HLA gene variations and mycotoxin toxicity: Four case reports.

Mycotoxins are produced by certain molds that can cause many health effects. We present four human cases of prolonged consistent mycotoxins exposure linked to genetic variations in human leukocyte antigen (HLA) alleles. The HLA-DR/DQ isotype alleles are linked to mycotoxins susceptibility due to the lack of proper immune response; individuals with these alleles are poor eliminators of mycotoxins from their system. Four subjects with variations in their HLA-DR alleles were exposed to mycotoxins from living in mold-infested houses and experienced persistent mold-related symptoms long after moving out from the mold-infested houses and only exposed to the levels of molds found in the ambient air. From one of the subjects, two urine samples were collected ~ 18 months apart after the cessation of exposure. Urinary elimination rate was extremely slow for two of the mycotoxins (ochratoxin A [OTA] and mycophenolic acid [MPA]) detected in both samples. In 18 months, decline in OTA level was only ~ 3-fold (estimated t½ of ~ 311 days) and decline in MPA level was ~ 11-fold (estimated t½ of ~ 160 days), which was ~ 10- and ~ 213-fold slower than expected in individuals without HLA-DR alleles, respectively. We estimated that ~ 4.3 and ~ 2.2 years will be required for OTA and MPA to reach < LLQ in urine, respectively. Three other subjects with variations in HLA-DR alleles were members of a family who lived in a mold-infested house for 4 years. They kept experiencing mold-related issues >2 years after moving to a non-mold-infested house. Consistent exposure was confirmed by the presence of several mycotoxins in urine >2 years after the secession of higher than background (from outdoor ambient air) exposure. This was consistent with the extremely slow elimination of mycotoxins from their system. Variations in HLA-DR alleles can, consequently, make even short periods of exposure to chronic exposure scenarios with related adverse health effects. It is, therefore, important to determine genetic predisposition as a reason for prolonged/lingering mold-related symptoms long after the cessation of higher than background exposure. Increased human exposure to mycotoxins is expected from increased mold infestation that is anticipated due to rising CO2, temperature, and humidity from the climate change with possibly increased adverse health effects, especially in individuals with genetic susceptibility to mold toxicity.

Biomonitoring of Dietary Mycotoxin Exposure and Associated Impact on the Gut Microbiome in Nigerian Infants.

Mycotoxins are toxic chemicals that adversely affect human health. Here, we assessed the influence of mycotoxin exposure on the longitudinal development of early life intestinal microbiota of Nigerian neonates and infants (NIs). Human biomonitoring assays based on liquid chromatography tandem mass spectrometry were applied to quantify mycotoxins in breast milk (n = 68) consumed by the NIs, their stool (n = 82), and urine samples (n = 15), which were collected longitudinally from month 1-18 postdelivery. Microbial community composition was characterized by 16S rRNA gene amplicon sequencing of stool samples and was correlated to mycotoxin exposure patterns. Fumonisin B1 (FB1), FB2, and alternariol monomethyl ether (AME) were frequently quantified in stool samples between months 6 and 18. Aflatoxin M1 (AFM1), AME, and citrinin were quantified in breast milk samples at low concentrations. AFM1, FB1, and ochratoxin A were quantified in urine samples at relatively high concentrations. Klebsiella and Escherichia/Shigella were dominant in very early life stool samples (month 1), whereas Bifidobacterium was dominant between months 3 and 6. The total mycotoxin levels in stool were significantly associated with NIs' gut microbiome composition (PERMANOVA, p < 0.05). However, no significant correlation was observed between specific microbiota and the detection of certain mycotoxins. Albeit a small cohort, this study demonstrates that mycotoxins may influence early life gut microbiome composition.

Associated factors with mycotoxin exposure in Spanish population.

