MS method for anabolic steroids in nutritional supplements

Keywords: Chromatographic analysis, Chromatography -LC/MS Nutritional supplements, Anabolic steroids, Anabolic steroids esters, Prohormones, LC-MS-MS analysis

Validation of a LC-MS-MS method for anabolic steroids in nutritional supplements

Simona Martello, Marialinda Felli, Marcello

Introduction

Several doping cases have been reported in official doping tests taken during sports competitions worldwide. In some cases athletes denied the use of doping agents and claimed to have inadvertently or passively absorbed the drug, for example by the ingestion of food or products sold as nutritional supplements that contained prohibited substances [ ?? et al. 2004]. Nutritional supplements seem to modify the body appearance and to promote more consistent and intensive training for athletes by allowing a speedy recovery between training sessions and by enhancing competitive performance. Most of these products do not contain doping agents, but studies have demonstrated that some dietary supplements may contain substances that are not declared on the label, and have been prohibited by the International Olympic Committee/World Anti-Doping Agency [START_REF] Parr | Analytical strategies for the detection of non-labelled anabolic androgenic steroids in nutritional supplements[END_REF][START_REF] De Cock | Detection and determination of anabolic steroids in nutritional supplements[END_REF][START_REF] Delbeke | Prohormones and sports[END_REF][START_REF] Pipe | Nutritional supplements and doping[END_REF][START_REF] Ayotte | Sport nutritional supplements : quality and doping controls[END_REF]. Banned substances such as ephedrine, caffeine, steroids and prohormones were found in some "non-hormonal" nutritional supplements [START_REF] Geyer | Analysis of nonhormonal nutritional supplements for anabolic-androgenic steroids-results of an international study[END_REF]. It is not known whether the reason is faulty production control or intentional addition [START_REF] Maughan | Dietary supplements[END_REF], Ziegenfuss et al. 2002[START_REF] Broeder | Oral and ro-related prohormone supplementation[END_REF][START_REF] Millman | Steroid and nutritional supplement use in professional athletes[END_REF].

In spite of the widespread use of nutritional supplements among sportsmen, the European Community promulgated the first Council Directive on dietary supplements (2002/46/CE), but supplements commonly used by sportsmen (BCCA, amino acids, proteins, etc) are not mentioned.

In doping control, present testing methods for anabolic androgenic steroids in nutritional supplements rely on GC/MS [START_REF] Parr | Analytical strategies for the detection of non-labelled anabolic androgenic steroids in nutritional supplements[END_REF][START_REF] De Cock | Detection and determination of anabolic steroids in nutritional supplements[END_REF][START_REF] Delbeke | Prohormones and sports[END_REF] or tandem mass spectrometry [START_REF] De Cock | Detection and determination of anabolic steroids in nutritional supplements[END_REF][START_REF] Van Thuyne | Validation of a GC-MS screening method for anabolizing agents in solid nutritional supplements[END_REF]]. The methods developed using LC-MS [START_REF] Joos | anabolic steroid analysis-optimization and comparison of three techniques: electrospray ionization, atmospheric pressure chemical ionization and atmospheric pressure photoionization[END_REF] and LC-MS-MS [Leinonen et al. 2002[START_REF] Ho | Comprehensive screening of anabolic steroids, corticosteroids, and acidic drugs in horse urine by solid-phase extraction and liquid chromatography-mass spectrometry[END_REF][START_REF] Guan | Detection, quantification, and confirmation of anabolic steroids in equine plasma by liquid chromatography and tandem mass spectrometry[END_REF], Devebter et al. 2006, Politi et al. 2006] have not been applied to nutritional supplements. Only Reilly and Crouch report a LC-MS-MS method applied to 5α-androst-1-en-3,17- dione and/or 5α-androst-1-en-3β,17β-diol (1AD), used as nutritional supplements and analysed in plasma and urine [START_REF] Reilly | Analysis of the nutritional supplements 1AD, its metabolite, and related endogenous hormones in biological matrices using liquid chromatographytandem mass spectrometry[END_REF].

This study describes the validation of a qualitative LC-MS-MS method for the determination of 8 doping substances. The anabolic steroids selected were those most commonly found in nutritional supplements, as shown by litterature data [START_REF] De Cock | Detection and determination of anabolic steroids in nutritional supplements[END_REF], Geyer et al. 2004]. Moreover in nutritional supplements ephedrine was detected by GC/MS and was confirmed by LC/MS/MS. The use of LC-MS-MS provides several advantages over current methods because this technique allows the direct separation and identification without the need for derivatization, reducing the time of analysis, eliminating predictable sources of error and decreasing the use of hazardous and expensive reagents [Leinonen et al. 2002].

Materials and Methods

Nutritional supplements

64 nutritional supplements were purchased from shops and judical proceeding. The ingredients contained in the products has been classified as following: 4 vitamins/minerals, 7 glutamine/creatine, 9 amino acids, 12 proteins, 8 banned substances, 12 herbal extracts, 4 other. The manufacturer's recommended doses vary according to the supplement.

Chemicals and reagents

The following reference standards were used: ephedrine, testosterone, testosterone decanoate, nandrolone, nandrolone decanoate, testosterone-d 3 from Sigma (Saint Louis, Missouri USA); boldenone,metandienone,oxandrolone,stanozolol,trenbolone,testosterone propionate,testosterone acetate,testosterone enanthate, (1mg/ml) were prepared by dissolving 5 mg of each standard in 5 ml of methanol. Working standard solutions were made by dilution with methanol to the appropriate concentration (10 µg/ml and 1 µg/ml).

All solvents were HPLC grade and were obtained from Merck (Darmstadt, Germany); formic acid (extra pure) was obtained from Sigma-Aldrich-Riedel-de Haen (Seelze, Germany).

Materials and apparatus

The GC-MS analyses were performed on a Thermo electron gas chromatography coupled toa DSQ quadrupole mass spectrometer. Each sample was run through the following temperature program: initial temperature 70° C for 1 minute, then 10° C/min incremnets to 280° C, followed by a final isotherm of 5 minutes. The inject port and the source temperature were set at 250° C. precursor ions were selected and fragmented acquiring the full scan MS-MS spectra. At last the higher product ions were chosen and each compound was analysed in SRM mode to obtain the best sensitivity and specificity. Fragmentation of the selected precursor ions was performed by collisioninduced dissociation (CID) with helium, which fills the ion trap. The helium pressure in mass analyzer cavity was maintained at approximately 0.1 Pa.

Extraction procedure of nutritional supplements

The extraction procedure followed the validated method of Van Thuyne et al. ( 2004), modified for liquid chromatography detection [Van Thuyne [START_REF] Van Thuyne | Validation of a GC-MS screening method for anabolizing agents in solid nutritional supplements[END_REF]. Nutritional supplement (1 g) was added with the internal standard (testosterone-d3 100 ng/g). The residue was reconstituted with 5 ml MeOH and shaken by the vortex. 1 µl of the methanolic solution was injected into the GC-MS to detect the presence of ephedrine. Then the methanol was evaporated to dryness, mixed with 2 ml NaOH (1 M) and shaken with 5 ml n-pentane:diethylether (9:1) for 1 hr. After centrifugation the organic layer was separated and evaporated under nitrogen flow at 40°C. The residue was reconstituted with 50 µl of mobile phase (methanol:water with acetic acid 0.03%, 50:50) and injected into the LC-MS-MS system.

Validation Method

The method was validated using a blank nutritional supplement containing branched chain amino acids. This nutritional supplement was chosen to represent the spectrum of nutritional supplements because it is commonly used by body builders. After the validation, the method was applied to 64 unknown nutritional supplements. The analytical method for the determination of 8 anabolic compounds was validated in accordance to the Eurachem Guide suggestions [Eurachem.1998, 61].

According to Van Thuyne and [START_REF] Van Thuyne | Validation of a GC-MS screening method for anabolizing agents in solid nutritional supplements[END_REF], the method has only been just validated qualitatively as anabolic androgenic steroids and their esters in nutritional supplements are absolutely prohibited, with no limits of concentration allowed. The Limit of Detection (LoD) was calculated by qualitative measurements spiking a blank nutritional supplement with a reference mixture at concentrations of 1, 5, 10, 25 and 50 ng/g. Each level was performed on five different replicates of the blank supplement. Repeatability (intra-assay precision) for LoDs level was in the range 8-14%. The selected compounds of the reference mixture were: 4-androsten-3,17-dione, 4-estren-3,17-dione, 5-androsten-3β-ol-17-one (DHEA), boldenone, nandrolone, nandrolone decanoate, testosterone and testosterone decanoate. The specificity was tested during the validation procedure by extracting 10 aliquots of the blank nutritional supplement, to look for possible matrix interferences.

Results and Discussion

The relative retention time, the precursor ion and the product ions for each standard compound are given in Table I. The LC-MS-MS chromatograms of a blank nutritional supplement and a nutritional supplement spiked at 50 ng/g, extracted as above, are shown in Figure 1 and2, respectively.

