Deoxynivalenol exposure assessment in a cohort of pregnant women from Bradford,UK

Deoxynivalenol (DON) is a ubiquitous contaminant of cereal crops in temperate regions of the world. It causes growth faltering and immune suppression in animals. Limited information is available on DON exposure in UK subpopulations. The objective of this study was to provide DON exposure assessment in a subset of pregnant women scheduled for an elective caesarean in a large multi-ethnic mother/infant birth cohort from Bradford, UK. Women aged 16–44 years (n?=?85) provided a urine sample for DON analysis in the last trimester of pregnancy, and concurrently completed a food-frequency questionnaire (FFQ). The urinary DON biomarker was detected in all measured samples (geometric mean (GM)?=?10.3?ng?DON?mg^?1 creatinine, range?=?0.5?116.7?ng?mg^?1). Levels were higher in women classified as South Asian in origin (GM: 15.2?ng?mg^?1; 95% CI?=?10.7?21.5?ng?mg^?1) compared with non-South Asians (GM?=?8.6?ng?mg^?1; 95% CI?=?6.6?11.8?ng?mg^?1), p?=?0.02). Estimated DON intake from FFQ data and typical levels of DON contamination of food suggest that this was mainly due to higher levels of exposure from bread, particularly daily intake of DON from chapattis in South Asians (estimated mean?=?2.4?µg?day^?1; 95% CI?=?1.2, 3.7?µg?day^?1) compared with non-South Asians (estimated mean?=?0.2?µg?day^?1; 95% CI?=?0?0.4?µg?day^?1), p?<?0.001. This is the first biomarker demonstration of DON exposure in pregnant women, and several urinary DON levels were the highest ever recorded in any study. A larger survey within this birth cohort is warranted to investigate any potential risk to mothers and their babies, from DON exposure during pregnancy.     

Urine metabolite analysis as a function of deoxynivalenol exposure: an NMR-based metabolomics investigation

Deoxynivalenol (DON) is a toxic fungal metabolite that frequently contaminates cereal crops including wheat, maize and barley. Despite knowledge of frequent exposure through diet, our understanding of the potential consequences of human exposure remains limited, in part due to the lack of validated exposure biomarkers. In this study, we interrogated the urinary metabolome using nuclear magnetic resonance (NMR) spectroscopy to compare individuals with known low and high DON exposure through consumption of their normal diet. Urine samples from 22 adults from the UK (seven males, 15 females; age range = 21–59 years) had previously determined urinary DON levels using an established liquid chromatography-mass spectrometry (LC-MS) assay. Urine samples were subsequently analysed using an NMR-based metabolomics approach coupled with multivariate statistical analysis. Metabolic profiling suggested that hippurate levels could be used to distinguish between groups with low (3.6 ng DON mg^?1 creatinine: 95% CI = 2.6, 5.0 ng mg^?1) and high (11.1 ng mg^?1: 95% CI = 8.1, 15.5 ng mg^?1) DON exposure, with the concentration of hippurate being significantly (1.5 times) higher for people with high DON exposure than for those with low DON exposure (p = 0.047). This, to our knowledge, is the first report of a metabolomics-derived biomarker of DON exposure in humans.     

Survey of deoxynivalenol and its conjugates deoxynivalenol-3-glucoside and 3-acetyl-deoxynivalenol in 374 beer samples

Beer is one of the most popular beverages worldwide. Malted cereal grains are among the basic ingredients and hence mycotoxin contamination might occur. Previous studies reported the presence of the Fusarium mycotoxins deoxynivalenol (DON) and 3-acetyl-deoxynivalenol (3ADON), as well as of the masked mycotoxin deoxynivalenol-3-glucoside (D3G) in beer. In the present survey, 374?beer samples from 38?countries with a focus on Austrian (156) and German (64) beers were analysed for the presence of D3G, DON and 3ADON. Beers were assigned to the following six categories: pale (217), wheat (46), dark (47), bock (20), nonalcoholic beers (19) and shandies (25). In total, 348 and 289 beers (93 and 77%, respectively) contained D3G and DON at the levels above the limit of detection, whereas 3ADON was not detected in any of the samples. Average concentrations of all beers were 6.9?µg?L^?1 for D3G and 8.4?µg?L^?1 in the case of DON. Nonalcoholic beers and shandies showed the lowest contaminations, 1.5 and 3.2?µg?L^?1 for D3G and 2.7 and 4.4?µg?L^?1 for DON, respectively. In bock beers characterised by a higher gravity, a significant trichothecene load of 14.8?µg?L^?1 D3G and 12.4?µg?L^?1 DON was found. The highest contamination (81?µg?L^?1 D3G, 89?µg?L^?1 DON) was detected in a pale beer from Austria, underlining the importance of this study for food safety. The molar D3G to DON ratio ranged between 0.11 and 1.25 and was 0.56 on average. Concluding, the average contamination of beer is not of toxicological concern for moderate beer drinkers. However, in the case of heavy beer drinkers, beer consumption may considerably contribute to the overall intake of DON, which might even lead to exceeding the maximum tolerable limits established for this Fusarium toxin.     

