Alternariol Methyl Ether Research Articles

A new alternariol glucoside from fungus Alternaria alternate cib-137

A new secondary metabolite, 2-O-methylalternariol 4-O-β-[4-methoxyl-glucopyranoside] (1), together with four known compounds alternariol methyl ether (2), altenuene (3), isoaltenuene (4) and 2-(2′S-hydroxypropyl)-5-methyl-7-hydroxychromone (5), was isolated from the fungus Alternaria alternate cib-137. Its structure was elucidated on the basis of spectroscopic data. Compounds 3 and 4 demonstrated moderate activity against Staphylococcus aureus.

Quantitation of multiple mycotoxins and cyanogenic glucosides in cassava samples from Tanzania and Rwanda by an LC-MS/MS-based multi-toxin method

A multi-mycotoxin method based on liquid chromatography/tandem mass spectrometry (LC-MS/MS) was used for a mycotoxin survey in 627 samples of processed cassava collected from different districts across Tanzania and Rwanda after the method performance for this matrix had been determined. Matrix effects as well as extraction efficiencies were found to be similar to most other previously investigated matrices with the exception of distinct matrix effects in the negative ionisation mode for early eluting compounds. Limits of detection were far below the regulatory limits set in the European Union for other types of commodities. Relative standard deviations were generally lower than 10% as determined by replicates spiked on two concentration levels. The sample-to-sample variation of the apparent recoveries was determined for 15 individually spiked samples during three different analytical sequences. The related standard deviation was found to be lower than 15% for most of the investigated compounds, thus confirming the applicability of the method for quantitative analysis. The occurrence of regulated mycotoxins was lower than 10% (with the exception of zearalenone) and the related limits were exceeded only in few samples, which suggests that cassava is a comparatively safe commodity as regards mycotoxins. The most prevalent fungal metabolites were emodin, kojic acid, beauvericin, tryptophol, 3-nitropropionic acid, equisetin, alternariol methylether, monocerin, brevianamide F, tenuazonic acid, zearalenone, chrysophanol, monilifomin, enniatins, apicidin and macrosporin. The related concentrations exceeded 1 mg kg–1 only in few cases. However, extremely high levels of cyanogenic plant toxins, which had been previously added to the method, were observed in few samples, pointing out the need for improved post-harvest management to decrease the levels of these compounds.

Distortion from planarity in arenes produced by internal rotation of one single hydroxyl hydrogen: The case of alternariol

Alternariol (AOH) and alternariol methyl ether (AME) are compounds toxic to farm animals and humans produced by ubiquitous fungi of the Alternaria genus. Information on chemical and physical properties of these compounds is rather scarce although their X-ray structures are known. AOH and AME are composed of three aromatic rings with three hydroxyl groups. However, the crystal structure of AOH is nonplanar whereas that of AME which differs only in one methyl group substituting one hydroxyl hydrogen is planar. By means of quantum calculations we find that the internal rotation of that single hydrogen affects the structure of AOH producing deviations from planarity near 5°. We also show that although quantum calculations predict energy differences about 5kcal/mol between planar and perpendicular conformations of that hydrogen, its rotation has no noticeable effects on electron density properties that could indicate modifications of aromaticity features. Based upon these results, we suggest an explanation to the nonplanarity of the AOH crystal solely in terms of the spatial arrangement of molecules in the crystal interconnected through a network of hydrogen bonds involving rotatable hydroxyl hydrogens that produce distortions from planarity.

Co-occurrence of multiple mycotoxins in dry chilli (Capsicum annum L.) samples from the markets of Sri Lanka and Belgium