Human exposure to mycotoxins is a global concern since filamentous fungi can contaminate food and feed from crops to ready-to-eat meals. Human urine biomonitoring is a widely used technique to evaluate mycotoxins exposure, as an alternative to food correlation studies. The aim of this study is to describe human exposure to mycotoxins and to investigate the associated sociodemographic, lifestyle and dietary variables. Participants were 540 women from the Valencia (Spain) cohort of the Spanish Childhood and Environment Project (INMA). A validated multi-mycotoxin method using HPLC-Q-TOF-MS was applied to determine the concentration of ten selected mycotoxins: Enniatin A, Enniatin B, Enniatin A1, Enniatin B1, Beauvericine, Aflatoxin B1, Aflatoxin B2, Aflatoxin G1, Aflatoxin G2 and Ochratoxin A. A simultaneous untargeted screening of mycotoxins and their metabolites has been performed. Mycotoxins associations were assessed by bivariate and multivariate regression models using participants' sociodemographic, lifestyle and dietary data collected through questionnaires. Mycotoxins were detected in 81% of urine samples. The method quantified mycotoxins concentrations in up to 151 samples. Most quantified mycotoxins were: Enniatin B [% of detection (concentration range)] = 26% (1.0-39.7 ng/mg) and Enniatin B1 = 7% (0.5-14.4 ng/mg). Besides the ten-targeted mycotoxins, other mycotoxins and metabolites were studied, and higher incidence was observed for Deepoxy-deoxynivalenol (45%), Ochratoxin B (18%) and Ochratoxin α (17%). Higher mycotoxins concentrations were associated with rural areas as well as with participants belonged to lower social class, beer, light sodas and fruit juice consumers. On the contrary, higher processed meat intake was related to lower mycotoxins' levels. Studies are required to better evaluate the exposure to mycotoxins from food and their environmental relationships.

Human biomonitoring of deoxynivalenol (DON) - Assessment of the exposure of young German adults from 1996 - 2021.

The mycotoxin deoxynivalenol (DON) is a frequently found contaminant in cereals and cereal-based products. As a German contribution to the European Joint Programme HBM4EU, we analysed the total DON concentration (tDON) in 24-h urine samples from the German Environmental Specimen Bank (ESB). In total, 360 samples collected in 1996, 2001, 2006, 2011, 2016, and 2021 from young adults in Muenster (Germany), were measured by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) after enzymatic deconjugation of the glucuronide metabolites. tDON was found in concentrations above the lower limit of quantification (0.3 µg/L) in 99% of the samples. Medians of the measured concentrations and the daily excretion were 4.3 µg/L and 7.9 µg/24 h, respectively. For only nine participants, urinary tDON concentrations exceeded the provisional Human biomonitoring guidance value (HBM GV) of 23 µg/L. Urinary tDON concentrations were significantly higher for male participants. However, 24-h excretion values normalized to the participant's body weight did not exhibit any significant difference between males and females and the magnitude remained unchanged over the sampling years with exception of the sampling year 2001. Daily intakes were estimated from excretion values. Exceedance of the tolerable daily intake (TDI) of 1 µg/kg bw per day was observed for less than 1% of all participants. TDI exceedances were only present in the sampling year 2001 and not in more recent sampling years while exceedance of the HBM guidance value was also observed in 2011 and 2021.

Maternal exposure to multiple mycotoxins and adverse pregnancy outcomes: a prospective cohort study in rural Bangladesh.

There is limited and inconsistent evidence, primarily from cross-sectional studies, linking mycotoxins to adverse birth outcomes. This study investigates the potential role of maternal dietary exposure to multiple mycotoxins in the development of several adverse pregnancy and birth outcomes. We analyzed data from 436 singleton pregnancies enrolled in a prospective cohort study in the rural Habiganj district, Bangladesh, between July 2018 and November 2019. Thirty-five urinary mycotoxin biomarkers were quantified using liquid chromatography coupled with tandem mass spectrometry and used to estimate dietary mycotoxin exposure. Multivariable regression models, adjusted for potential confounding and clustering, were fitted to assess the associations between maternal exposure to frequently occurring mycotoxins (ochratoxin A-OTA, citrinin- CIT, and Deoxynivalenol- DON) and pregnancy loss, preterm birth (PTB), low birth weight (LBW), born small-for-gestational-age (SGA) and small-vulnerable newborn. The results indicate that only in 16 of 436 pregnancies (4%) were urine samples free from all investigated mycotoxins. Biomarkers for six major mycotoxins were detected in the urine samples. OTA (95%), CIT (61%), and DON (6%) were most frequently detected, with at least two mycotoxins co-occurring in the majority of women (63%). There was evidence that maternal dietary intake of OTA was associated with higher odds of having an LBW baby, with the odds increasing in a dose-dependent manner. We found no evidence of associations between pregnancy loss, PTB, SGA, small-vulnerable newborns, and maternal dietary exposure to OTA, CIT, and DON, albeit with large confidence intervals, so findings are consistent with protective as well as large harmful effects. Exposure to multiple mycotoxins during pregnancy is widespread in this rural community and represents a health risk for mothers and babies. Tailored public health policies and interventions must be implemented to reduce mycotoxin exposure to the lowest possible level.