In order to test the specificity 10 aliquots of the blank supplement were analysed and no matrix interference was found at the retention time of the 8 analytes and the internal standard. The relative retention time of the analyte should correspond to that of the standard analyte, from a spiked sample, with a tolerance of ±2%. The relative intensities of the detected ions, expressed as a percentage of the intensity of the most intense transition, must correspond to those of the standard analyte, from a spiked sample with the relative tolerance of ±25%, as suggested by the World Anti-Doping Agency [The WADA Technical Document -TD2003IDCR]. All these criteria were applied to all the spiked samples for the LoD determination. According to the Eurachem guidelines [Eurachem 1998, 61] the LoD for qualitative measurements is a concentration threshold below which specificity becomes unreliable. It was defined as the concentration level where a compound could be detected in all five of the tested samples. Table II shows how this threshold concentration was determined for each standard compounds and figure 3 reports the relative LODs chromatograms According to our experimental data six compounds can be detected at or below 10 ng/g, two at 25 ng/g. The validated method was applied to 64 nutritional supplements, containing ingredients as described above. 12.5% of the nutritional supplements tested contained substances not declared on the label. The positive nutritional supplements are reported in table III, in which we reported: the producer, the ingredients declared in the label and the banned substances. Sample 1, 2, 4, 7, 8, 9 and 12 contain banned substances not declared; sample 5 and 6 confirm the presence of ephedrine and in sample 3 we found ephedrine but the label reports only the plant source. Finally for sample 17 we could not confirm the presence of ephedrine as reported on the label. In figure 4 a nutritional supplement positive to 4-estren-3,17-dione and 4-androsten-3,17-dione is shown.

Conclusions

The method reported here is sufficiently sensitive, specific and selective for the detection and confirmation of 4-androsten-3,17-dione, 4-estren-3,17-dione, 5-androsten-3β-ol-17-one (DHEA), boldenone, nandrolone, nandrolone decanoate, testosterone and testosterone decanoate in nutritional supplements, in accordance to the validation criteria suggested by Eurachem guidelines. This LC-MS-MS method was applied to 64 nutritional supplements and 12.5% of them were found positive.

The low levels of the compounds found in the samples (in the range of ng/g) may indicate an accidental contamination and not an intentional admixture. However, athletes should consider only purchasing from companies performing quality tests on prohormones and testing for possible contamination during production.

  from Steraloids Inc. (Newport R.I. USA). The stock standard solutions

  About LC-MS-MS parameters, the analytes were separated on a Discovery HS C 18 column (150 × 4.6 mm, 5 µm, Supelco, Bellefonte PA). A Discovery HS C 18 guard column (20 × 4.6 mm, 5 µm) was used prior to the analytical column. The mobile phase was a mixture of methanol (A) and acetic acid 0.03% (B) with a flow of 1.2 ml/min. The gradient program of the mobile phase started with A:B (60:40, v/v), was maintained for 3 minutes, then changed to A:B (95:5, v/v) over 12 min. Then the column was set back to the initial mobile phase conditions in 10 min. A sample loop of 20 µl was used.A Spectra System P4000 pump coupled with LCQ Advantage ion trap mass spectrometer (Thermoelectron Corp., San José, CA, USA) was used for the LC-MS-MS analyses. The mass spectrometer was equipped with an atmospheric pressure chemical ionisation (APCI) probe operating in positive ion mode. The source parameters were as follows: nitrogen gas used as sheath and auxiliary gas at flow 1.2 L/min and 6 L/min respectively; vaporizer temperature 450° C; spray voltage 4500 V; capillary voltage and capillary temperature 5 V and 220° C respectively; and tube lens offset of 50 V. The mass spectra of the standard compounds were first acquired in full scan mode (150-460 m/z) by infusion of a reference solution of 1 µg/ml. From these spectra the

  Figure 1

Table I :

 I Relative retention times, precursor ions and product ions of each compound of the standard mixture.

	Compound 4-estren-3,17-dione Boldenone 4-androsten-3,17-dione Nandrolone Testosterone-d3 Testosterone 5-androsten-3β β β β-ol-17-one Nandrolone decanoate F o r P e e r R e v i e w O RRT Parent Ion (m/z) Product Ions (m/z) 0.761 273 255, 237, 197 0.828 287 269, 173, 135 0.884 287 269, 251 0.900 275 257,239 1.000 292 274, 256 1.003 289 271, 253 1.014 271 271, 253 2.226 429 257, 239, 275
	Testosterone decanoate	2.387	443	271, 253

n l y Number of positive and negative findings in spiked samples at different analyte concentrations

	Concentration	4-estren-	Boldenone	4-androsten-	Nandrolone Testosteron	DHEA Nandrolone	Testosterone
	(ng/g)	3,17-dione		3,17-dione		e		decanoate	decanoate
	50	5/0	5/0	5/0	5/0	5/0	5/0	5/0	5/0
	25	5/0	5/0	5/0	5/0	5/0	5/0	5/0	5/0
	10	5/0	5/0	5/0	5/0	5/0	2/3	5/0	3/2
	5	5/0	2/3	5/0	5/0	5/0	0/5	3/2	1/4
	1	2/3	1/4	2/3	3/2	5/0	0/5	1/4	0/5
	LOD	5 ng/g	10 ng/g	5 ng/g	5 ng/g	1 ng/g	25 ng/g	10 ng/g	25 ng/g

Table II Page 17 of 18 http

 II

			Food Additives and Contaminants	Page 18 of 18
	N.	Producer	Ingredients declared on the label	Banned substances
	1	A	L-glutamine	4-estren-3,17-dion 4-androsten-3,17-dion
	2	A	Proteins, carbohydrates	4-estren-3,17-dion
	3	B	Ephedra sinica/Ma Huang; Guaranà; White willow bark; Chromium	ephedrine
			picolinate
	4	B	L-carnitine; Garcinia Cambogia; Vitamin B6; Chromium	ephedrine
	5	Ma Huang (ephedrine alkaloids); Guaranà extract; Green tea extract; o F (sinephrine alkaloids); Herbal blend r C L-phenylalanine; L-tyrosine; White willow bark; Citrus aurantium	ephedrine
	6 7 8 9 10 11 12 13	C D D D E F G G	(ginger, passion flower, yohimbe) P Ephedrine alkaloids; Guaranà e extract; Yerba mate extract; citrimax extract; L-carnitine; L-tyrosine e r Branched-Chain Amino Acids (BCCA) L-carnitine R Amino acids e Branched-Chain Amino Acids (BCCA) v Ephedrine; guaifenesin i e Dehydroepiandrosteron (DHEA) w Dehydroepiandrosteron (DHEA)	ephedrine nandrolone decanoate testosterone decanoate testosterone 4-androstendion ephedrine DHEA testosterone DHEA
	14 15 16 17	H I I C	Ephedrine; guaifenesin Testosterone decanoate prohormones Ephedrine (from herbal extract); guarana extract; yerba mate extract; O Testosterone decanoate ephedrine 4-androstendion 4-estrendion n l y ephedrine declared on the citrimax; white willlow bark; label not found
			yohimbe; bioperine
			Table III

://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Food Additives and Contaminants

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


Lubomir Svoboda
email: svobodalubomir@post.cz

Vladislav Chrastny

Lubomír Svoboda

Vladislav Chrastný

Contents of eight trace elements in edible mushrooms from a rural area

Keywords: Trace elements, Trace elements (nutritional) edible mushrooms, trace elements, arsenic, heavy metals, barium, cobalt, copper, rubidium, silver, thallium, vanadium

Eight trace elements were determined using ICP-MS in 78 samples of fruiting bodies of 22 edible mushroom species. The mushrooms were collected from four sites in a rural area unpolluted by human activities. Median values (dry matter) were as follows:-arsenic (As) 1.45 mg kg -1 , barium (Ba) 1.41 mg kg -1 , cobalt (Co) 0.28 mg kg -1 , copper (Cu) 47.0 mg kg -1 , rubidium (Rb) 130 mg kg -1 , silver (Ag) 2.95 mg kg -1 , thallium (Tl) 0.02 mg kg -1 and vanadium (V) 0.25 mg kg -1 . Higher trace element accumulation was observed within samples of Macrolepiota procera, Macrolepiota rhacodes, Lycoperdon perlatum, Lycoperdon gigantea and Xerocomus chrysenteron for As and Cu, and within samples of Cantharellus cibarius and of genera Boletus and Suillus for Rb.

Introduction

Extensive research has been carried out since the 1970´s on the occurrence of trace elements (mainly heavy metals) in mushrooms (higher fungi, macrofungi). The research has had mainly two aims: screening of mushroom fruiting bodies as bioindicators of environmental pollution and searching for edible species accumulating high levels of some trace elements. As detected, many wild growing species accumulate high concentrations, especially of cadmium, mercury, lead and copper, at levels considerably exceeding those in plants and in foods generally (for a review see Kalač & Svoboda 2000).

Knowledge of the roles of trace elements in physiology of higher fungi has been limited.

Concentrations of the elements in fruiting bodies are generally species-dependent. Substrate composition is an important factor, but great differences exist in the uptake of individual metals [START_REF] Tyler | Accumulation and exclusion of metals in Collybia peronata and Amanita rubescens[END_REF][START_REF] Gast | Heavy metals in mushrooms and their relationship with soil characteristics[END_REF][START_REF] Michelot | Update of metal content profiles in mushrooms -toxicological implications and tentative approach to the mechanisms of bioaccumulation[END_REF].

The age of the fruiting body or its size are of less importance. Some authors reported higher metal concentrations in younger fruiting bodies. This is explained by the transport of a metal from mycelium to the fruiting body during the start of fructification. During the following increase of the fruiting body mass, the metal concentrations decrease. The proportion of metal concentrations from atmospheric deposition seems to be of less importance due to a short lifetime of fruiting bodies of the most of edible species, which is usually 10-14 days. In our opinion, metal levels in fruiting bodies of wild growing mushrooms are considerably affected by the age of mycelium and by the interval between the fructifications. All these factors cause a very wide variability in the trace element concentrations within a species, commonly to one order of magnitude.

