Benedikt Warth

Quantitation of Mycotoxins in Food and Feed from Burkina Faso and Mozambique Using a Modern LC-MS/MS Multitoxin Method

In this study an LC-MS/MS multitoxin method covering a total of 247 fungal and bacterial metabolites was applied to the analysis of different foods and feedstuffs from Burkina Faso and Mozambique. Overall, 63 metabolites were determined in 122 samples of mainly maize and groundnuts and a few samples of sorghum, millet, rice, wheat, soy, dried fruits, other processed foods and animal feeds. Aflatoxin B(1) was observed more frequently in maize (Burkina Faso, 50% incidence, median = 23.6 μg/kg; Mozambique, 46% incidence, median = 69.9 μg/kg) than in groundnuts (Burkina Faso, 22% incidence, median = 10.5 μg/kg; Mozambique, 14% incidence, median = 3.4 μg/kg). Fumonisin B(1) concentrations in maize were higher in Mozambique (92% incidence, median = 869 μg/kg) than in Burkina Faso (81% incidence, median = 269 μg/kg). In addition, ochratoxin A, zearalenone, deoxynivalenol, nivalenol, and other less reported mycotoxins such as citrinin, alternariol, cyclopiazonic acid, sterigmatocystin, moniliformin, beauvericin, and enniatins were detected. Up to 28 toxic fungal metabolites were quantitated in a single sample, emphasizing the great variety of mycotoxin coexposure. Most mycotoxins have not been reported before in either country.

Assessment of human deoxynivalenol exposure using an LC–MS/MS based biomarker method

The Fusarium toxin deoxynivalenol (DON) is one of the most abundant mycotoxins worldwide and poses many adverse health effects to human and animals. Consequently, regulatory limits and a provisional maximum tolerable daily intake (PMTDI) for this important type B-trichothecene were assigned. We conducted a pilot survey to investigate the level of DON exposure in Austrian adults by measurements of DON and its glucuronide conjugates (DON-GlcAs), as biomarkers of exposure, in first morning urine. The average concentration of total DON (free DON+DON-GlcAs) was estimated to be 20.4±2.4 μg L⁻¹ (max. 63 μg L⁻¹). Surprisingly, we found that one third of the volunteers (n=27) exceeded the established PMTDI when consuming regular diet. DON-GlcAs were directly quantified by LC-MS/MS and the results were compared with indirect quantification after enzymatic hydrolysis and confirmed the suitability of the direct method. Moreover, we investigated the in vivo metabolism of DON in humans and were able to determine two closely eluting DON-GlcAs in naturally contaminated urine samples for the first time. In contrast to previous findings we have tentatively identified DON-15-glucuronide as a major DON metabolite in human urine based on the analysis of these samples. About 75% of total glucuronides were derived from this metabolite while DON-3-glucuronide accounted for approximately 25%. The reported new findings clearly demonstrate the great potential of suitable biomarkers to critically assess exposure of humans and animals to DON.

New insights into the human metabolism of the Fusarium mycotoxins deoxynivalenol and zearalenone

This study reports on the detailed investigation of human deoxynivalenol (DON) and zearalenone (ZEN) in vivo metabolism through the analysis of urine samples obtained from one volunteer following a naturally contaminated diet containing 138μg DON and 10μg ZEN over a period of four days. Based on the mycotoxin intake and the concentrations of mycotoxin conjugates in urine, a mass balance was established. The average rates of DON excretion and glucuronidation were determined to be 68 and 76%, respectively. The investigation of formed glucuronides revealed DON-15-glucuronide as main conjugation product besides DON-3-glucuronide. Furthermore, for the first time in human urine a third DON-glucuronide was detected and the fate of ingested masked DON forms (3-acetyl-DON and DON-3-glucoside) was preliminary assessed. The mean excretion rate of ZEN was determined to be 9.4%. ZEN was mainly present in its glucuronide form and in some samples ZEN-14-glucuronide was directly determined 3-10h after exposure. For the first time concrete figures have become available for the excretion pattern of DON and ZEN-glucuronides throughout a day, the comparison of total DON in 24h and first morning urine samples and the urinary excretion rate of total ZEN in humans following exposure through naturally contaminated food. Therefore, valuable preliminary information has been obtained through the chosen experimental approach although the study involved only one single individual and needs to be confirmed in larger monitoring studies. The presented experiment contributes to a better understanding of human DON and ZEN in vivo metabolism and thereby supports advanced exposure and risk assessment to increase food safety and examine the relationship between these mycotoxins and potentially associated chronic diseases in the future.