Assessment of multiple mycotoxin exposure and its association with food consumption: a human biomonitoring study in a pregnant cohort in rural Bangladesh.

Aflatoxins (AFs), ochratoxin A (OTA), citrinin (CIT), fumonisin B1 (FB1), zearalenone (ZEN), and deoxynivalenol (DON) are mycotoxins that may contaminate diets, especially in low-income settings, with potentially severe health consequences. This study investigates the exposure of 439 pregnant women in rural Bangladesh to 35 mycotoxins and their corresponding health risks and links their exposure to certain foods and local stimulants. Overall, 447 first-morning urine samples were collected from pregnant women between July 2018 and November 2019. Mycotoxin biomarkers were quantified by DaS-HPLC-MS/MS. Urinary concentration of frequently occurring mycotoxins was used to estimate dietary mycotoxin exposure. Median regression analyses were performed to investigate the association between the consumption of certain foods and local stimulants, and urinary concentration of frequently occurring mycotoxins. Only in 17 of 447 urine samples (4%) were none of the investigated mycotoxins detected. Biomarkers for six major mycotoxins (AFs, CIT, DON, FB1, OTA, and ZEN) were detected in the urine samples. OTA (95%), CIT (61%), and DON (6%) were most frequently detected, with multiple mycotoxins co-occurring in 281/447 (63%) of urine samples. Under the lowest exposure scenario, dietary exposure to OTA, CIT, and DON was of public health concern in 95%, 16%, and 1% of the pregnant women, respectively. Consumption of specific foods and local stimulants-betel nut, betel leaf, and chewing tobacco-were associated with OTA, CIT, and DON urine levels. In conclusion, exposure to multiple mycotoxins during early pregnancy is widespread in this rural community and represents a potential health risk for mothers and their offspring.

High-Throughput Determination of Major Mycotoxins with Human Health Concerns in Urine by LC-Q TOF MS and Its Application to an Exposure Study.

Human biomonitoring constitutes a suitable tool to assess exposure to toxins overcoming the disadvantages of traditional methods. Urine constitutes an accessible biological matrix in biomonitoring studies. Mycotoxins are secondary metabolites produced naturally by filamentous fungi that produce a wide range of adverse health effects. Thus, the determination of urinary mycotoxin levels is a useful tool for assessing the individual exposure to these food contaminants. In this study, a suitable methodology has been developed to evaluate the presence of aflatoxin B2 (AFB2), aflatoxin (AFG2), ochratoxin A (OTA), ochratoxin B (OTB), zearalenone (ZEA), and α-zearalenol (α-ZOL) in urine samples as exposure biomarkers. For this purpose, different extraction procedures, namely, the Solid Phase Extraction (SPE); Dispersive Liquid-Liquid Microextraction (DLLME); and Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) methods were assessed, followed by Liquid Chromatography coupled to Quadrupole Time of Flight Mass Spectrometry with Electrospray Ionization (LC-ESI-QTOF-MS) determination. Then, the proposed methodology was applied to determine mycotoxin concentrations in 56 human urine samples from volunteers and to estimate the potential risk of exposure. The results obtained revealed that 55% of human urine samples analyzed resulted positive for at least one mycotoxin. Among all studied mycotoxins, only AFB2, AFG2, and OTB were detected with incidences of 32, 41, and 9%, respectively, and levels in the range from

Assessment of Human Exposure to Five Alternaria Mycotoxins in China by Biomonitoring Approach.