Information on levels of detrimental metals and metaloids has been required for countries with a traditionally high consumption of wild growing edible mushrooms. This study follows our previous work [START_REF] Nováčková | Contents of mercury, cadmium and lead in edible mushrooms and in uderlying substrates from a rural area with an occurrence of serpentines and amphiboles[END_REF]), the primary aim of which was to consider the contents of mercury, cadmium and lead in wild edible mushrooms growing in a site with specific geological ground, namely with the occurrence of serpentines and amphiboles. No significant differences were observed between the level of three studied metals in mushrooms from the tested and background unpolluted areas. The objective of the present work was to determine the potential health risk contents of eight trace elements, including those with

Materials and methods

Sampling

The study was carried out at four sites over an area of 0.7-2.7 ha in a rural unpolluted region near a small town Moravský Krumlov, south-western Moravia, Czech Republic, about 30 km south-west from the city of Brno. The sites were up to 15 km apart, around the nature reserve "Serpentine steppe" near Mohelno, at an altitude of 230 -460 m above sea level. Nuclear power station Dukovany (4 x 440 MW), in operation since 1985, lies near the sites. No roads with extensive traffic have crossed the tested area.

Mushrooms were collected in mixed forests with prevailing oak, beech, acacia, spruce and pine during the period 2001 -2003. One complete fruiting body was used as a sample. The bodies were cleaned of all surface contamination by a stainless steel knife. No washing or cap peeling was applied. The bodies were sliced and dried at an ambient temperature in a dustfree room in a manner typical for mushroom preparation for culinary purposes and then powdered. In total, 78 samples of 22 mushroom species were collected and analysed.

Analytical procedures

About 0.3 g of an air dried mushroom sample was wet digested with 5 ml of suprapure concentrated nitric acid (Merck, Germany) in a closed Teflon ® vessel in a microwave oven MDS 2000 (CEM Corp., USA). The digest was diluted to 25 ml with deionized water (MILLI-Q Element, Millipore, France) and filtrated using glass microfibre filter GF/C (Whatman, UK).

Each of the mushroom samples was analysed in triplicate. The determination of the metal concentrations was performed using a mass spectrometer with inductively coupled plasma (ICP-MS) PQExCell (VG Elemental, UK) under standard conditions with a Meinhard concentric nebulizer. For all measurements presented, quantification was performed using an aqueous multi-element standard solution Merck VI (CertiPUR, Merck, Germany). The calibration procedure consisted of ten measurement replicates of the instrumental blank and five measurement replicates of the differently diluted standard solutions. To eliminate non- , barium, cadmium, cobalt, copper, lead, rubidium, silver, thallium and vanadium (calculated as 10-fold of standard deviation from ten replicates of the instrumental blank solution) 10, 20, 2.5, 75, 2.5, 15, 2 and 2.5 µg kg -1 dry matter, respectively. A reference material CRM BCR No 60, Trace elements in an aquatic plant Lagarosiphon major (Commission of the European Communities, Community Bureau of Reference, Belgium) and epiphytic lichen Evernia prunastri (International Atomic Energy Agency, Wienna, Austria) was used for the evaluation of the measurement precision and accuracy. Differences between determined and certified contents were for all analysed elements below 10 %.

Statistical method

Outlying results were identified by Grubbs' test and were not statistically processed by Student´s test. For all statistical procedures, values below the limits of quantification were used for the calculations as halves of the limits of quantification. Differences in the element contents among species (given in Table II) were tested by Duncan´s multiple range test with alpha levels for critical ranges P < 0.05, using program Statistica for Windows, StatSoft, Inc., USA.

Results and discussion

In total, 78 samples of 22 mushroom species were collected and analysed. Contents of the individual metals are given in Table I. Element concentrations are expressed as mg kg -1 dry matter. There exists a consensus that dry matter content of mushrooms is 10 %.

High values of standard deviation, even higher several times than mean values (Table II), were caused by a highly abnormal metal content distribution. A very wide range of metal contents within a species is quite common in mushrooms (for review see Kalač & Svoboda 2000). Such a situation is different from that in plants. Basic Statistical characteristics of the determined trace elements for eight mushroom species with at least four five samples are given in Table II. Statistical testing by Duncan´s test revealed no differences in four elements (arsenic, barium, silver and vanadium) and limited differences in three elements (cobalt, rubidium and thallium). Copper was the only element with three different content levels. The results of the test are affected with wide variations of the element contents within the individual species.

Arsenic

High contents of metalloid arsenic (median 1.45 mg kg -1 dry matter, Table I) were found in Macrolepiota rhacodes, M. procera, Lycoperdon perlatum and Xerocomus chrysenteron.

Several accumulating species such as Agaricus spp., Laccaria amethystina, L. fraterna, L. laccata, Macrolepiota procera and Telephora terrestris were reported [START_REF] Vetter | Comparison of mineral elements in Agaricus and Pleurotus fruit bodies[END_REF][START_REF] Stijve | Arsenic in edible mushrooms[END_REF][START_REF] Parisis | Antimony uptake and correlation with other mushrooms species[END_REF][START_REF] Byrne | Arsenobetaine and other arsenic species in mushrooms[END_REF][START_REF] Slekovec | Uptake of arsenic by mushrooms from soil[END_REF][START_REF] Šlejkovec | Arsenic compounds in higher fungi[END_REF][START_REF] Weeks | Multi-element survey of wild edible fungi and blackberries in the UK[END_REF]. Moreover, an extreme arsenic content was found in inedible Sarcosphaera coronaria with maximum values of 2130 and 7090 mg kg -1 dry matter [START_REF] Stijve | Arsenic accumulation in some higher fungi[END_REF][START_REF] Borovička | New locality of Sarcosphaera coronaria[END_REF], respectively.

Food and feed plants, in contrast to marine animal organisms, contain only a very limited level of arsenic. Cereal and potatoes exhibit medium content of 0.05 mg kg -1 fresh matter and fruits and vegetables between 0.02-0.04 mg kg -1 fresh matter [START_REF] Weigert | Metal loads of food of vegetable origin including mushrooms[END_REF].

Barium

The mean barium content of all analysed samples was 2.15 mg kg -1 dry matter. As compared with other metals, barium contents were relatively equal without a tendency of its accumulation within certain genera of mushrooms. A similar situation was observed also for cobalt and vanadium.

There is a lack of limited information on contents of barium in mushrooms. Both [START_REF] Vetter | Comparison of mineral elements in Agaricus and Pleurotus fruit bodies[END_REF] in Agaricus spp. and Pleurotus spp. and [START_REF] Arguete | Accumulation of several heavy metals and lanthanides in mushrooms (Agaricales) from the Chicago region[END_REF] in Amanita rubescens, A. flavorubescens and Russula pectinatoides [START_REF] Bakken | Accumulation of radiocesium in fungi[END_REF], [START_REF] Vetter | Comparison of mineral elements in Agaricus and Pleurotus fruit bodies[END_REF] and [START_REF] Arguete | Accumulation of several heavy metals and lanthanides in mushrooms (Agaricales) from the Chicago region[END_REF] found mean contents of barium in order of a maximum few mg per kilogram of dry matter. Our results are in a good agreement with their findings. Shiraishi (2004) performed an analysis of dietary intakes of 19 elements (including barium, cobalt, copper, and rubidium) in eighteen food categories by Japanese subjects: For barium, two categories had high concentrations: seaweeds and nuts and seeds (4.61 and 5.91 mg kg -1 fresh matter, 6 respectively). For mushrooms, average content of 0.26 mg kg -1 fresh matter was reported (Shiraishi 2004).

Cobalt

For cobalt we obtained median and maximum value of 0.28 and 3.25 mg kg -1 dry matter, respectively. Usual contents of cobalt in mushrooms are in order of tenth, maximally a few mg per kilogram of dry matter [START_REF] Młodecki | Mushrooms like source of cobalt in food[END_REF][START_REF] Drbal | Content of cobalt in some edible mushrooms[END_REF][START_REF] Mutsch | Trace elements in higher fungi[END_REF][START_REF] Kalač | Contents of mercury, lead and cadmium in mushrooms[END_REF][START_REF] Vetter | Comparison of mineral elements in Agaricus and Pleurotus fruit bodies[END_REF][START_REF] Barcan | Absorption of heavy metals in wild berries and edible mushrooms in an area affected by smelter emissions[END_REF][START_REF] Sesli | Levels of trace elements in the fruiting bodies of macrofungi growing in the East black Sea region of Turkey[END_REF][START_REF] Siobud-Dorocant | Multivariate analysis of metal concentration profiles in mushrooms[END_REF][START_REF] Demirbaş | Concentrations of 21 metal in 18 species of mushrooms growing in the East Black Sea region[END_REF][START_REF] Mendil | Determination of trace elements on some wild edible mushrooms samples from Kastamonu, Turkey[END_REF][START_REF] Yeşil | Level of heavy metals in some edible and poisonous macrofungi from Batman of south east Anatolia, Turkey[END_REF][START_REF] Tüzen | Determination of heavy metals in soil, mushrooms and plant samples by atomic absorption spectrometry[END_REF][START_REF] Işiloğlu | Concentrations of trace elements in wild edible mushrooms[END_REF], Solyak et al. 2005). An extraordinary high value of 148 mg kg -1 dry matter found [START_REF] Michelot | Metal content profiles in mushrooms collected in primary forests of Latin America[END_REF] in Calocera viscosa. Mean contents of cobalt in mushrooms are comparable with foodstuffs like cereals, potatoe, and fruits, where these range from 0.01 to 0.1 mg kg -1 fresh matter [START_REF] Weigert | Metal loads of food of vegetable origin including mushrooms[END_REF]. Similar range was determined by Shiraishi (2004).

