Horst Klaffke

Proposal of a comprehensive definition of modified and other forms of mycotoxins including “masked” mycotoxins

As the term “masked mycotoxins” encompasses only conjugated mycotoxins generated by plants and no other possible forms of mycotoxins and their modifications, we hereby propose for all these forms a systematic definition consisting of four hierarchic levels. The highest level differentiates the free and unmodified forms of mycotoxins from those being matrix-associated and from those being modified in their chemical structure. The following lower levels further differentiate, in particular, “modified mycotoxins” into “biologically modified” and “chemically modified” with all variations of metabolites of the former and dividing the latter into “thermally formed” and “non-thermally formed” ones. To harmonize future scientific wording and subsequent legislation, we suggest that the term “modified mycotoxins” should be used in the future and the term “masked mycotoxins” to be kept for the fraction of biologically modified mycotoxins that were conjugated by plants.

Human CYP2E1 mediates the formation of glycidamide from acrylamide

Regarding the cancer risk assessment of acrylamide (AA) it is of basic interest to know, as to what amount of the absorbed AA is metabolized to glycidamide (GA) in humans, compared to what has been observed in laboratory animals. GA is suspected of being the ultimate carcinogenic metabolite of AA. From experiments with CYP2E1-deficient mice it can be concluded that AA is metabolized to GA primarily by CYP2E1. We therefore examined whether CYP2E1 is involved in GA formation in non-rodent species with the focus on humans by using human CYP2E1 supersomes™, marmoset and human liver microsomes and in addition, genetically engineered V79 cells expressing human CYP2E1 (V79h2E1 cells). Special emphasis was placed on the analytical detection of GA, which was performed by gas chromatography/mass spectrometry. The results show that AA is metabolized to GA in human CYP2E1 supersomes™, in marmoset and human liver microsomes as well as in V79h2E1 cells. The activity of GA formation is highest in supersomes™; in human liver it is somewhat higher than in marmoset liver. A monoclonal CYP2E1 human selective antibody (MAB-2E1) and diethyldithiocarbamate (DDC) were used as specific inhibitors of CYP2E1. The generation of GA could be inhibited by MAB-2E1 to about 80% in V79h2E1 cells and to about 90% in human and marmoset liver microsomes. Also DDC led to an inhibition of about 95%. In conclusion, AA is metabolized to GA by human CYP2E1. Overall, the present work describes (1) the application and refinement of a sensitive methodology in order to determine low amounts of GA, (2) the applicability of genetically modified V79 cell lines in order to investigate specific questions concerning metabolism and (3) the involvement, for the first time, of human CYP2E1 in the formation of GA from AA. Further studies will compare the activities of GA formation in genetically engineered V79 cells expressing CYP2E1 from different species.

A basic tool for risk assessment : A new method for the analysis of ergot alkaloids in rye and selected rye products

As a basis for the collection of occurrence and exposure data of ergot alkaloids in food, an HPLC method coupled with fluorimetric detection (HPLC-FLD) for the determination of 12 pharmacologically active ergot alkaloids in rye and rye products was developed. Samples were extracted with a mixture of ethyl acetate, methanol, and aqueous ammonia, followed by centrifugation and purification by solid phase filtration (SPF) with basic alumina. After solvent adjustment, the samples were analyzed by HPLC-FLD using a phenyl-hexyl-column. Recoveries for five major alkaloids were between 89.3% (ergotamine) and 99.8% (alpha-ergokryptine) with a maximum LOQ of 3.3 microg/kg (ergometrine). Precision expressed as RSD ranged from 2.8% (ergocristine) to 12.4% (alpha-ergokryptine) for repeatability, and from 6.5% (ergocornine) to 14.9% (ergotamine) for within-laboratory reproducibility, respectively. In a survey of 39 rye product samples, ergocristine and ergotamine were found to be the major alkaloids in commercially available rye products with contents of 127 microg/kg (ergocristine), and 134 microg/kg (ergotamine) in rye flour, and 152.5 and 117.8 microg/kg in coarse meal, respectively.