This biomonitoring study was conducted to investigate the concentration levels of five Alternaria mycotoxins in urine samples from 269 healthy volunteers living in the Yangtze River Delta, China. Alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA) and tentoxin (TEN) were detected in 38.3%, 48.7%, 63.9% and 23.4% of urine samples with the concentrations ranging from 0.057 to 45.8 ng/mL, 0.020 to 0.802 ng/mL, 0.050 to 80.6 ng/mL and 0.021 to 0.939 ng/mL, respectively. Altenuene (ALT) was not detected in any urine sample. Based on the urinary concentrations, the probable daily intake (PDI) values of Alternaria mycotoxins were calculated, and 100%, 99.2-100%, 0.372% and 1.12% of participants exceeded the threshold of toxicological concern (TTC) values for AOH, AME, TeA and TEN, respectively. This study revealed high potential health risks related to the contaminations of major Alternaria mycotoxins in China and highlighted the necessity for more toxicological studies to provide better basis for further comprehensive risk assessments.

Assessment of Mycotoxin Exposure in a Rural County of Chile by Urinary Biomarker Determination.

Aflatoxin B1 (AFB1), ochratoxin A (OTA), zearalenone (ZEN), and deoxynivalenol (DON) are frequent mycotoxins that may cause carcinogenic, mutagenic, estrogenic, or gastrointestinal effects. The aim of this study was to assess the exposure to and risk from AFB1, OTA, ZEN, and DON in 172 participants of the Maule Cohort (MAUCO) by a biomarker analysis in urine and to associate their exposure with food consumption and occupation. Mycotoxins in the first morning urine were analyzed by solid-phase extraction and quantified by Ultra-High-Performance Liquid Chromatography with a mass-mass detector. Participants' information regarding food consumption, occupation, and other characteristics was obtained from a baseline and 2-year follow-up survey of the cohort. The prevalence and mean levels of mycotoxins in the urine were as follows: DON 63%, 60.7 (±78.7) ng/mL; AFB1 8%, 0.3 (±0.3) ng/mL; α-zearalenol (α-ZEL) 4.1%, 41.8 (±115) ng/mL; ß-ZEL 3.5%, 17.4 (±16.1) ng/mL; AFM1 2%, 1.8 (±1.0) ng/mL; OTA 0.6% (1/172), 1.3 ng/mL; and ZEN 0.6%, 1.1 ng/mL. These results were translated into exposures of DON, ZEN, and aflatoxins of public health concern. Participants who consumed coffee and pepper the day before had a significantly greater presence of DON (OR: 2.3, CI95 1.17-4.96) and total ZEL (OR: 14.7, CI95 3.1-81.0), respectively, in their urine. Additionally, we observed associations between the habitual consumption of beer and DON (OR: 2.89, CI95 1.39-6.42). Regarding the levels of mycotoxins and the amount of food consumed, we found correlations between DON and nuts (p = 0.003), total ZEL and cereals (p = 0.01), and aflatoxins with capsicum powder (p = 0.03) and walnuts (p = 0.03). Occupation did not show an association with the presence of mycotoxins in urine.

Determination of Urinary Mycotoxin Biomarkers Using a Sensitive Online Solid Phase Extraction-UHPLC-MS/MS Method.

In the course of assessing the human exposure to mycotoxins, biomarker-based approaches have proven to be important tools. Low concentration levels, complex matrix compositions, structurally diverse analytes, and the large size of sample cohorts are the main challenges of analytical procedures. For that reason, an online solid phase extraction-ultra high-performance liquid chromatography-tandem mass spectrometry (online SPE-UHPLC-MS/MS) method was developed, allowing for the sensitive, robust, and rapid analysis of 11 relevant mycotoxins and mycotoxin metabolites in human urine. The included spectrum of analytes comprises aflatoxin M1 (AFM1), altenuene (ALT), alternariol monomethyl ether (AME), alternariol (AOH), citrinin (CIT) and its metabolite dihydrocitrinone (DH-CIT), fumonisin B1 (FB1), ochratoxin A (OTA), and zearalenone (ZEN) as well as α- and ß-zearalenol (α- and ß-ZEL). Reliable quantitation was achieved by means of stable isotope dilution, except for ALT, AME and AOH using matrix calibrations. The evaluation of method performance displayed low limits of detection in the range of pg/mL urine, satisfactory apparent recovery rates as well as high accuracy and precision during intra- and interday repeatability. Within the analysis of Zimbabwean urine samples (n = 50), the applicability of the newly developed method was shown. In addition to FB1 being quantifiable in all analyzed samples, six other mycotoxin biomarkers were detected. Compared to the occurrence rates obtained after analyzing the same sample set using an established dilute and shoot (DaS) approach, a considerably higher number of positive samples was observed when applying the online SPE method. Owing to the increased sensitivity, less need of sample handling, and low time effort, the herein presented online SPE approach provides a valuable contribution to human biomonitoring of mycotoxin exposure.