Copper

The mean copper content of all analysed samples 74.7 mg kg -1 dry matter (Table II) was the second highest value from all analysed metals, with maximum values 451 and 502 mg kg -1 dry matter found in Xerocomus chrysenteron. However, these two concentrations were identified by Grubbs' test like the outliers. Other high levels were observed within the genera Macrolepiota and Lycoperdon. Usual copper contents in mushrooms are in order of tens mg kg -1 dry matter. In accumulating species like Agaricus spp., Lycoperdon spp., Macrolepiota procera and M. rhacodes the contents could be between 100-300 mg kg -1 dry matter [START_REF] Wondratschek | Monitoring of heavy metals in soils by higher fungi[END_REF][START_REF] Alonso | The concentrations and bioconcentration factors of copper and zinc in edible mushrooms[END_REF], Kalač & Svoboda 2000). However, higher contents could be found in heavy polluted areas such us in the close vicinity of copper smelters. [START_REF] Svoboda | Concentration of mercury, cadmium, lead and copper in fruiting bodies of edible mushrooms in an emission area of a copper smelter and a mercury smelter[END_REF]. The average copper content in fruits and vegetables ranges between 0.6 and 1.0 mg kg -1 fresh material [START_REF] Weigert | Metal loads of food of vegetable origin including mushrooms[END_REF], which is consistent with findings of Shiraishi (2004).

Rubidium

The mean rubidium content of all analysed samples 174 mg kg -1 dry matter (Table II) have always been the highest from all analysed metals, with maximum value 3009 mg kg -1 dry matter found in Xerocomus chrysenteron. However, this extraordinary concentration was identified by Grubbs' test like an outlier. Above-average contents 355 and 351 mg kg -1 dry matter of rubidium were found in two available samples of Cantharellus cibarius and in the [START_REF] Hedrich | Short-time activation analysis of some Austrian mushrooms[END_REF] reported also the highest content of rubidium in a sample of C. cibarius 911 mg kg -1 dry matter. Řanda et al. (2005) observed the extraordinary high content of 1250 mg kg -1 dry matter in Hydnum repandum and the second highest values of 590 mg kg -1 dry matter was also found in C. cibarius. [START_REF] Tyler | Metals in sporophores of Basidiomycetes[END_REF] determined the highest contents of 1190 and 1070 mg kg -1 dry matter in H. repandum and C. cibarius, respectively and a high level in all species of Lactarius and in most samples of Amanita. [START_REF] Klán | Investigation of non-radioactive Rb, Cs, and radiocaesium in higher fungi[END_REF] besides high content in C. cibarius and in samples of the Boletaceae family reported the highest level in Lactarius rufus. [START_REF] Tyler | Accumulation and exclusion of metals in Collybia peronata and Amanita rubescens[END_REF] performed stepwise regression statistics for contents of twelve metals including of rubidium in Amanita rubescens and Collybia peronata and characteristics of their growing substrate. He concluded that metal concentration, acidity and certain physical properties of the substrates in most cases are of much less importance in regulating the uptake of metals by the two examined mushroom species than by the vascular plants. However, there is an exception in rubidium, since there is a relatively high positive correlation of its level between fruiting body and substrate and negative correlation between pH or degree of metal ion saturation. [START_REF] Kośla | Rubidium -a microelement[END_REF] mentioned the similar situation in plants, where its uptake is increasing with decreasing pH of substrate. Shiraishi (2004) found a high concentration of rubidium in the categories of nuts and seeds, potatoes, mushrooms, meats, and seaweeds (13.0, 7.46, 7.46, 5.21 and 4.14 mg kg -1 fresh matter, respectively).

Silver

For silver, the determined contents ranged very widely from values under the limit of quantification 0.015 mg kg -1 dry matter to maximum concentration 315 mg kg -1 dry matter in a sample of Lycoperdon perlatum. We did not observe any evident ability to accumulate silver in a certain taxonomic group like genus or family. However, in contrary to barium, cobalt and vanadium, silver contents are highly fluctuating with a high variation within the individual mushroom species. Overall RSD for silver calculated from all samples regardless of their taxonomic species was the highest (224 %) from all eleven eight investigated metals (Table II). Very similarly wide-ranging contents were reported by Falandysz et al. (1994a), Arugguete (1998), [START_REF] Siobud-Dorocant | Multivariate analysis of metal concentration profiles in mushrooms[END_REF] and Řanda et al. (2005). Nevertheless, it is very difficult to perform an objective conclusion about the accumulation of silver in certain mushrooms species. A higher number of samples would be thus useful for the comparison of silver-accumulation ability of certain mushrooms species. [START_REF] Hedrich | Short-time activation analysis of some Austrian mushrooms[END_REF] found a high bioaccumulation potency for silver in Agaricus augustus and Falandysz et al. (1994b) According to [START_REF] Petering | Silver[END_REF], silver is present in plants in the range of 0.006 to 0.28 mg kg -1 . In fungi and bacteria, they reported silver content of about 29 to 210 mg kg -1 , respectively. Aquatic plants tend to concentrate silver from their environments several hundredfold.

Thallium

A very high variance of content, similar as to that observed for silver, was also determined for thallium. This observation is consistent with findings from other authors. Mean thallium content of all analysed samples 0.045 mg kg -1 dry matter (Table II) was the lowest one among the determined elements. Usual levels of thallium in mushrooms have been in order of hundredth or maximally a few tenth mg kg -1 dry matter [START_REF] Seeger | Thallium in higher mushrooms[END_REF][START_REF] Bakken | Accumulation of radiocesium in fungi[END_REF][START_REF] Parisis | Antimony uptake and correlation with other mushrooms species[END_REF][START_REF] Yoshida | Accumulation of radiocesium and trace elements in mushrooms collected from Japanese forests[END_REF][START_REF] Falandysz | ICP/MS and ICP/AES elemental analysis (38 elements) of edible wild mushroom growing in Poland[END_REF][START_REF] Řanda | Trace elements in higher fungi (mushrooms) determined by activation analysis[END_REF]. No considerable inter-species differences in thallium concentration were observed. However, [START_REF] Seeger | Thallium in higher mushrooms[END_REF] and [START_REF] Yoshida | Accumulation of radiocesium and trace elements in mushrooms collected from Japanese forests[END_REF] mentioned a somewhat higher accumulation ability of Agaricus spp. Extraordinary high values 4.79 and 3.48 mg kg -1 dry matter were registered in Lactarius deliciosus and Suillus luteus, respectively, growing near a flotation waste reservoir serving a mining and metallurgical works [START_REF] Dmowski | Thallium contamination of selected plants and fungi in the vicinity of the Bolesław zinc smelter in Bukowno (S. Poland). Preliminary study[END_REF]). An absolutely maximum reported content of 5.5 mg kg -1 dry matter found [START_REF] Seeger | Thallium in higher mushrooms[END_REF] in inedible Peziza badia (Ascomycetes).

The usual mean thallium contents of cereals, fruits, and vegetables are about 0.05-0.1 mg kg -1 fresh material [START_REF] Weigert | Metal loads of food of vegetable origin including mushrooms[END_REF].

Vanadium

Toxic Amanita muscaria has been well-known vanadium accumulator since the 1930s. [START_REF] Koch | Occurrence of amavadin in mushrooms of Amanita species[END_REF] and Řanda et al. (2005) confirmed high accumulation ability of A. muscaria, with content in order of magnitude of hundreds mg kg -1 dry matter. Usual levels of vanadium in other species are much more lower -typically in order of tenth mg per kilogram of dry matter [START_REF] Hedrich | Short-time activation analysis of some Austrian mushrooms[END_REF][START_REF] Parisis | Antimony uptake and correlation with other mushrooms species[END_REF][START_REF] Valiulis | Metal accumulation in some fungi species growing in Lithuania[END_REF][START_REF] Vetter | Vanadium content of some common edible, wild mushroom species[END_REF]. Our results are in a good agreement with the literature data. According to [START_REF] Reilly | Metal Contamination of Food: Its Significance for Food Quality and Human Health[END_REF], levels in most foods appear to be low, between 1 and 30 mg kg -1 . Skimmed milk, lobster and some other sea foods, vegetable oils, certain vegetables, and some cereals are reported among the richest food source of vanadium [START_REF] Repinc | Determination of vanadium in biological and environmental samples by RNNA with emphasis on quality control[END_REF].

  in fruiting bodies of widely consumed mushroom species growing in an area known by an increased occurrence of serpentines and amphiboles.

  internal standardisation was used. The results were within limits of quantification for arsenic

  genera Boletus and Suillus.

  and A. bisporus.[START_REF] Byrne | Silver accumulation by Fungi[END_REF] determined the highest silver contents in samples of Agaricus spp. and above-average levels in genera Lycoperdon and Boletus.

Table I . Content of eight analysed elements (mg kg -1 dry matter) in mushroom samples.