In this study a multi-mycotoxin method was applied to screen seventeen chemically divergent mycotoxins from dry chilli samples (Capsicum annum L.) collected from the markets in Sri Lanka (n = 86) and Belgium (n = 35) using a high performance liquid chromatography tandem mass spectrometer. In addition to aflatoxins (<LOQ–687 μg/kg) and ochratoxin A (OTA; <LOQ–282 μg/kg), the chilli samples were also found to be contaminated with sterigmatocystin (STERIG; <LOQ–32 μg/kg), fumonisin B2 (FB2; <LOQ–87 μg/kg), citrinin (<LOQ–2.1 mg/kg) and alternariol methyl ether (70 and 222 μg/kg). Aflatoxin B1 (AFB1) was the predominant mycotoxin contaminating almost 77% of the Sri Lankan samples. While OTA was found in 41% of the samples notably, 38% of them were contaminated with STERIG. Overall, 87% and 63% of the Sri Lankan and Belgian samples, respectively were contaminated at least with one mycotoxin. Remarkably, 67% of the Sri Lankan samples exceeded EU maximum level (ML) of 5 μg/kg for AFB1 and 44% of the samples exceeded the EU ML of 10 μg/kg for total aflatoxins. One third of the Sri Lankan chillies were contaminated with more than three different mycotoxins. Co-occurrence of different mycotoxins, AFB1–OTA (36%), AFB1–STERIG (28%), OTA–AFB1–STERIG (17%) and AFB1–FB2 (14%) was found in different forms of chillies. Higher frequency of mycotoxins co-occurrence found in the processed chillies such as flakes and powder could be due to the fraudulent usage of low quality grade chilli pods for processing. Noticeably, 9 of the 11 positive chilli samples from Belgium exceeded the EU ML for AFB1.

Alternaria mycotoxins associated with grape berries in vitro and in situ

The aim of this study was to assess the production of secondary metabolites of Alternaria species isolated from grape berries and determined the occurrence of Alternaria mycotoxins in vitro and in dried berries. Direct morphological examination on different media was used for identification and HPLC/MS method for quantification of Alternaria mycotoxins. We isolated A. alternata and A. tenuissima and randomly selected strains in vitro and in dried berries. In vitro we identified the production of Alternaria metabolites, included mycotoxins: alternariol, alternariol methylether, tenuazonic acid. Beside that one in dry berries were in measurable concentrations: macrosporin A, tentoxin and altenuete (I, II).

Occurrence and pathology of mycotoxins in commercial parrot feeds

Mycotoxins are toxic secondary metabolites of fungi. Animal feeds can be easily infected by fungi during production and storage, resulting in mycotoxin contamination. This study was performed to evaluate the possible health risks of mycotoxin-contaminated feed for cockatiels. The occurrence of mycotoxins in commercial parrot feeds (5 seed mixes and 5 pelleted feeds) was investigated by liquid chromatography tandem mass spectrometry. The following 12 mycotoxins were detected: zearalenone, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, fusarenon X, aflatoxin B1, sterigmatocystin, alternariol, alternariol methylether, fumonisin B1, fumonisin B3, and ochratoxin A. Zearalenone was the most prevalent. Pathological effects after 21 days feeding mycotoxin-contaminated diets were examined in anin vivo trial with 3 groups of 5 cockatiels: group 1 (control) was fed a non-contaminated pelleted feed; group 2 was fed a pelleted feed containing zearalenone, deoxynivalenol, 15-acetyldeoxynivalenol, and fumonisins; and group 3 was fed a pelleted feed containing fumonisins. Average body weight gain and relative organ weight were not significantly different between the treatment groups and the control group. Apoptosis of renal tubular cells, diarrhoea, reduced appetite, enlargement of liver, kidney and proventriculus were occasionally observed in the birds from groups 2 and 3. In summary, contamination with mycotoxins is common in parrot feeds. The mycotoxin levels did not reach toxic levels, but might pose a potential threat to some sensitive cockatiels.