Development and validation of a rapid multi‐biomarker liquid chromatography/tandem mass spectrometry method to assess human exposure to mycotoxins

RATIONALE Mycotoxins regularly occur in food worldwide and pose serious health risks to consumers. Since individuals can be exposed to a variety of these toxic secondary metabolites of fungi at the same time, there is a demand for proper analytical methods to assess human exposure by suitable biomarkers. METHODS This study reports on the development of a liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for the quantitative measurement of 15 mycotoxins and key metabolites in human urine using polarity switching. Deoxynivalenol (DON), DON-3-O-glucuronide, DON-15-O-glucuronide (D15GlcA), de-epoxy DON, nivalenol (NIV), T-2 toxin, HT-2 toxin, zearalenone, zearalenone-14-O-glucuronide, α- and β-zearalenol, fumonisins B(1) and B(2) (FB(1), FB(2)), ochratoxin A (OTA) and aflatoxin M(1) (AFM(1)) were determined without the need for any cleanup using a rapid and simple dilute and shoot approach. RESULTS Validation was performed in the range of 0.005-40 µg L(-1) depending on the analyte and expected urinary concentration levels. Apparent recoveries between 78 and 119% and interday precisions of 2-17% relative standard deviation (RSD) were achieved. The applicability of the method was demonstrated by the analysis of urine samples obtained from Cameroon. In naturally contaminated urine samples up to six biomarkers of exposure (AFM(1), DON, D15GlcA, NIV, FB(1), and OTA) were detected simultaneously. CONCLUSIONS We conclude that the developed LC/MS/MS method is well suited to quantify multiple mycotoxin biomarkers in human urine down to the sub-ppb range within 18 min and without any prior cleanup. The co-occurrence of several mycotoxins in the investigated samples clearly emphasizes the great potential and importance of this method to assess exposure of humans and animals to naturally occurring mycotoxins.

Mycotoxin exposure in rural residents in northern Nigeria: a pilot study using multi-urinary biomarkers.

A pilot, cross-sectional, correlational study was conducted in eight rural communities in northern Nigeria to investigate mycotoxin exposures in 120 volunteers (19 children, 20 adolescents and 81 adults) using a modern LC-MS/MS based multi-biomarker approach. First morning urine samples were analyzed and urinary biomarker levels correlated with mycotoxin levels in foods consumed the day before urine collection. A total of eight analytes were detected in 61/120 (50.8%) of studied urine samples, with ochratoxin A, aflatoxin M1 and fumonisin B1 being the most frequently occurring biomarkers of exposure. These mycotoxin biomarkers were present in samples from all age categories, suggestive of chronic (lifetime) exposures. Rough estimates of mycotoxin intake suggested some exposures were higher than the tolerable daily intake. Overall, rural consumer populations from Nasarawa were more exposed to several mixtures of mycotoxins in their diets relative to those from Kaduna as shown by food and urine biomarker data. This study has shown that mycotoxin co-exposure may be a major public health challenge in rural Nigeria; this calls for urgent intervention.

Bio-monitoring of mycotoxin exposure in Cameroon using a urinary multi-biomarker approach.

Bio-monitoring of human exposure to mycotoxin has mostly been limited to a few individually measured mycotoxin biomarkers. This study aimed to determine the frequency and level of exposure to multiple mycotoxins in human urine from Cameroonian adults. 175 Urine samples (83% from HIV-positive individuals) and food frequency questionnaire responses were collected from consenting Cameroonians, and analyzed for 15 mycotoxins and relevant metabolites using LC-ESI-MS/MS. In total, eleven analytes were detected individually or in combinations in 110/175 (63%) samples including the biomarkers aflatoxin M1, fumonisin B1, ochratoxin A and total deoxynivalenol. Additionally, important mycotoxins and metabolites thereof, such as fumonisin B2, nivalenol and zearalenone, were determined, some for the first time in urine following dietary exposures. Multi-mycotoxin contamination was common with one HIV-positive individual exposed to five mycotoxins, a severe case of co-exposure that has never been reported in adults before. For the first time in Africa or elsewhere, this study quantified eleven mycotoxin biomarkers and bio-measures in urine from adults. For several mycotoxins estimates indicate that the tolerable daily intake is being exceeded in this study population. Given that many mycotoxins adversely affect the immune system, future studies will examine whether combinations of mycotoxins negatively impact Cameroonian population particularly immune-suppressed individuals.