 I

	Xerocomus badius		0.49	1.54	0.25	30.6	452	0.75	ND	0.41
			0.59	4.42	0.42	51.7	102	0.70	0.01	0.71
	Species		As 0.39	Ba 2.39	Co 0.26	Cu 53.0	Rb 316	Ag 2.24	Tl 0.20	V 0.09
	Cantharellus cibarius		0.48 0.82	6.68 0.71	1.22 0.09	50.4 32.9	355 294	ND 2.06	0.03 0.09	0.15 0.08
			0.31 0.87	2.62 0.87	0.47 0.03	34.1 61.1	351 344	0.07 2.25	0.02 0.02	0.37 0.09
	Armillaria mellea		0.01 0.53	0.20 3.07	0.02 0.40	16.2 65.1	158 510	2.53 63.6	ND 0.09	0.23 0.11
	Lepista nuda Xerocomus subtomentosus		3.07 0.43	1.44 1.26	0.17 0.04	91.6 17.2	3.51 112	0.61 ND	ND 0.03	0.37 0.08
	Marasmius oreades		55.5 0.49	2.98 0.99	0.09 0.32	50.1 31.0	77.0 152	ND 3.44	0.03 0.04	0.67 0.12
			2.35 0.23	5.15 0.44	1.20 0.59	104 24.2	8.14 42.0	3.51 2.49	0.02 0.01	1.16 0.06
	Amanita rubescens		0.69 0.53	0.68 1.72	0.49 0.11	44.8 15.1	67.8 87.3	ND 2.52	0.01 0.02	0.11 0.33
	Agaricus campestris Agaricus macrosporus Macrolepiota rhacodes F o r Xerocomus chrysenteron F o r	0.95 0.35 0.70 4.76 2.60 2.64 30.8 1.57 24.3 2.66 1.16 1.87	3.83 1.25 5.55 0.26 2.63 1.39 0.65 4.75 21.1 1.18 1.91 2.16	0.77 0.09 0.45 0.05 0.24 0.30 0.12 0.24 3.25 0.08 0.56 0.53	58.3 18.3 41.1 11.3 47.0 91.7 140 93.0 451 30.8 69.4 37.3	541 104 192 278 3.92 21.3 30.3 205 3009 82.9 81.3 279	11.2 2.50 0.06 6.33 17.0 23.7 6.67 7.27 3.00 ND 3.47 7.3	0.08 0.02 0.04 0.30 0.02 0.03 0.01 0.03 0.71 0.02 0.01 0.04	0.39 0.36 0.84 0.07 0.70 0.32 0.19 0.25 1.12 0.09 0.06 0.15
	Macrolepiota procera Russula cyanoxantha	30.2 8.61 2.57 2.07 2.84 4.68 1.45 4.48 P e e 0.57 0.81 0.64 0.73 0.60 3.66 2.98 2.03 r 1.75 1.14 1.12 0.53 5.09 9.17 P 0.21 1.29 0.27 2.50 e 0.13 1.62 0.31 1.72 0.28 5.58 e r	0.34 0.16 0.70 0.41 0.34 8.27 0.40 0.89 0.24 0.13 1.04 0.29 0.73 0.81 1.11 0.65	123 117 203 205 226 238 188 269 35.8 34.5 502 25.2 39.0 51.3 42.8 33.0	37.3 22.0 27.2 9.43 53.0 10.5 18.4 24.3 139 165 160 36.9 33.0 128 64.7 160	8.94 6.39 129 1.58 1.71 5.39 2.02 151 0.495 0.235 11.9 ND 0.38 1.60 119 0.33	0.01 ND 0.02 0.02 0.02 0.02 0.01 0.01 0.04 0.01 0.02 0.01 ND 0.01 ND 0.01	0.42 0.23 0.20 0.69 0.11 0.52 0.25 0.53 0.60 0.05 0.68 0.09 0.08 0.07 0.09 1.22
	Suillus grevillei Suillus granulatus Russula aeruginea Leccinum scabrum Boletus erythropus Boletus aestivalis Lycoperdon perlatum		3.21 0.16 0.40 1.02 0.20 0.41 0.10 0.22 1.21 1.44 0.79 0.86 1.85 3.48 0.51 3.02 2.97 3.68 3.61 27.6 4.32 1.40	1.83 0.38 0.31 10.2 R 0.12 0.12 0.06 1.02 e 3.10 0.66 5.37 1.81 2.89 0.73 R 1.15 0.69 e 0.64 0.15 0.55 0.09 1.41 0.06 0.55 0.06 1.24 0.10 1.32 0.46 2.17 0.28 v 25.1 14.8 37.4 60.6 38.3 53.6 64.0 38.9 22.0 29.6 24.0 23.7 25.2 33.6 60.4 i e w 795 130 318 248 90.6 148 65.9 211 179 173 206 308 341 318 67.2 2.86 0.21 177 11.9 1.02 0.42 132 16.9 3.11 0.07 108 11.3 1.05 0.21 202 22.1 0.72 0.07 220 12.9 v i e 2.57 0.98 53.9 25.5 1.28 0.09 23.0 237 w	0.77 0.40 1.67 61.2 0.36 0.53 8.00 0.93 2.02 0.30 1.82 1.87 7.36 8.42 0.62 151 15.6 1.94 10.2 3.18 5.24 2.95	0.01 0.01 0.03 0.01 0.02 0.01 ND 0.06 0.01 0.02 0.03 0.06 0.16 0.24 0.01 0.02 0.01 0.01 0.01 0.02 0.01 0.02	0.72 0.09 0.33 0.72 0.83 0.05 0.10 1.66 0.40 0.10 0.07 0.31 0.09 0.15 0.61 0.27 0.14 0.39 0.45 0.50 0.52 0.65
	1.96 1.40 5.32 1.48 2.62 1.68 6.03 4.66 8.14 Bold face type is used for marking of outlying values (Grubbs' test) 0.23 0.06 15.1 145 0.08 0.25 14.4 161 1.67 0.62 53.4 868 Boletus edulis 0.88 0.90 22.0 489 1.59 0.20 52.1 341 0.53 0.10 24.9 216 O n 7.79 40.9 15.1 6.84 13.5 3.99 l 6.93 0.49 310 22.7 75.0 Lycoperdon gigantea 0.82 0.08 41.3 1.88 10.6 5.02 2.09 252 11.2 315 O n l 2.17 0.79 0.16 36.1 304 9.92 y 0.19 0.11 0.64 0.04 0.05 0.11 0.01 0.01 0.02 0.07 y	0.04 0.04 0.16 0.13 0.11 0.39 0.29 1.07 0.15 0.14
			7.34	2.62	0.18	84.7	574	21.0	0.74	0.38
	Table I continued

Table II . Basic statistical characteristics of the trace element contents (mg kg -1 dry matter) of all analysed samples.

 II . number of samples; x ... mean value; S x ... standard deviation; RSD ... relative standard deviation Outlying values identified by Grubbs´ test were not used for statistical calculations.

		As	Ba	Co	Cu	Rb	Ag	Tl	V
	n	77	77	77	76	77	77	76	77
	x	3.35	2.15	0.45	74.7	174	14.4	0.05	0.34
	S x	6.19	2.02	0.52	70.6	179	32.3	0.09	0.29
	RSD (%)	185	94.3	115	94.5	103	224	194	87.1
	Median	1.45	1.41	0.28	47.0	130	2.95	0.02	0.25
	x min	0.01	0.08	0.02	14.4	1.88	ND	ND	0.04
	x max	30.8	10.2	3.25	310	868	151	0.64	1.22
	1 st quartile 3 rd quartile n ..F 0.49 3.07 o r 0.73 2.86	0.11 0.62	30.7 91.7	30.3 278	0.75 8.94	0.01 0.04	0.10 0.50
			P
			e
			e r
				R
					e
					v i e
						w
							O n l
								y

Table III . II. Statistical data of trace element contents (mg kg -1 dry matter) in mushroom species with at least five samples. Different letters in a column mean significant difference at P < 0.05. The letters are given in alphabetical order with increasing content of the metal. Outlying values identified by Grubbs´ test were not used for statistical calculations

 III Outlying values identified by Grubbs´ test were not used for statistical calculations.

	Page 17 of 17
		As	Ba	Co	Cu	Rb	Ag	Tl	V
	Macrolepiota procera (n = 6)
	x	3.02 a	1.77 a	0.55 a,b 221 c	23.8 a	48.6 a	0.01 a	0.38 a
	S x	1.30	1.33	0.24	29.3	16.0	71.4	0.01	0.23
	Median	2.71	1.38	0.41	215	21.4	3.71	0.02	0.38
	x min	1.45	0.60	0.34	188	9.43	1.58	0.01	0.11
	x max	4.68	3.66	0.89	269	53.0	151	0.02	0.69
	Boletus aestivalis (n = 6) x S x Median x min x max F o r 2.42 a 1.72 1.91 0.51 5.32	1.12 a 0.82 1.28 0.08 2.17	0.29 a 0.21 0.26 0.06 0.62	33.7 a 19.4 29.4 14.4 60.4	317 b 290 239 67.2 868	13.4 a 14.3 8.10 0.62 40.9	0.22 b 0.22 0.17 0.01 0.64	0.18 a 0.22 0.12 0.04 0.61
	Boletus edulis (n = 5; n = 4 for Tl) x 3.06 a P	1.28 a	0.31 a	44.0 a	385 b	11.0 a	0.07 a	0.18 a
	S x Median x min x max Xerocomus badius (n = 6)	2.44 2.17 e 0.85 0.88 1.48 0.53 7.34 2.62 e r 0.33 0.18 0.10 0.90	25.6 36.1 22.0 84.7	145 341 216 574	6.58 9.92 3.99 21.0	0.03 0.06 0.04 0.11	0.11 0.14 0.11 0.38
	x S x Median x min	0.61 a 0.19 0.56 0.39	2.17 a 1.42 1.97 R 0.24 a 0.16 0.26 0.71 0.03 e 49.1 a 14.4 52.3 30.6	336 b 142 330 102	11.9 a 25.3 2.15 0.70	0.07 a 0.08 0.06 ND	0.25 a 0.26 0.10 0.08
	x max Xerocomus subtomentosus (n = 6) 0.87 x 0.60 a S x 0.49	4.42 1.73 a 1.54	0.42 0.23 a 0.21 v 65.1 33.1 a 29.9 i e 510 117 a 56.0	63.6 3.04 a 2.37	0.20 0.02 a 0.01	0.71 0.21 a 0.14
	Median x min x max	0.46 0.23 1.57	1.25 0.44 4.75	0.17 0.04 0.59	21.2 15.1 93.0 w 108 42.0 205	2.51 ND 7.27	0.02 0.01 0.04	0.18 0.06 0.39
	Xerocomus chrysenteron (n = 7; n = 6 for Ba,Rb; n = 5 for Cu ) x 5.42 a 2.68 a 0.83 a 41.6 a S x 8.43 3.23 1.12 15.8 Median 1.87 1.55 0.53 35.8 x min 1.12 0.53 0.08 30.8 x max 24.3 9.17 3.25 69.4 Russula cyanoxantha (n=7)	151 a 72.5 149 81.3 279 O 3.77 a 0.02 a 4.40 0.01 3.00 0.02 ND 0.01 11.9 0.04 n l y	0.39 a 0.41 0.15 0.05 1.12
	x	0.26 a	3.03 a	0.87 b	40.4 a	94.6 a	17.5 a 0.01 a	0.44 a
	S x	0.09	1.78	0.48	9.91	52.2	44.9	0.01	0.48
	Median	0.27	2.50	0.73	39.0	90.6	0.38 0.01	0.10
	x min	0.13	1.29	0.29	25.2	33.0	ND	ND	0.07
	x max	0.41	5.58	1.81	53.6	160	119	0.02	1.22
	Lycoperdon perlatum (n=8)
	x	6.58 a 2.44 a	0.32 a	153 b	45.0 a	33.1 a 0.01 a	0.41 a
	S x	8.59	2.04	0.31	93.6	77.8	53.4	0.01	0.16
	Median	3.64	1.92	0.21	155	19.5	7.69 0.01	0.42
	x min	1.40	0.72	0.07	23.0	11.3	1.94 0.01	0.14
	x max	27.6	6.93	0.98	310	237	151	0.02	0.65