Determination of ergot alkaloids in rye and rye flour

An effective and timesaving analytical method was developed for the determination of 12 ergot alkaloids (ergometrine, ergotamine, ergocristine, α-ergokryptine, ergosine, ergocornine, and their respective -inine isomers) in rye and rye flour. Samples were extracted with dichloromethane/ethyl acetate/methanol/aqueous ammonia (25%) (50/25/5/1, v/v/v/v), and extracts were purified using a basic alumina column. The eluate was dried in the nitrogen stream and redissolved in acetonitrile/ ammonia carbamate-buffer (0.2 g/1), (1/1, v/v), and injected into an HPLC-FLD system (λEx 330 nm, λEm 415 nm), using the same mixture as mobile phase and a Phenyl-Hexyl column. Detection limits for the individual compounds ranged from 0.01 μg/kg to 0.5 μg/kg. In sample material spiked with a mixture of these compounds at two different levels (13 μg/kg and 27 μg/kg per compound), mean (n=5) recoveries were at 101% (sr 6.4%) and 89% (sr 3.1%), respectively.

Development of a high performance liquid chromatography tandem mass spectrometry based analysis for the simultaneous quantification of various Alternaria toxins in wine, vegetable juices and fruit juices.

An analytical method based on high performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS) detection for the simultaneous quantification of 12 Alternaria toxins in wine, vegetable juices and fruit juices was developed. Excellent chromatographic performance was demonstrated for tenuazonic acid (TeA) in a multi-analyte method. This comprehensive study is also the first to report the determination of TeA, alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN) and altenuene (ALT), altertoxin I (ATX-I), altertoxin II (ATX-II), altenuisol (ATL), iso-altenuene (isoALT), altenuic acid III (AA-III) and the AAL toxins TB1 und TB2 in samples from the German market. Several types of HPLC columns were tested for the liquid chromatographic separation of the toxins of interest that widely differ in their polarities. The focus was on gaining suitable retention while avoiding derivatization steps especially for TeA and AA-III. Three atmospheric pressure ionization techniques used with liquid chromatography (electrospray, chemical and photo ionization) were tested to obtain the best selectivity and sensitivity. Samples were diluted with sodium hydrogen carbonate buffer and extracted on a diatomaceous earth solid phase extraction cartridge. Method validation was carried out by using tomato juice, citrus juice and white wine as blank matrices. Limits of detection ranged from 0.10 to 0.59μgL(-1) and limits of quantification ranged from 0.4-3.1μgL(-1) depending on the toxin and matrix. Recoveries were around 100±9% for all toxins except stemphyltoxin III (STTX-III) and altenusin (ALS) due to instability during sample clean up. Matrix-induced effects leading to ion suppression especially for ATX-I, ATX-II and AA-III were investigated. Relative standard deviations of repeatability (RSDr) and intermediate reproducibility (RSDR) were ≤9.3 and ≤17.1, respectively, for the toxins in different matrices at levels of 5 and 30μgL(-1). Finally, 103 commercially obtained wine and juice samples from the German market in 2015 were analysed. TeA was found most frequently (68% of all analysed samples) in concentrations of up to 60.0μgL(-1). AOH, AME and TEN were detected in fewer samples (37%, 16% and 30%) at lower concentrations of up to 8.2, 1.5 and 10.3μgL(-1), respectively. AA-III and ATL were detected for the first time in 3% and 17% of food all samples, in concentrations of up to 6.0μgL(-1) and 5.9μgL(-1), respectively.

Spotlight on the Underdogs-An Analysis of Underrepresented Alternaria Mycotoxins Formed Depending on Varying Substrate, Time and Temperature Conditions.

Alternaria (A.) is a genus of widespread fungi capable of producing numerous, possibly health-endangering Alternaria toxins (ATs), which are usually not the focus of attention. The formation of ATs depends on the species and complex interactions of various environmental factors and is not fully understood. In this study the influence of temperature (7 °C, 25 °C), substrate (rice, wheat kernels) and incubation time (4, 7, and 14 days) on the production of thirteen ATs and three sulfoconjugated ATs by three different Alternaria isolates from the species groups A. tenuissima and A. infectoria was determined. High-performance liquid chromatography coupled with tandem mass spectrometry was used for quantification. Under nearly all conditions, tenuazonic acid was the most extensively produced toxin. At 25 °C and with increasing incubation time all toxins were formed in high amounts by the two A. tenuissima strains on both substrates with comparable mycotoxin profiles. However, for some of the toxins, stagnation or a decrease in production was observed from day 7 to 14. As opposed to the A. tenuissima strains, the A. infectoria strain only produced low amounts of ATs, but high concentrations of stemphyltoxin III. The results provide an essential insight into the quantitative in vitro AT formation under different environmental conditions, potentially transferable to different field and storage conditions.