LC-MS/MS-based multibiomarker approaches for the assessment of human exposure to mycotoxins

AbstractMycotoxins are toxic fungal secondary metabolites that frequently contaminate food and feed worldwide, and hence represent a major hazard for food and feed safety. To estimate human exposure arising from contaminated food, so-called biomarker approaches have been developed as a complementary biomonitoring tool besides traditional food analysis. The first methods based on radioimmunoassays and enzyme-linked immunosorbent assays as well as on liquid chromatography were developed in the late 1980s and early 1990s for the carcinogenic aflatoxins and in the last two decades further tailor-made methods for some major mycotoxins have been published. Since 2010, there has been a clear trend towards the development and application of multianalyte methods based on liquid chromatography–electrospray ionization tandem mass spectrometry for assessment of mycotoxin exposure made possible by the increased sensitivity and selectivity of modern mass spectrometry instrumentation and sophisticated sample cleanup approaches. With use of these advanced methods, traces of mycotoxins and relevant breakdown and conjugation products can be quantified simultaneously in human urine as so-called biomarkers and can be used to precisely describe the real exposure, toxicokinetics, and bioavailability of the toxins present. In this article, a short overview and comparison of published multibiomarker methods focusing on the determination of mycotoxins and relevant excretion products in human urine is presented. Special attention is paid to the main challenges when analyzing these toxic food contaminants in urine, i.e., very low analyte concentrations, appropriate sample preparation, matrix effects, and a lack of authentic, NMR-confirmed calibrants and reference materials. Finally, the progress in human exposure assessment studies facilitated by these analytical methods is described and an outlook on probable developments and possibilities is presented. FigureMycotoxin exposure assessment: traditional food analysis compared to the innovative, complementary biomarker approach

Investigation of the hepatic glucuronidation pattern of the Fusarium mycotoxin deoxynivalenol in various species.

Deoxynivalenol (DON) is one of the most abundant mycotoxins contaminating food and feed worldwide. Upon absorption, the major portion of the toxin is excreted by humans and animal species as glucuronide. However, consistent in vitro data on DON glucuronidation are lacking. In the present study, the metabolism of DON was investigated using liver microsomes from humans and six different animal species. It was shown that all animal and human liver microsomes led to the formation of up to three different mono-O-glucuronides with significant interspecies differences. While the activity of human liver microsomes was low (0.8 to 2.2 pmol·min(-1)·mg(-1)), bovine liver and rat liver microsomes conjugated DON with activities of 525 pmol·min(-1)·mg(-1) and 80 pmol·min(-1)·mg(-1), respectively.

Evaluation of software sensors for on-line estimation of culture conditions in an Escherichia coli cultivation expressing a recombinant protein

Software sensors for monitoring and on-line estimation of critical bioprocess variables have mainly been used with standard bioreactor sensors, such as electrodes and gas analyzers, where algorithms in the software model have generated the desired state variables. In this article we propose that other on-line instruments, such as NIR probes and on-line HPLC, should be used to make more reliable and flexible software sensors. Five software sensor architectures were compared and evaluated: (1) biomass concentration from an on-line NIR probe, (2) biomass concentration from titrant addition, (3) specific growth rate from titrant addition, (4) specific growth rate from the NIR probe, and (5) specific substrate uptake rate and by-product rate from on-line HPLC and NIR probe signals. The software sensors were demonstrated on an Escherichia coli cultivation expressing a recombinant protein, green fluorescent protein (GFP), but the results could be extrapolated to other production organisms and product proteins. We conclude that well-maintained on-line instrumentation (hardware sensors) can increase the potential of software sensors. This would also strongly support the intentions with process analytical technology and quality-by-design concepts.

Non-synergistic cytotoxic effects of Fusarium and Alternaria toxin combinations in Caco-2 cells.

Exposure of humans and animals to mycotoxins via food and feed generally involves a conglomeration of compounds contaminating the consumed products. Investigations on combinatory effects of mycotoxins are therefore of great importance. In this study, cytotoxic effects of binary mixtures of the Fusarium toxins enniatin B, aurofusarin, deoxynivalenol, nivalenol and zearalenone, and tenuazonic acid produced by Alternaria spp., were evaluated by the WST-1 assay in the colorectal carcinoma cell-line Caco-2 after 24h of incubation. The selection of these mycotoxins was based on typically occurring natural contamination patterns in grains. Aurofusarin, which can be found abundantly in contaminated foodstuff and has not been toxicologically characterized properly so far, showed pronounced cytotoxicity, decreasing the mitochondrial activity at 10μM to 51% compared to a solvent control. Combinations of other mycotoxins with aurofusarin showed additive effects. In contrast, binary mixtures of enniatin B, deoxynivalenol, nivalenol and zearalenone at cytotoxic concentrations, predominantly resulted in antagonistic effects. Binary combinations of these four Fusarium toxins with tenuazonic acid also revealed interacting effects leading to a decrease in cytotoxicity, compared to expected combinatory effects. Especially in combination with deoxynivalenol, tenuazonic acid was found to significantly reduce the cytotoxicity of this mycotoxin in Caco-2 cells. Synergistic effects were not observed for any toxin combination under the chosen conditions.