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Food Additives and Contaminants

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


Elke Anklam
email: fac@tandf.co.uk

Update on the Progress in Acrylamide and Furan Research

Presented during the EC-DG SANCO 1 /CIAA 2 organised Workshop "Acrylamide" (16/17 March 2006), and Joint EC-SANCO/EFSA 3 / EC-DG JRC 4 Workshop "Furan in Food" (19 May, 2006).

Acrylamide in food was discovered by Swedish researchers in April 2002, and raised significant concern due to its classification as a probable human carcinogen. After the initial announcement in Sweden, many researchers and research groups around the world immediately began investigations into acrylamide, at first focusing on the development of suitable analytical methods, and then determining the content in different foods for exposure estimates. Laboratories and food research institutes also conducted intensive studies on the formation pathways, and the link to the Maillard reaction was identified fairly rapidly by several independent research groups already in September 2002. The knowledge of precursors (asparagine and reducing sugars) opened the avenue to mitigation studies, resulting in many patents and peer-reviewed scientific papers elaborating on the different interventions and their degree of success.

Exceptional to the work around acrylamide is that from the very early stages all stakeholders convened to share their knowledge and ongoing work in an open and transparent manner. The European Food and Drink Federation (CIAA) established a Technical Acrylamide Expert Group in 2003 with the aim to share research initiatives and disseminate this knowledge by the fastest and most efficient way. This marked the birth of the "Acrylamide Toolbox", that today represents an up-to-date summary of the most promising avenues and achievements to reduce acrylamide levels in the concerned foods (potato products, bread & bakery wares, breakfast cereals, and coffee).

The Acrylamide Toolbox is updated on a continuous basis as the science progresses. The EC-DG SANCO/CIAA Workshop on Acrylamide held in March 2006 in Brussels was intended to present new findings from academic and industry research to be integrated into the Toolbox, addressing analytical aspects, formation pathways, mitigation (academic research and industry efforts), home cooking and catering, and risk assessment.

Working groups (WGs) were established to tackle common themes for each of the key food categories, i.e. potatoes, cereals and coffee, as well as "home cooking". The defined framework of the WGs comprised (a) the key knowledge and achievements to date (b) results of current studies and projects (c) future opportunities, gaps and constraints, and (d) future outlook.

The outcomes of the working groups as well as selected presentations of the invited experts are reported in this Special Issue. In terms of mitigation concepts, emphasis has so far clearly been on the manufacturing side, identifying several options at recipe and processing stages. Acrylamide can, however, also be "controlled" by lowering either the reducing sugar or asparagine content of the crops, and in this Issue important genetic/agronomic options at crop level are elaborated. Several risk management options can be considered, ranging from legal measures for sugars in potatoes, to the German signal level approach, that represents a voluntary scheme followed by manufacturers in Germany. A final summary of the acrylamide workshop has been compiled by Dr. David Lineback and follows on the editors foreword. 2 Key aspects of the Joint EC-DG SANCO/EFSA/ DG JRC Workshop "Furan in Food", held on 19th May 2006 in Brussels, are also presented. Furan and acrylamide have certain commonalities. Firstly, both are generated during the thermal treatment of food, and have consequently been a natural part of our diet for many thousands of years. Secondly, both compounds are a potential concern with regard to human health, and furan is considered a possible human carcinogen. The occurrence of low amounts of furan in some foods has been known for several decades, but its possible risk to health was first raised by the US FDA in Spring 2004 after their investigations showed its presence in many different products, especially those that are canned or jarred.

Reliable analytical methods are required to gather occurrence data of furan in food that would then allow for an exposure assessment. Several papers on "state-of-the-art" analytical methodologies as well as the occurrence of furan in different foods comprise the second part of this Special Issue. A mechanistic paper on the formation of furan from ascorbic acid is also presented. However, several routes of formation of furan can be envisaged in foods, and such fundamental studies are pivotal to understanding the determinants in the furan formation pathway(s) and their contribution to the furan concentration in the final products.

In summary, this Special Issue of Food Additives and Contaminants captures the key advances in research pertaining to the formation and mitigation of acrylamide in food. For furan, it highlights the challenges in developing reliable methodologies and the gaps to be addressed prior to establishing a reliable exposure assessment. This is, however, only a fragment of the ongoing research into acrylamide and furan on a global scale, reflecting the intensive efforts of all stakeholders to ensure that the foods we consume are safe.

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1 European Commission, Directorate General Health and Consumer Protection 2 European Food and Drink Federation 3 European Food Safety Authority 4 European Commission, Directorate General Joint Research Centre Page 1

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Belén Patiño

Angel Medina

Domenech Mirian

González-Jaén Mª

Misericordia Jiménez

Vázquez Covadonga

Ángel Medina

Mirian Doménech

Mª Teresa González-Jaén

Covadonga Vázquez

PCR diagnosis

Keywords: Penicilliun brevicompactum, Mycophenolic acid, PCR-diagnosis, 15 grapes, LC-ion trap-mass spectrometry. 16

come

Introduction 1

Penicillium brevicompactum Dierckx is a ubiquitous fungus which has been 2 isolated from air (Wu et al., 2003), apple production, cheese [START_REF] Kure | Mould contamination in production of semi-14 hard cheese[END_REF]; 3 [START_REF] Serra | Black Aspergillus species 17 as ochratoxin A producers in Portuguese wine grapes[END_REF] cashew [START_REF] Freire | Mycoflora and mycotoxins of 19 Brazilian cashew kernels[END_REF], pepper and nuts (Freire et al., 4 2000) and grapes (Barkai-Golan, 1980; [START_REF] Abrunhosa | Mycotoxin 2 production from fungi isolated from grapes[END_REF][START_REF] Serra | Black Aspergillus species 17 as ochratoxin A producers in Portuguese wine grapes[END_REF]; 5 [START_REF] Serra | Mycotoxin-producing and other fungi isolated 14 from grapes for wine production, with particular emphasis on ochratoxin A[END_REF]. Some P. brevicompactum strains from diverse commodities 6 have been described either as mycophenolic acid, brevianamide A, (Paterson et 7 al., 1990;[START_REF] Rundberget | The presence of Penicillium and 8 Penicillium mycotoxins in food wastes[END_REF] or patulin producers [START_REF] Paterson | Novel use 9 of the isoepoxydon dihydrogenase gene probe of the patulin metabolic 10 pathway and chromatography to test penicillia isolates from apple 11 production systems for the potential to contaminate apple juice with patulin[END_REF]. 8

Mycophenolic acid-producing strains of P. brevicompactum have been isolated 9 from different origins (Vinokurova et al., 2005) such as ginger (Overy & Frisvad, 10 2005), but no reports of mycophenolic acid-producing strains from grapes have 11 been published so far. The mycophenolic acid has been found to affect many 12 physiological processes. It shows antibiotic, anti-tumour, anti-psoriasis and 13

immunosuppressive features, and it is currently used in Medicine (Bentely, 2000). 14

Additionally, it might be relevant in secondary mycotoxicosis (bacterial infection 15 caused by intake of immunosuppressive mycotoxins). Since P. brevicompactum 16 produces diverse toxic compounds such as mycophenolic acid, brevianamide A 17 and patulin and it is an ubiquitous fungus, it should be considered as a threat to 18 commodity system [START_REF] Paterson | 24 terverticillate penicilia using biochemical, physiological and morphological 2 features III. An evaluation of pectinase and amylase isoenzymes for species 3 characterization[END_REF]. criteria, such as micromorphology, macromorphology and colours produced in the 2 mycelium or diffused into the medium [START_REF] Raper | Manual of the penicillia[END_REF]Ramírez, 1982;Pitt, 3 1991;Samson & Frisvad, 2004). However many authors have shown that some of 4 these characteristics (i.e. colony texture) are highly variable and therefore do not 5 always allow unambiguous classification [START_REF] Frisvad | Classification of terverticillate penicillia based on 4 profiles of mycotoxins and other secondary metabolites[END_REF]. As an 6 alternative to secondary metabolites profiling [START_REF] Frisvad | Classification of terverticillate penicillia based on 4 profiles of mycotoxins and other secondary metabolites[END_REF], 7 detection of specific enzyme activity and isoenzyme electrophoretic patterns 8 (Frisvad, 1981;Paterson et al., 1989), have been used. More recently genotypic 9 characterization (RFLPs, rRNA genes sequence, RAPDs) has proven its utility 10 [START_REF] Dupont | Molecular tools for identification of 16 Penicillium starter cultures used in food industry[END_REF][START_REF] Pianzzola | Characterization of Penicillium isolates 20 associated with blue mold on apple in Uruguay[END_REF]. However, although phylogenetic 11 studies of P. brevicompactum and related species have been recently developed 12 [START_REF] Peterson | Phylogenetic analysis of Penicillium species based on ITS 14 and LSU-RNA nucleotide sequences[END_REF]