A limited survey of mycotoxins in traditional maize based opaque beers in Malawi

A total of 9 traditional maize based opaque beers were randomly collected from tribal (chewa) rituals and commercial village brewers from Lilongwe and Dowa districts, Malawi in August 2012 for evaluation of mycotoxins using a multi-mycotoxin method based on liquid chromatography-tandem mass spectrometry. Samples were analyzed for aflatoxins B1 (AFB1), AFB2, AFG1, AFG2, fumonisin B1 (FB1), FB2, FB3, zearalenone, ochratoxin A, nivalenol, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, neosolaniol, fusarenon-X diacetoxyscirpenol, HT-2 toxin, T-2 toxin, alternariol, alternariol methyl ether, and sterigmatocystin. All beer samples were positive for fumonisins with FB1+FB2 and FB1+FB2+FB3 content of 1745±1294μg/kg (mean±std. dev.) and 1898±1405μg/kg respectively. With exception of one beer sample, all the beers also contained aflatoxins at a mean concentration of 90±95μg/kg. Consumption of 1.0–6.0L of the traditional beer from this study translates to daily FB1+FB2 exposure of 29–174μg/kg body weight (bw)/day (i.e. >provisional maximum tolerable daily intake of 2μg/kg bw/day set by the Joint FAO/WHO Expert Committee on Food Additives) and aflatoxin exposure of 1.5–9.0μg/kg bw/day for a 60kg adult. The study demonstrated that the consumption of the maize based traditional home-made beer in Malawi can significantly enhance aflatoxins and fumonisin dietary exposure among beer consumers.

Mycotoxins in corn and wheat silage in Israel

Silage is an important feed source for intensive dairy herds worldwide. Fungal growth and mycotoxin production before and during silage storage is a well-known phenomenon, resulting in reduced nutritional value and a possible risk factor for animal health. With this in mind, a survey was conducted to determine for the first time the occurrence of mycotoxins in corn and wheat silage in Israel. A total of 30 corn and wheat silage samples were collected from many sources and analysed using a multi-mycotoxin method based on LC-MS/MS. Most mycotoxins recorded in the present study have not been reported before in Israel. Overall, 23 mycotoxins were found in corn silage; while wheat silage showed a similar pattern of mycotoxin occurrence comprising 20 mycotoxins. The most common post-harvest mycotoxins produced by the Penicillium roqueforti complex were not found in any tested samples, indicative of high-quality preparation and use of silage. Moreover, none of the European Union-regulated mycotoxins – aflatoxin B1, ochratoxin, T-2 toxin, diacetoxyscirpenol and deoxynivalenol – were found above their limits of detection (LODs). The Alternaria mycotoxins – macrosporin, tentoxin and alternariol methyl ether – were highly prevalent in both corn and wheat silage (>80%), but at low concentrations. The most prominent (>80%) Fusarium mycotoxins in corn silage were fusaric acid, fumonisins, beauvericin, monilifomin, equisetin, zearalenone and enniatins, whereas in wheat silage only beauvericin, zearalenone and enniatins occurred in more than 80% of the samples. The high prevalence and concentration of fusaric acid (mean = 765 µg kg–1) in Israeli corn silage indicates that this may be the toxin of highest potential concern to dairy cow performance. However, more data from different harvest years and seasons are needed in order to establish a more precise evaluation of the mycotoxin burden in Israeli silage.

アルタナリア属菌と穀類黒目粒のマイコトキシン

Mycotoxins are secondary metabolites produced by several fungal species and represent a great concern for the economical and healthy implications on food and feed chain. Cereals are the primary source of human diet, wheat being the third most produced grain worldwide. Although Fusarium still represents the main source of mycotoxin contamination of wheat, in recent years, due also to evident climate changes that influence agricultural environment, other mycotoxingenic fungi have been pointed out as important wheat contaminants. Among these a disease called Black Point, caused by Alternaria spp., is increasing it importance as re-emerging risk. Diseases and mycotoxins (alternariol, altenuene, alternariol methyl-ether and tenuazolic acid) associated with Alternaria infection have been reported in several countries suggesting to deepen the knowledge about this genus. This paper summarizes the recent findings on wheat contamination by Alternaria spp and their related toxins.

Occurrence of alternariol and alternariol monomethyl ether in beverages from the Entre Rios Province market, Argentina