[Determination of amanita peptide toxins in human urine by TurboFlow online clean-up-liquid chromatography-tandem mass spectrometry].

Amanita peptide toxins are cyclic polypeptide mushroom toxins that can cause acute liver damage. The fatality rate associated with these toxins is very high. Monitoring the concentration of amanita peptide toxins in human urine can provide valuable information for early clinical diagnosis and treatment. Therefore, a TurboFlow online clean-up-liquid chromatography-triple quadrupole mass spectrometry (TF-LC-MS/MS) method was established for the simultaneous quantitative determination of five amanita peptide toxins (α-amanitin, ß-amanitin, γ-amanitin, phallacidin, and phalloidin) in human urine. After pre-treatment with high-speed centrifugation, urine samples were analyzed using TF-LC-MS/MS. The main factors influencing purification efficiency, including the TF column, loading solution, eluting solution, transfer flow, and transfer time, were optimized in this study. Under the optimized experimental conditions, the analytes were purified using a TurboFlowTM Cyclone column (50 mm×0.5 mm) and separated on a Hypersil GOLD C18 column (100 mm×2.1 mm) using the mobile phases of methanol and 4 mmol/L aqueous ammonium acetate solution with gradient elution. The analytes were detected in selected reaction monitoring (SRM) mode via positive electrospray ionization. Matrix-matched external standard calibration was used for quantitation. The linear range of the method ranged from 1.0 µg/L to 50.0 µg/L for all five amanita peptide toxins, with correlation coefficients (r2) higher than 0.997. The limits of detection were 0.15-0.3 µg/L and the limits of quantification (LOQs) were 0.5-1.0 µg/L for the five amanita peptide toxins in urine. The intra-day and inter-day recoveries of amanita peptide toxins were 87.0%-108.6% and 86.8%-112.7%, respectively, at the spiked levels of 2.0, 5.0, and 10.0 µg/L. The intra-day and inter-day relative standard deviations (RSDs) were less than 14.5%. The method is accurate, rapid, sensitive, easy to operate, and can satisfy the requirements of public health emergency testing or clinical poisoning testing.

Biomonitoring of Multiple Mycotoxins in Urine by GC-MS/MS: A Pilot Study on Patients with Esophageal Cancer in Golestan Province, Northeastern Iran.

A pilot study to investigate the occurrence of 10 mycotoxins (deoxynivalenol, DON; 3-acetyldeoxynivalenol, 3-ADON; 15-acetyldeoxynivalenol, 15-ADON; fusarenon-X, FUS-X; diacetoxyscirpenol, DAS; nivalenol, NIV; neosolaniol, NEO; zearalenone, ZON; zearalanone, ZAN; T-2 toxin, T-2; and HT-2 toxin, HT-2) in esophageal cancer patients was performed with the urinary biomarkers approach in Golestan, Iran. Urine multimycotoxin analysis was performed by dispersive liquid-liquid microextraction and gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis, and values were normalized with urinary creatinine (µg/g). Four mycotoxins, namely NEO (40%), HT-2 (17.6%), DON (10%), and HT-2 (5.8%), were detected in the analyzed urine samples. DON was only detected in the control group (5.09 µg/g creatinine), while T-2 (44.70 µg/g creatinine) was only present in the esophageal cancer group. NEO and HT-2 were quantified in both control and case groups, showing average of positive samples of 9.09 and 10.45 µg/g creatinine for NEO and 16.81 and 29.09 µg/g creatinine for HT-2, respectively. Mycotoxin co-occurrence was observed in three samples as binary (NEO/HT-2 and T-2/HT-2) and ternary (DON/NEO/HT-2) combinations, reaching total concentrations of 44.58, 79.13, and 30.04 µg/g creatinine, respectively. Further investigations are needed to explore a causal association between mycotoxin contamination and esophageal cancer. For this pilot study in Golestan, the low sample size was a very limiting factor.

