
	Figure 2a and 2b : Matrix blank (2a) and spiked matrix blank (2b) in crustaceans, 0,2
	spike level at 0.08 ng/g (m/z 471 referring to the internal standard, the other
	ion traces to CAP)
	F o r Figure 3: Results of the degradation study (CAP_002), concentration in ng/g F o 0,15 Measurement values [ng/g] r Figure 4: Summary of the z-scores of the participating laboratories (dark triangles 0,1
	P indicating results out of the accepted tolerance of +/-2) P
				e e
	0,05				e r r e
	Run 01 0,06	Run 02	Run 03	Run 04 0,08	Run 05	Run 06 0,1	R e e Run 07 Run 08 Run 09 Concentration [ng/g] Run 10 R 0,14 0,12	Run 11	Run 12	Run 13 0,16	Run 14	Run 15 0,18	Run 16	0,2
	v i e w v i Figure 1: Confidence interval of the 16 calibration curves calculated from the 16 e w different runs (factor-level-combinations) according to table 1.
	CAP001		Median [µg/kg] 0.619		s R 0.133 = 21.5 %	Median O n l O n l [µg/kg] 0.477 0.101 = 21.2 % s R y y
	CAP002		0.525		0.128 = 24.4 %		0.469		0.075 = 16.9 %
	CAP003		0.425		0.090 = 21.2 %		0.427		0.067 = 15.7 %
	CAP005		0.503		0.155 = 30.8 %		0.486		0.067 = 13.8 %
															1 2

uk Food Additives and Contaminants

	1
	2
	3 4 5	0010 (LC)	CAP001 Degradation study (Concentration v s. Time) CAP002 CAP003 CAP005 -2,146
	6	0029 (LC)
	7	0,60 0032 (LC)
	8	0038 (GC)
	9 10	0041 (LC) 0,50 0053 (LC)
	11 12	0056 (LC) 0,40 0058 (GC)
	13	0059 (GC)
	14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 F 0,30 0061 (GC) 0067 (GC) 0,1 -80°C Figure 3: Results of the degradation study (CAP_002), concentration in ng/g P e e r R e v i e w O n l y 1 10 100 weeks -30 °C +4°C + 20°C (dark) P e e r R e v i e w O n l y -2 0 2 -2 0 2 -2 0 2 -2 0 2 0124 (GC) 0138 (LC) 0145 (LC) o r 0,00 0,10 0,20 0080 (GC) 0086 (GC) 2,467 3,366 F 0089 (LC) 0095 (LC) o 0104 (GC) r 0111 (GC) 2,148
			4 5

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