One hundred and eighty five samples of red, white and rosé wines and different juices purchased in Entre Rios, Argentina, were analyzed for the Alternaria mycotoxins alternariol (AOH) and alternariol methyl ether (AME). White wines were analyzed after removal of alcohol by a nitrogen stream and concentrated. AOH in red wines was cleaned up by solid-phase extraction columns in series (octadecyl and amino propyl modified silica) and AME quantified directly on the sample. The juices were filtered and concentrated, and then all sample extracts were quantified by high performance liquid chromatography with photodiode array detector that allows confirmation through UV spectra. Method validation revealed a good sensitivity with adequate LOD and LOQ for AME and less sensitivity for AOH (i.e. white wine: AME 0.8 and 1.4ng/mL, AOH 2 and 3.3ng/mL; red wine: AME 0.1 and 0.2ng/mL, AOH 4.5 and 7.5ng/mL; apple juice: AME 1.7 and 2.8ng/mL, AOH 5 and 9ng/mL; other juices: AME 2.0 and 3.1ng/mL, AOH 6 and 10ng/mL). Recoveries in all cases were greater than 80%. Four of 53 white wine samples were contaminated with AOH with a maximum level of 18ng/mL, 6 of 56 samples of red wine had a maximum of 13ng/mL, and 3 of 68 samples of juices had traces of AOH. AME was less frequently detected than AOH, and the LOD and LOQ for AME are smaller than for AOH. Only three samples of white wine and one of red wine were contaminated, but in only one white wine sample (AME 225ng/mL) did the toxin level exceed the LOQ.

Alternaria toxins: Altertoxin II is a much stronger mutagen and DNA strand breaking mycotoxin than alternariol and its methyl ether in cultured mammalian cells

Altertoxin II (ATX II) is one of the several mycotoxins produced by Alternaria fungi. It has a perylene quinone structure and is highly mutagenic in Ames Salmonella typhimurium, but its mutagenicity in mammalian cells has not been studied before. Here we report that ATX II is a potent mutagen in cultured Chinese hamster V79 cells, inducing a concentration-dependent increase of mutations at the hypoxanthine guanine phosphoribosyltransferase gene locus at concentrations similar to that of the established mutagen 4-quinoline-N-oxide. Thus, ATX II is at least 50-times more potent as a mutagen than the common Alternaria toxins alternariol (AOH) and alternariol methyl ether (AME). In contrast to AOH and AME, ATX II does not affect the cell cycle of V79 cells. ATX II also causes DNA strand breaks in V79 cells, with a potency again exceeding that of AOH and AME. The high mutagenic and DNA strand breaking activity of ATX II raises the question of whether this Alternaria toxin poses a risk for public health, and warrants studies on the occurrence of ATX II and other perylene quinone-type mycotoxins in food and feed.

Toxigenic Alternaria species from Argentinean blueberries

Blueberries are traditionally consumed in North America, some European countries and Japan. In Argentina, the blueberry crop is profitable because production starts in November, when the northern hemisphere lacks fresh fruit. Fungal contaminants can grow and produce mycotoxins in fresh fruit. The aims of this work were to identify the main genera of the mycobiota of blueberries grown in Argentina and to determine the toxicogenic potential, pathogenicity and host specificity of the species isolated. The genus Alternaria was the main component of the blueberry mycobiota (95%); minor proportions of Phoma spp. (4%) and Penicillium spp. (1%) were also isolated. According to their sporulation patterns, 127 Alternaria isolates belonged to the Alternaria tenuissima species-group, 5 to the Alternaria alternata species-group and 2 to the Alternaria arborescens species-group. The last mentioned species-group was not isolated at 5°C. Of the 134 isolates, 61% were toxicogenic in autoclaved rice; 97% of these produced alternariol (AOH) in a range from 0.14 to 119.18mg/kg, 95% produced alternariol methylether (AME) in a range from 1.23 to 901.74mg/kg and 65% produced tenuazonic acid (TA) in a range from 0.13 to 2778mg/kg. Fifty two isolates co-produced the three mycotoxins. According to the size of the lesion that they caused on blueberries, the isolates were classified as slightly pathogenic, moderately pathogenic and very pathogenic. No significant differences in pathogenicity were found on different blueberry varieties.In this work, high incidence and toxicogenic potential of the Alternaria isolates from blueberries were demonstrated. Thus, more studies should be done to evaluate the health risk posed by the presence of the Alternaria toxins in blueberries and in the manufactured by-products.