Analysis of Multiple Mycotoxins in the Qatari Population and Their Relation to Markers of Oxidative Stress.

Mycotoxins are naturally occurring food toxins worldwide that can cause serious health effects. The measurement of mycotoxin biomarkers in biological fluids is needed to assess individuals' exposure. The aim of this study was to investigate the incidence of mycotoxins in the Qatari population. Serum samples from 412 adults and urinary samples from 559 adults were analyzed for the presence of mycotoxin biomarkers. Multimycotoxin approaches have been applied, using liquid chromatography mass spectrometry methods. Samples were further analyzed for the oxidative stress markers and compared with regard to the incidence of mycotoxins. The presence of mycotoxins was identified in 37% of serum samples and in less than 20% of urine samples. It was found that 88% of positive of the samples were positive for only one mycotoxin, while 12% of positive samples had two or more mycotoxins. Trichothecenes and zearalenone metabolites were most commonly detected mycotoxins, followed by aflatoxins, roquefortine C and mycophenolic acid. The presence of mycotoxins was found to positively correlate with oxidative stress markers. The obtained results illustrate the importance of mycotoxin biomonitoring studies in humans and the need to elucidate the underlying mechanisms of mycotoxin-induced toxicity.

Case-Control Study of Nodding Syndrome in Acholiland: Urinary Multi-Mycotoxin Screening.

This case-control study adds to the growing body of knowledge on the medical, nutritional, and environmental factors associated with Nodding Syndrome (NS), a seizure disorder of children and adolescents in northern Uganda. Past research described a significant association between NS and prior history of measles infection, dependence on emergency food and, at head nodding onset, subsistence on moldy maize, which has the potential to harbor mycotoxins. We used LC-MS/MS to screen for current mycotoxin loads by evaluating nine analytes in urine samples from age-and-gender matched NS cases (n = 50) and Community Controls (CC, n = 50). The presence of the three mycotoxins identified in the screening was not significantly different between the two groups, so samples were combined to generate an overall view of exposure in this community during the study. Compared against subsequently run standards, α-zearalenol (43 ± 103 µg/L in 15 samples > limit of quantitation (LOQ); 0 (0/359) µg/L), T-2 toxin (39 ± 81 µg/L in 72 samples > LOQ; 0 (0/425) µg/L) and aflatoxin M1 (4 ± 10 µg/L in 15 samples > LOQ; 0 (0/45) µg/L) were detected and calculated as the average concentration ± SD; median (min/max). Ninety-five percent of the samples had at least one urinary mycotoxin; 87% were positive for two of the three compounds detected. While mycotoxin loads at NS onset years ago are and will remain unknown, this study showed that children with and without NS currently harbor foodborne mycotoxins, including those associated with maize.

Exposure Assessment of Multiple Mycotoxins and Cumulative Health Risk Assessment: A Biomonitoring-Based Study in the Yangtze River Delta, China.

The extensive exposure to multiple mycotoxins has been demonstrated in many countries; however, realistic assessments of the risks related to cumulative exposure are limited. This biomonitoring study was conducted to investigate exposure to 23 mycotoxins/metabolites and their determinants in 227 adults (aged 20-88 years) in the Yangtze River Delta, China. Eight mycotoxins were detected in 110 urine samples, and multiple mycotoxins co-occurred in 51/227 (22.47%) of urine samples, with deoxynivalenol (DON), fumonisin B1 (FB1), and zearalenone (ZEN) being the most frequently occurring. For single mycotoxin risk assessment, FB1, ZEN, aflatoxin B1 (AFB1), and ochratoxin A (OTA) all showed potential adverse effects. However, for the 12 samples containing DON and ZEN, in which none had a hazard risk, the combination of both mycotoxins in two samples was considered to pose potential endocrine disrupting risks to humans by hazard index (HI) method. The combined margin of exposure (MOET) for AFB1 and FB1 could constitute a potential health concern, and AFB1 was the main contributor. Our approach provides a blueprint for evaluating the cumulative risks related to different types of mycotoxins and opens a new horizon for the accurate interpretation of epidemiological health outcomes related to multi-mycotoxin exposure.