Materials and Methods 2

Fungal Isolates and culture conditions 3

All the isolates used in this study, along with their sources, are given in Table I. 4

The isolates were obtained in our laboratory from grapes collected from Spanish The ITS1-5.8S-ITS2 region of the nuclear ribosomal DNA from several Penicillium 13 strains, including the P. brevicompactum strains isolated from grapes, was 14 amplified using primers ITS1 (5´ TCCGTAGGTGAACCTGCGG 3´) and ITS4 (5´ 15 TCCTCCGCTTATTGATATG 3´) described elsewhere (White et al., 1990). The 16 PCR reaction was performed in an Eppendorf Mastercycler Gradient (Eppendorf, 17

Hamburg, Germany) using 10 pg-10 ng of genomic DNA. The amplification 18 I was used for PCR amplification 1 using the primers BRE1/BRE2 in the conditions described above. The genomic 2 DNA from all the strains was tested for PCR amplification using primers ITS1 and 3 ITS4 according to [START_REF] Henry | Identification of Aspergillus species using 11 internal transcribed spacer regions 1 and 2[END_REF] using the same master mix used for specific 4 PCR amplification except primers. 5 6

Reagents and standards for mycotoxin assays 7

Mycophenolic acid standards were purchased from Sigma (Sigma-Aldrich, 8

Alcobendas, Spain). Acetonitrile, chloroform, acetic acid, methanol (all LC grade) 9

and phosphoric acid (85%, A.R.) were from J.T. Baker (Deventer, Holland). Pure 10 water was obtained from a Milli-Q Plus apparatus (Millipore, Billerica, Ma., USA) 11 and was used when water was required. Yeast extract and sucrose were 12 purchased from Panreac (Montcada i Reixac, Barcelona, Spain) 13 14

Mycotoxin production 15

One Penicillium brevicompactum strain (CECT 2316) and two isolates of this 16 species (A and U) from Spanish grapes (Table I) were tested for mycophenolic 17 acid production using Yeast Extract Sucrose (YES) broth (Singh et Two specific primers to detect P. brevicompactum were designed on the basis of 2 the alignment of ITS1-5.8S-ITS2 regions performed with a representative sample 3 of Penicillium species available in our laboratory, including A and U isolates from 4 grapes and closely related species: P. oslonii and P. biourgeianum (Peterson et al., 5 2004), the latter considered a synonym of P. bialowiezense according to Frisvad & 6 Samson (2004). Additionally, 26 sequences of P. brevicompactum from the 7

GenBank were also used. Figure 1 The ability of P. brevicompactum strains A, U and CECT 2361 to produce 1 mycophenolic acid was tested by chromatographic analysis. The chromatographic 2 analysis of culture media using the Phenomenex Gemini ® C 18 column showed the 3 presence of a peak with a retention time of 5.4 min that was tentatively identified as 4 mycophenolic acid. The identity of mycophenolic acid was confirmed by the UV 5 spectrum that showed absorption maxima at 226.2 nm, 250.9 nm and 305.2 nm, 6 which matched to the absorption maxima shown by the standard. The 7 mycophenolic acid peak was found in the cultures of the three P. brevicompactum 8 strains (Figure 3). We have developed the first PCR detection assay specific for P. brevicompactum. 2

The specificity of this assay has been successfully tested in a wide and diverse 3 sample of P. brevicompactum strains associated to different substrates, other 4

Penicillium species and other fungal species often associated to grapes and 5 related commodities. This PCR method represents a good alternative to 6 conventional identification methods, especially difficult in the case of terverticillate 7 penicillia [START_REF] Paterson | 24 terverticillate penicilia using biochemical, physiological and morphological 2 features III. An evaluation of pectinase and amylase isoenzymes for species 3 characterization[END_REF], and does not require highly trained specialists or 8 expensive equipment [START_REF] Paterson | 24 terverticillate penicilia using biochemical, physiological and morphological 2 features III. An evaluation of pectinase and amylase isoenzymes for species 3 characterization[END_REF]. The PCR assay described in this 9 work also represents an advantage in terms of time of analysis and specificity in 10 comparison with the conventional methods and will facilitate detection of this 11 species in future surveys of agro-products. 12 13

The assay developed in this study targeted the multicopy ITS1-5.8S-ITS2 region in 14 order to improve the sensitivity of the detection. The detection limit of ITS 15 amplification product, defined as the clearly visible product on agarose gels 16 containing ethidium bromide, has been estimated between 1 to 10 pg of DNA 17 template in Fusarium [START_REF] Bluhm | Multiplex Polymerase 12 Chain Reaction Assay for the Differential Detection of Trichothecene-and 13 Fumonisin-Producing Species of Fusarium in Cornmeal[END_REF]. The sensitivity of our PCR assay based 18 on ITS sequences would be in the same order of magnitude and, therefore, more 19 sensitive than primers based on single copy gene, estimated between 0.1 and 1 ng 20 of DNA template per reaction [START_REF] Bluhm | Multiplex Polymerase 12 Chain Reaction Assay for the Differential Detection of Trichothecene-and 13 Fumonisin-Producing Species of Fusarium in Cornmeal[END_REF]. 21

22

Those features are very important since P. brevicompactum is a ubiquitous and 23 cosmopolitan fungi that has been isolated from widespread substrates. 24 from different origins, pointed out that 60 % of the strains tested were able to 5 produce mycophenolic acid. In the present work, we also report production of 6 mycophenolic acid by P. brevicompactum isolated from grapes for the first time. 7

The high incidence of P. brevicompactum strains capable to produce mycophenolic 8 acid stresses the relevance of this species in terms of food safety. The occurrence of P. brevicompactum strains in grapes and their ability to produce 1 mycophenolic acid reported here increases the number and importance of the 2 dietary foods and derivates susceptible to be contaminated by this metabolite and, 3 therefore, we suggest that its inclusion in future survey programs should be 4 considered by food safety organisations as well as more exhaustive studies on the 5 effects of mycophenolic acid on the immune response in humans (Overy & Frisvad, 6 2005).

19 20 4

 194 Penicillium brevicompactum Dierckx (Section Coronata) is included in the 21 important group of terverticillate penicillia which also includes the Sections 22 Roqueforti, Chrysogena, Penicillium, Digitata and Viridicata. The classification of 23 this group has been in a state of change during the history of the genus. 24 Taxonomic keys to identify Penicillium spp. are based primarily on morphological 1

7 1 ( 6 7 8 Genomic

 7168 program used was described by[START_REF] Henry | Identification of Aspergillus species using 11 internal transcribed spacer regions 1 and 2[END_REF] and it is as follows: 1 cycle of4 19 min 30 s at 95 ºC, 40 cycles of 30 s at 95 ºC (denaturalization), 30 s at 50 ºC 20 (annealing), 60 s at 72 ºC (extension) and finally 1 cycle of 3 min at 72 ºC. The 21 PCR products generated were purified using the High Pure PCR Product 22 Purification Kit (Roche, Mannheim, Germany) and cloned into the cloning vector 23 pCR4-TOPO with the TOPO TA Cloning Kit for Sequencing (Invitrogen, U.K.). 24Plasmids were sequenced in both directions using the ABI PRISM DNA Sequencer Applied Biosystems, Foster City, USA) according to the manufacturer's 2 instructions in the Genomic Unit of the University Complutense of Madrid (Spain). 3The ITS1-5.8S-ITS2 sequences of the P. brevicompactum strains U, A, and CECT 4 2361 were deposited in the EMBL database under accession numbers AJ877044, 5 AJ877045, and AJ877046, respectively.Development and evaluation of species-specific PCR primers. 8The ITS1-5.8S-ITS2 DNA sequences of P. brevicompactum and related species 9 obtained in our laboratory and retrieved from data bases (GenBank and EMBL) 10 were analysed and aligned by Clustal method using Dnastar (Lasergene, Madison, 11 Wis., USA). Primers were designed on the basis of these DNA alignments. The 12 primers obtained were named BRE1 (5´GCTGCCGGGGGACATCTGTCC 3') and 13 BRE2 (5´GGCGCCGGCCAGTCCT ACAAAG 3'). The PCR amplification protocol 14 used was as follows: 1 cycle of 4 min 30 s at 95 ºC, 25 cycles of 30 s at 95 ºC, 20 s 15 at 69.5 ºC, 30 s at 72 ºC and finally 1 cycle of 5 min at 72 ºC. All amplification 16 reactions were carried out in volumes of 25 µL containing 3 µL (10 pg-10 ng) of 17 template DNA, 1.50 µL of each primer (20 µM), 2.5 µL of 10 x PCR buffer, 1 µL of 18 MgCl 2 (50 mM), 0.25 µL of dNTPs (100 mM) and 0.2 µL of Taq DNA polymerase (5 19 U/µL) supplied by the manufacturer (Ecogen, Barcelona, Spain). PCR products 20 were detected in 2% agarose ethidium bromide gels in TAE 1 X buffer (Tris-acetate 21 40 mM and EDTA 1.0 mM). The DNA ladder "Real escala nº2" (Durviz, Valencia, 22 Spain) was used as molecular size marker. DNA from the strains indicated in Table