Effects of temperature and incubation period on production of toxic metabolites by Alternaria radicina and A. alternata

The production of toxic metabolites by four isolates of &lt;i&gt;Alternaria radicina&lt;/i&gt; and two isolates of &lt;i&gt;A. alternata&lt;/i&gt; in rice grains and carrot discs at 1, 10 and 20&lt;sup&gt;°&lt;/sup&gt;C was investigated. Incubation lasted 21 and 35 days or 14 and 28 days for rice grains and carrot discs, respectively. Accumulation of toxins in inoculated carrot roots stored for 24 weeks and in inoculated dried carrots stored for 48 weeks was also determined. It was found that &lt;i&gt;A. radicina&lt;/i&gt; produced radicinin (RAD) and &lt;i&gt;epi&lt;/i&gt;-radicinol (&lt;i&gt;epi&lt;/i&gt;-ROH), whereas tenuazonic acid (TeA), altertoxin I (ATX I), alternariol (AOH) and alternariol methyl ether (AME) were produced by &lt;i&gt;A. alternata&lt;/i&gt;. Although the isolates tested were capable of producing toxins in rice grains at 1&lt;sup&gt;°&lt;/sup&gt;C, none of them was detected in carrot discs. Accumulation of &lt;i&gt;epi&lt;/i&gt;-ROH was observed in carrot roots stored for 24 weeks, whereas decreased amounts of RAD and &lt;i&gt;epi&lt;/i&gt;-ROH were observed in dried carrots stored for 48 weeks. No &lt;i&gt;A. alternata&lt;/i&gt; toxins were detected in stored carrot roots, whereas trace amounts of AOH were recorded in dried carrots after 32 and 48 weeks of storage.

Mouse hepatoma cell lines differing in aryl hydrocarbon receptor-mediated signaling have different activities for glucuronidation

For studies on the aryl hydrocarbon receptor (AhR)-dependent toxicity of the mycotoxins alternariol (AOH) and alternariol methyl ether (AME), three mouse hepatoma (Hepa-1) cell lines with intact and with compromised AhR signaling were compared with respect to their activities for hydroxylation, methylation, and glucuronidation. Whereas the activities of cytochrome P450-mediated monooxygenase and catechol-O-methyl transferase were very low and did not differ between the three cell lines, a pronounced difference was observed for UDP-glucuronosyl transferase activity, which was much higher in Hepa-1c1c4 than in c1c7 and c1c12 cells. In all three cell types, the rate of glucuronidation of AOH was about four times higher than that of AME. Whereas AME caused a concentration-dependent G2/M arrest in each cell line, AOH arrested Hepa-1c1c7 and c1c12 cells but not c1c4 cells. However, Hepa-1c1c4 cells were arrested by AOH when β-glucuronidase was added to the incubation medium in order to reverse the formation of AOH glucuronides. We conclude that the failure of AOH to cause cell cycle inhibition in Hepa-1c1c4 cells is due to its efficient glucuronidation. The considerable UDP-glucuronosyl transferase activity of Hepa-1c1c4 cells should be taken into account when other compounds are studied in this cell line. Moreover, we demonstrate that differences in glucuronide formation between cell types can be overcome by the addition of β-glucuronidase to the cell culture medium.

The Alternaria mycotoxins alternariol and alternariol methyl ether induce cytochrome P450 1A1 and apoptosis in murine hepatoma cells dependent on the aryl hydrocarbon receptor

The Alternaria mycotoxins alternariol (AOH) and alternariol methyl ether (AME) are potential carcinogens. As planar compounds, AOH and AME are preferentially metabolized by cytochrome P450 (CYP) 1A1 and 1A2. The most prominent regulator of CYP1A1 is the dimeric transcription factor complex AhR/ARNT, which is activated by planar ligands. Therefore, we studied the activation of AhR/ARNT by AOH and AME and monitored CYP1A1 induction in murine hepatoma cells (Hepa-1c1c7). Indeed, AOH and AME enhanced the levels of CYP1A1 in Hepa-1c1c7 cells but not in cells with inactivated AhR (Hepa-1c1c12) or ARNT (Hepa-1c1c4). AOH and AME did not increase the production of reactive oxygen species but reduced cell counts in Hepa-1c1c7 cells after 24 and 48 h. This effect, however, was independent of AhR/ARNT. At 48 h, AOH and AME increased apoptosis dependent on AhR and ARNT. In conclusion, AOH and AME are novel inducers of the AhR/ARNT pathway, which mediates induction of CYP1A1 and apoptosis and might thereby contribute to the toxicity of these mycotoxins.