Development of a simple and rapid LC-MS/MS method for the simultaneous quantification of five Alternaria mycotoxins in human urine.

Alternaria mycotoxins, such as tenuazonic acid (TeA), altenuene (ALT), alternariol (AOH), tentoxin (TEN) and alternariol monomethyl ether (AME) are frequently found in foods and may pose a potential risk to human health. Human biomonitoring can help measure our exposure to these mycotoxins, and help us determine if the exposure is changing over time. In this study, a simple liquid-liquid extraction sample preparation procedure followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous analysis of five Alternaria mycotoxins in human urine. High recoveries (92.7-103.2%) were obtained for all the tested mycotoxins with relative standard deviations (RSDs, %) of less than 6.4%. The limits of quantification (LOQs) for the analytes in urine ranged from 0.001 to 0.05 ng/mL. The method was successfully applied to investigate the levels of five Alternaria mycotoxins from 135 volunteers. In all of the samples, at least one Alternaria mycotoxin was detected. TeA, AME and AOH were the predominant Alternaria mycotoxins, and the detection rates were 85.9%, 96.3% and 51.9%, respectively.

Development of a Sensitive and Reliable UHPLC-MS/MS Method for the Determination of Multiple Urinary Biomarkers of Mycotoxin Exposure.

A variety of mycotoxins from different sources frequently contaminate farm products, presenting a potential toxicological concern for animals and human. Mycotoxin exposure has been the focus of attention for governments around the world. To date, biomarkers are used to monitor mycotoxin exposure and promote new understanding of their role in chronic diseases. The goal of this research was to develop and validate a sensitive UHPLC-MS/MS method using isotopically-labeled internal standards suitable for accurate determination of 18 mycotoxin biomarkers, including fumonisins, ochratoxins, Alternaria and emerging Fusarium mycotoxins (fumonisin B1, B2, and B3, hydrolyzed fumonisin B1 and B2, ochratoxin A, B, and alpha, alternariol, alternariol monomethyl ether, altenuene, tentoxin, tenuazonic acid, beauvericin, enniatin A, A1, B, and B1) in human urine. After enzymatic digestion with ß-glucuronidase, human urine samples were cleaned up using HLB solid phase extraction cartridges prior to instrument analysis. The multi-mycotoxin and analyte-specific method was validated in-house, providing satisfactory results. The method provided good linearity in the tested concentration range (from LOQ up to 25-500 ng/mL for different analytes), with R2 from 0.997 to 0.999. The limits of quantitation varied from 0.0002 to 0.5 ng/mL for all analytes in urine. The recoveries for spiked samples were between 74.0% and 133%, with intra-day precision of 0.5%-8.7% and inter-day precision of 2.4%-13.4%. This method was applied to 60 urine samples collected from healthy volunteers in Beijing, and 10 biomarkers were found. At least one biomarker was found in all but one of the samples. The high sensitivity and accuracy of this method make it practical for human biomonitoring and mycotoxin exposure assessment.

Development and application of a strategy for analyzing eight biomarkers in human urine to verify toxic mushroom or ricinus communis ingestions by means of hydrophilic interaction LC coupled to HRMS/MS.