9 7 8 11 12 20 (Food 10 µm 4 ( 5 8 9 16 17

 9711201045816 al., 1991; Overy 18 & Frisvad, 2005) and grape must:water (20:80 v/v). Grape must:water (20:80 v/v) 19was used to find out whether this substrate had any effect on the fungal 20 development and/or production of the studied mycotoxins, as two isolates were 21 from grapes grown in Spain.Liquid medium (50 mL) was poured into 100 mL Erlenmeyer flasks. The flasks 1 were plugged with cotton, covered with aluminium foil and autoclaved at 115 ºC for 2 30 min. Sterilised liquid media were inoculated with 1 mL of a spore suspension 3 made in aqueous Tween 80 (0.5%) having 10 5 spores/mL and then they were 4 incubated at 25 ºC for 2 weeks. Three flasks per isolate were assayed for 5 mycotoxin accumulation. Controls consisted of non-inoculated sterilized liquid 6 media incubated under the same conditions.Mycotoxin extraction, clean-up and analysis 9Extraction of mycophenolic acid and HPLC analysis was carried out according to 10 the method described by[START_REF] Medina | Bee pollen, a 16 substrate that stimulates ochratoxin A production by Aspergillus ochraceus 17 Wilh[END_REF] with slight modifications.Five ml of culture medium were mixed with 500 µL of a 0.1 M solution of 13 phosphoric acid, extracted with 2 x 5 mL chloroform and, after evaporation of the 14 organic phase, the residue was dissolved in 0.5 mL of mobile phase 15 (acetonitrile:water:acetic acid 47:52:1 v/v/v). 16 17Chromatographic equipment 18The LC system consisted of a Waters 600 pump equipped with a Waters 717 19 automatic injector and a Waters 474 scanning fluorescence detector (FLD) Waters). A Waters 996 photodiode array detector (DAD) was usually set after the 21 fluorescence detector. Millennium 32® software ver. 4.0 (Waters) was used to 22 control the chromatograph and to process the signals. Separations were performed 23 on a stainless steel LiChrospher 100 C 18 reversed-phase column (250 x 4 mm, 5 24 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk particle size) (Agilent Technologies, Waldbronn, Germany) connected to a 1 guard column (4 x 4 mm, 5 µm particle size) filled with the same phase. The 2 column was kept at 40 °C. Alternatively, separation was performed on a 3 Phenomenex Gemini ® C 18 column (150 x 4.6 mm, 5 µm particle size) Phenomenex, Torrance, Ca., USA) connected to a Phenomenex Gemini ® C 18 SecurityGuard cartridge (4 x 3 mm, 5 µm particle size). The column was kept at 35 6 °C. The flow rate was 1 mL/min and the mobile phase was acetonitrile:water:acetic 7 acid (47:52:1 v/v/v). Mycophenolic acid was quantified by DAD at 254 nm.Toxin confirmation by LC-MS 10LC-ion trap-mass spectrometry was used to unambiguously confirm the possible 11 presence of mycophenolic acid. The analysis was carried out on an Agilent 1100 12 liquid chromatograph (Agilent Technologies), equipped with a Zorbax SB-C 18 13 column (150 x 4.6 mm, 5 µm particle size) (Agilent Technologies) and coupled to a 14 Bruker Esquire 3000 Plus ion trap mass spectrometer (Bruker Instruments, 15 Billerica, Ma., USA).For the analysis of mycophenolic acid the mobile phase was 18 acetonitrile:water:acetic acid (50:49:1, v/v/v). The ionization method was 19 electrospray ionization (ESI) in positive mode by using the following ionization 20 source parameters: N 2 nebulizer gas at 10 psi, dry gas at 5 L/min, dry temperature 21 at 250 o C, and capillary voltage at 4.5 kV.

11 12[insert figure 1 about here] 13 14 21 [

 111321 shows ITS1-5.8S-ITS2 sequence of P. 8 brevicompactum isolate A (AJ877045) indicating the position of primers BRE-1 and 9 BRE-2. Primer BRE1 was located within the ITS1, at the position +77, and the 10 primer BRE2 at the position +445 (within the ITS2).All the Penicillium strains listed in TableIwere tested for amplification using primer 15 pair BRE1/BRE2. A single fragment of about 390 bp was amplified only when 16 genomic DNA from P. brevicompactum strains was used. No product was 17 observed with genomic DNA from other Penicillium isolates, nor in the case of 18 species belonging to other genera (Figure2). Control amplifications of the genomic 19 DNA with primers ITS1 and ITS4 were positive for all the strains analysed. 20

9 10[ 11 12 18 19

 91118 Insert figure 3 about here]The presence of mycophenolic acid in cultures was confirmed by LC-MS based on 13 the protonated molecule [M+H] + and the most abundant product ion [(M+H) + + 14 H 2 O)] whose m/z ratios were 321 and 338, respectively. The confirmation was also 15 made using the second fragmentation of ion 321 (LC-MS-MS), obtaining in both 16 standard and sample an ion with m/z 303 corresponding to the fragmentation 17[(M+H) + -H 2 O].Production of mycophenolic acid was compared in two different media, YES and 20 grape must:water (20:80 v/v). The results obtained did not show significant 21 differences between both media (

  P. brevicompactum strains isolated from commodities other 1 than grapes were capable to produce mycophenolic acid. Some authors have 2 described production of this metabolite by all the P. brevicompactum isolates 3 tested[START_REF] Overy | Mycotoxin production and postharvest storage rot of 19 ginger (Zingiber officinale) by Penicillium brevicompactum[END_REF] while Vinokurova et al. (2005), studying isolates 4

9 10 16 17

 916 The levels produced by our isolates grown under the conditions used in this work 11were relatively low if compared with levels reported by other authors who found in 12 vitro production of this compound around 300 mg/L (Vinokurova et al., 2005). In 13 their work, however, these authors suggested a strong influence of culture media 14 on the mycophenolic acid production. Therefore, more studies are necessary to 15propose an optimised culture medium to evaluate this metabolite.The cultivation of P. brevicompactum collected from grapes on a grape-like media 18 (grape must: water, 20:80 v/v) did not result in enhanced levels of mycophenolic 19 acid production in comparison with YES medium. However, more studies are 20 needed to confirm that production of mycophenolic acid by P. brevicompactum 21 isolates would not be positively influenced by grape substrate.

7 8 10 11 " 1 2005FoodFigure 1 .Figure 2 .Figure 3 .

 8111123 Figure 1. Sequence of ITS1-5.8S-ITS2 of Penicillium brevicompactum isolate A

  ; Peterson et al 2004) a specific method to detect P.

	1
		F o
		r
		P
		e e r
	13 14 15 16	brevicompactum based on PCR has not been reported. The objectives of this work were to analyse the ability to produce mycophenolic R e v i e w
	17 18	acid by P. brevicompactum strains isolated from grapes and to design specific primers, based on ITS region, and the corresponding PCR assay to detect this O n
	19	species.	l y
		5

Sabouraud liquid medium (Scharlau Chemie, Barcelona, Spain). 16 Cultures were inoculated with mycelial disks cut from the plates and incubated at 17

  Collections of Micro-organisms) and the ARS culture collection (NRRL, United 8

	1	by Pitt (1999). The strains were inoculated in Czapek yeast extract agar (CYA),
	2	incubated for 7 days at 5 ºC, 26 ºC and 37 ºC, in Malt extract agar (MEA) at 26 ºC
	3	and in 25% glycerol nitrate agar (G25N) at 26 ºC. All identifications were confirmed
	4	by amplification of 28S rRNA gene (598 bp) and subsequent sequencing. The
	5 5	sequences showed 100% similarity to P. brevicompactum strains from the NCBI
	6 7 6 7 8	vineyards or from culture collections: the Spanish Type Culture Collection (Spain), and MycoBank (CBS) data bases. F o the Agroindustrial fungi & yeasts collection (MUCL, The Belgian Co-ordinated r DNA extraction and PCR amplification.
	9 10 11 9 10 11	States Department of Agriculture). [Insert table I about here] P Genomic DNA of the strains listed in Table I was obtained using either the genomic e DNA Extraction Kit (Genomix, Talent, Trieste, Italy), according to the e r manufacturer's instructions, or the protocol described by Querol et al. (1992).
	12 13 14 15 18 12	Cultures were maintained on potato dextrose-agar (PDA) medium (Scharlau Chemie, Barcelona, Spain) at 4º C and stored as spore suspension in 15 % glycerol at -80 ºC. The isolates were cultured in 100 mL Erlenmeyer flasks O n containing 20 mL 25 ºC, 150 rpm. Mycelia from 2-days-old cultures were harvested by filtration w e i v e R
	19	through Whatman paper nº 1 and kept at -80 ºC for DNA isolation. l y
	20
	21	Fungal Identification
	22	The strains A and U isolated from Spanish grapes (Tempranillo variety) were
	23	identified by the Spanish Type Culture Collection following the protocol described
		6

Table II

 II

	22	).
	23	[insert table II about here]

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	1	Discussion
		F o
		r
		P
		e e r
		R e v i e w
		O n
		l y
		13

Table II :

 II Mycophenolic acid production by strains of P. brevicompactum isolated from grapes grown in Spain. RSD: relative standard deviation.

					Mycophenolic acid (µg/ml)
		Isolate YES medium RSD % Grape must:water	RSD %
	3,00						(20:80 v/v)
	2,50	2316		148.878	7.30		90.910			11.37
	1,50 2,00	F o r A 130.528 U 183.256 F o r F o r	7.38 1.70	115.309 134.350			7.18 4.81
			P P P
				e e e
		1,00	2,00	3,00	4,00 e 5,00 r 6,00 e r e r	Minutes 7,00 8,00	9,00	10,00	11,00	12,00	13,00	14,00	15,00
						R R R
						e e e
						v i e v i e v i e
							w w w
								O n l O n l O n l
										y y y

Microbiology 41: 568-579.

Environmental Microbiology 58: 2948-2953.

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Acknowledgements

9