PCR-based assay for the detection of Alternaria species and correlation with HPLC determination of altenuene, alternariol and alternariol monomethyl ether production in tomato products

Alternaria spp. contamination and subsequent production of mycotoxins is a common problem in vegetable crops. Identification of Alternaria species by traditional methods requires specific skills and may not detect toxigenic moulds inactivated by food processing. By using molecular methods such as PCR the detection of Alternaria spp. becomes possible directly from the food or feed samples. In this study, a PCR method based on the Internal Transcribed Spacer (ITS) genetic marker has been used for detection of Alternaria spp. in raw and processed commercial tomato samples. Occurrence of altenuene, alternariol and alternariol methyl ether in the samples was analysed by high-performance liquid chromatography (HPLC) in order to assess the ability of the PCR assay to identify tomato samples containing Alternaria mycotoxins. The PCR assay revealed the presence of Alternaria spp. DNA in 41 out of 90 commercial samples (45.6%), while HPLC detected at least one of the Alternaria mycotoxins within 31 of the PCR positive samples. Detection of Alternaria DNA correlated well with the presence of the analysed Alternaria mycotoxins, indicating that the PCR protocol developed in this work for detection of Alternaria spp. DNA could be used as an indirect marker of the presence of Alternaria mycotoxins in raw and processed tomato products.

Alternaria species associated with 'moldy heart' on peaches in Argentina

'Moldy heart' caused by Alternaria spp has been previously reported in apples in Argentina. However, according to our knowledge this is the first report of Alternaria spp in 'moldy heart' affected peaches in this country. Direct isolation of the pathogen was performed on Dichloran-Chloramphenicol-Malt Extract-Agar. Seven strains were isolated and identified as A. alternata and one as A. tenuissima. Their toxicogenic potential (alternariol (AOH), alternariol methyl ether (AME) and tenuazonic acid (TA) production) was determined. The toxins were extracted with chloroform and determined by TLC. All strains produced AOH and AME while six of them were TA producers. The fungicides tested on agar were captan and mancozeb. At 2.5g/L (commercial concentration) both fungicides inhibited the germination of spores. Captan completely inhibited mycelial growth. The effect of the fungicide on toxin production was variable according to fungicide type, doses, strains and toxins considered.

Occurrence of Mycotoxins in Feed as Analyzed by a Multi-Mycotoxin LC-MS/MS Method

Crops used for animal feed can be easily contaminated by fungi during growth, harvest, or storage, resulting in the occurrence of mycotoxins. Because animal feed plays an important role in the food safety chain, the European Commission has set maximum levels for aflatoxin B1 and recommended maximum levels for deoxynivalenol, zearalenone, ochratoxin A, and the sum of fumonisin B1 and B2. A multimycotoxin LC-MS/MS method was developed, validated according to Commission Decision 2002/657/EC and EN ISO 17025 accredited for the simultaneous detection of 23 mycotoxins (aflatoxin-B1, aflatoxin-B2, aflatoxin-G1, aflatoxin-G2, ochratoxin A, deoxynivalenol, zearalenone, fumonisin B1, fumonisin B2, fumonisin B3, T2-toxin, HT2-toxin, nivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, diacetoxyscirpenol, fusarenon-X, neosolaniol, altenuene, alternariol, alternariol methyl ether, roquefortine-C, and sterigmatocystin) in feed. The decision limits of the multimycotoxin method varied from 0.7 to 60.6 microg/kg. The apparent recovery and the results of the precision study fulfilled the performance criteria as set in Commission Decision 2002/657/EC. The analysis of three different feed matrices (sow feed, wheat, and maize) provided a good basis for the evaluation of the toxin exposure in animal production. In total, 67 samples out of 82 (82%) were contaminated; type B-trichothecenes and fumonisins occurred most often. The majority of the infected feed samples (75%) were contaminated with more than one type of mycotoxin.