The analytical proof of a toxic mushroom and/or plant ingestion at an early stage of a suspected intoxication can be crucial for fast therapeutic decision making. Therefore, comprehensive analytical procedures need to be available. This study aimed to develop a strategy for the qualitative analysis of α- and ß-amanitin, psilocin, bufotenine, muscarine, muscimol, ibotenic acid, and ricinine in human urine by means of hydrophilic interaction liquid chromatography-high resolution MS/MS (HILIC-HRMS/MS). Urine samples were prepared by hydrophilic-phase liquid-liquid extraction using dichloromethane and subsequent solid-phase extraction and precipitation, performed in parallel. Separation and identification of the biomarkers were achieved by HILIC using acetonitrile and methanol as main eluents and Orbitrap-based mass spectrometry, respectively. The method was validated as recommended for qualitative procedures and tests for selectivity, carryover, and extraction recoveries were included to also estimate the robustness and reproducibility of the sample preparation. Limits of identification were 1 ng/mL for α- and ß-amanitin, 5 ng/mL for psilocin, bufotenine, muscarine, and ricinine, and 1500 ng/mL and 2000 ng/mL for ibotenic acid and muscimol, respectively. Using γ-amanitin, l-tryptophan-d5, and psilocin-d10 as internal standards, compensation for variations of matrix effects was shown to be acceptable for most of the toxins. In eight urine samples obtained from intoxicated individuals, α- and ß-amanitin, psilocin, psilocin-O-glucuronide, muscimol, ibotenic acid, and muscarine could be identified. Moreover, psilocin-O-glucuronide and bufotenine-O-glucuronide were found to be suitable additional targets. The analytical strategy developed was thus well suited for analyzing several biomarkers of toxic mushrooms and plants in human urine to support therapeutic decision making in a clinical toxicology setting. To our knowledge, the presented method is by far the most comprehensive approach for identification of the included biomarkers in a human matrix.

Dispersive Solid-Phase Extraction using Magnetic Carbon Nanotube Composite for the Determination of Emergent Mycotoxins in Urine Samples.

Dispersive magnetic solid-phase extraction (DMSPE) has received growing attention for sample treatment preconcentration prior to the separation of analytes due to its many advantages. In the present work, the potential of DMSPE for the determination of emergent mycotoxins (enniatins A, A1, B and B1, and beauvericin) is investigated for the first time. Different magnetic nanoparticles were tested and a magnetic multiwalled carbon nanotube (Fe3O4@MWCNT) composite was selected for the extraction and preconcentration of the five target mycotoxins in human urine samples before their analysis by ultrahigh performance liquid chromatography coupled to high resolution mass spectrometry (UHPLC-HRMS). The nanocomposite was characterized by energy dispersive X-ray spectrometry, scanning electron microscopy, Fourier transform infrared spectrophotometry, and X-ray diffraction. Several parameters affecting the adsorption and desorption of DMSPE steps were optimized and the method was fully validated. Due to a matrix effect, matrix-matched calibration curves were necessary to carry out quantification. In this way, limits of quantification of between 0.04 and 0.1 µg/L, relative standard deviation values lower than 12% and recoveries between 89.3% and 98.9% were obtained. Finally, a study of the reuse of the Fe3O4@MWCNT composite was carried out, confirming that it can be reused at least four times.

Identification of ochratoxin-N-acetyl-L-cysteine as a new ochratoxin A metabolite and potential biomarker in human urine.

Ochratoxin A (OTA) is a nephrotoxic mycotoxin with nephrocarcinogenic potential found in a broad spectrum of food commodities. The mode of action of this compound, as well as its metabolism, is still not fully understood. To determine whether the conjugation of OTA with glutathione plays an important role in human OTA metabolism, an ochratoxin-glutathione conjugate (OTB-GSH), as well as the corresponding urinary metabolite ochratoxin-N-acetyl-L-cysteine (OTB-NAC), were synthesized and their structures confirmed by NMR spectroscopy. By means of synthesized stable isotope-labeled d5-OTB-GSH and d5-OTB-NAC references, a sensitive HPLC-MS/MS method has been developed and applied for the screening of human urine samples. OTB-NAC could be detected in 11 of the analyzed 18 urine samples and was quantified in 5 urine samples in the range between 0.023 and 0.176 ng mg-1 creatinine. OTB-GSH has not been detected in the urine samples. In OTB-NAC positive samples, this metabolite contributed to a comparable concentration range to the total OTA excretion as the parent compound. This is the first direct analysis of an OTA phase 2 metabolite in humans.