LC-MS/MS multi-analyte method for mycotoxin determination in food supplements

A multi-analyte method for the liquid chromatography-tandem mass spectrometric determination of mycotoxins in food supplements is presented. The analytes included A and B trichothecenes (nivalenol, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, neosolaniol, fusarenon-X, diacetoxyscirpenol, HT-2 toxin and T-2 toxin), aflatoxins (aflatoxin-B1, aflatoxin-B2, aflatoxin-G1 and aflatoxin-G2), Alternaria toxins (alternariol, alternariol methyl ether and altenuene), fumonisins (fumonisin-B1, fumonisin-B2 and fumonisin-B3), ochratoxin A, zearalenone, beauvericin and sterigmatocystin. Optimization of the simultaneous extraction of these toxins and the sample pretreatment procedure, as well as method validation were performed on maca (Lepidium meyenii) food supplements. The results indicated that the solvent mixture ethyl acetate/formic acid (95:5, v/v) was the best compromise for the extraction of the analytes from food supplements. Liquid–liquid partition with n-hexane was applied as partial clean-up step to remove excess of co-extracted non-polar components. Further clean-up was performed on Oasis HLB™ cartridges. Samples were analysed using an Acquity UPLC system coupled to a Micromass Quattro Micro triple quadrupole mass spectrometer equipped with an electrospray interface operated in the positive-ion mode. Limits of detection and quantification were in the range of 0.3–30 ng g−1 and 1–100 ng g−1, respectively. Recovery yields were above 60% for most of the analytes, except for nivalenol, sterigmatocystine and the fumonisins. The method showed good precision and trueness. Analysis of different food supplements such as soy (Glycine max) isoflavones, St John's wort ( Hypericum perforatum), garlic (Allium sativum), Ginkgo biloba, and black radish (Raphanus niger) demonstrated the general applicability of the method. Due to different matrix effects observed in different food supplement samples, the standard addition approach was applied to perform correct quantitative analysis. In 56 out of 62 samples analysed, none of the 23 mycotoxins investigated was detected. Positive samples contained at least one of the toxins fumonisin-B1, fumonisin-B2, fumonisin-B3 and ochratoxin A.

Stable Isotope Dilution Assays of Alternariol and Alternariol Monomethyl Ether in Beverages

Stable isotope dilution assays (SIDAs) for the determination of the most important mycotoxins of the black mold Alternaria, namely, alternariol and alternariol monomethyl ether, have been developed. For this purpose, deuterated alternariol and alternariol methyl ether were synthesized by palladium catalyzed protium-deuterium exchange from the unlabeled toxins. Reaction conditions were chosen in such a manner that the formation of the [(2)H(4)]-isotopologues was favored. The synthesized products were characterized by LC-MS, NMR, and UV-spectroscopy. On the basis of the use of [(2)H(4)]-alternariol and [(2)H(4)]-alternariol methyl ether as internal standards, SIDAs were developed and applied to the determination of alternariol and alternariol methyl ether in beverages using LC-MS/MS. Method validation revealed a high sensitivity, i.e., low limits of detection (alternariol, 0.03 microg/kg; alternariol methyl ether, 0.01 microg/kg) and limits of quantitation (alternariol, 0.09 microg/kg; alternariol methyl ether, 0.03 microg/kg), respectively. Recovery from spiked apple juice was 100.5 +/- 3.4% for alternariol (range 0.1-1 microg/kg) and 107.3 +/- 1.6% for alternariol methyl ether (range 0.05-0.5 microg/kg). Interassay precision (expressed as coefficient of variation, CEV) for alternariol was 4.0% (7.82 +/- 0.31 microg/kg; vegetable juice, naturally contaminated) and 4.6% (1.04 +/- 0.05 microg/kg; grape juice, naturally contaminated). For alternariol methyl ether, a CEV of 2.3% (0.79 +/- 0.02 microg/kg; vegetable juice, naturally contaminated) was obtained. Analysis of fruit juices showed low contamination with alternariol and alternariol methyl ether in general, but higher values of both toxins were found in wine and vegetable juices. The values for alternariol were higher than those for alternariol methyl ether in nearly any case. However, the developed SIDA has proven to be optimally suited for further studies on alternariol and alternariol methyl ether content in food samples to obtain further insight into possible health hazards for the consumer.