Determination of aflatoxin M1 in breast milk as a biomarker of maternal and infant exposure in Colombia

Chronic exposure to aflatoxins, and especially to aflatoxin B₁ (AFB1), causes hepatocellular carcinoma with prevalence 16–32 times higher in developing compared with developed countries. Aflatoxin M₁ (AFM1) is a monohydroxylated metabolite from AFB1 that is secreted in milk and which can be used as a biomarker of AFB1 exposure. This study aimed to determine AFM1 levels in human breast milk using immunoaffinity column clean-up with HPLC and fluorescence detection. Breast milk samples were obtained from 50 nursing mothers. Volunteers filled in a questionnaire giving their consent to analyse their samples as well as details of their socioeconomic, demographic and clinical data. The possible dietary sources of aflatoxins were assessed using a food frequency questionnaire. A total of 90% of the samples tested positive for AFM1, with a mean of 5.2 ng l^?1 and a range of 0.9–18.5 ng l^?1. The study demonstrated a high frequency of exposure of mothers and neonates to AFB1 and AFM1 in Colombia, and it points out the need to regulate and monitor continuously the presence of aflatoxins in human foods. Further research is needed in order to determine the presence of other mycotoxins in foods and in human samples as well as to devise protection strategies in a country where mycotoxins in human foods are commonly found.     

Signal amplification using colloidal gold in a biolayer interferometry-based immunosensor for the mycotoxin deoxynivalenol

Deoxynivalenol (DON) is a toxin produced by certain species of Fusarium fungi that can infest wheat, barley and corn. The fungi cause diseases in crops worldwide and some of the secondary metabolites, such as DON, can adversely affect animal health and food safety. To monitor DON in wheat rapidly, a biosensor using the principle of biolayer interferometry (BLI) was developed. The signal from the sensor was substantially amplified through the use of a primary antibody–colloidal gold conjugate. The amplification was much greater in the presence of wheat matrix than in buffered solution, suggesting matrix components may have contributed to the enhancement. The improved signal provided by the amplification allowed for the development of rapid qualitative and quantitative assays. The limit of detection of the method was 0.09?mg?kg^?1; the limit of quantitation was 0.35?mg?kg^?1. Recovery from wheat spiked over the range from 0.2 to 5?mg?kg^?1 averaged 103% (RSD?=?12%). The quantitative assay compared favourably (r ^2?=?0.9698) with a reference chromatographic method for 40 naturally contaminated wheats. The qualitative assay was able to classify accurately the same group of 40 samples as either above or below a 0.5?mg?kg^?1 threshold. These results suggest that the BLI technique can be used to measure DON in wheat rapidly.     

Pilot survey of aflatoxin–albumin adducts in sera from Egypt

Aflatoxins are potent liver carcinogens that frequently contaminate cereals in developing countries. Aflatoxin exposure has been predicted in Egypt but, to date, no studies have measured the level of aflatoxin–albumin (AF–alb) adducts as a validated biomarker to assess exposure. In this pilot survey, a limited number of sera samples, available from a hepatocellular carcinoma (HCC) case-control study in Egypt, were analysed. AF–alb was detected in 24/24 samples from HCC-negative individuals (geometric mean 9.0 pg mg^?1; range 3.5–25.8pg mg^?1), while 7/22 samples from HCC-positive cases had detectable AF–alb (geometric mean 2.6 pg mg^?1; range: non-detectable–32.8 pg mg^?1). These AF–alb data do not represent a case-control comparison due to inherent difficulties in comparing markers of dietary intake between controls and patients with disease. Although these data are limited, the potential health consequences of aflatoxin exposure in this region merit further investigation.     

Survey on the occurrence of aflatoxins in rice from different provinces of Iran

Aflatoxins were surveyed in 256 rice samples taken from retail markets in different provinces of Iran during October 2007 and July 2008. A methanol/water (80?:?20,?v/v) mixture and an aflatoxin immunoaffinity column (IAC) were used for extraction and clean-up. Mycotoxins were determined using HPLC with fluorescence detection and post-column derivatization using a photo-ionization cell. Levels of contamination ranged 0.0–5.8?ng?g^?1 (mean, 1.4?ng?g^?1) and 0.1–6.3?ng?g^?1 (mean, 1.6?ng?g^?1) for AFB1 and total aflatoxins, respectively. AFB1 was detected in almost all samples. Results showed that 55 samples (21.5%) were contaminated with more than 2?µg?kg^?1 of AFB1, while seven samples (2.7 %) contained more than 4?µg?kg^?1 total aflatoxins. The calculated probable daily intake of AFB1 from rice for Iranians ranged 1.4–5.8?ng AFB1 per kg body weight per day for average consumers and, hence. exceeding the estimated provisional maximum tolerable daily intake.     

Mycotoxin co-occurrence in rice,oat flakes and wheat noodles used as staple foods in Ecuador

The co-occurrence of aflatoxin B₁ (AFB₁), B₂ (AFB₂), G₁ (AFG₁) and G₂ (AFG₂), ochratoxin A (OTA), deoxynivalenol (DON), fumonisin B₁ (FB₁), zearalenone (ZEN), and HT-2 and T-2 toxins in the main Ecuadorian staple cereals (rice, oat flakes, and yellow and white wheat noodles) was evaluated. A ultra high performance liquid chromatography/time-of-flight mass spectrometry (UHPLC/TOFMS) method was developed and validated to screen for the presence of these mycotoxins in those cereal matrices. Matrix-matched calibration curves were used to compensate for ion suppression and extraction losses and the recovery values were in agreement with the minimum requirements of Regulation 401/2006/EC (70–110%). For most mycotoxins, the LODs obtained allowed detection in compliance with the maximum permitted levels set in Regulation EC/2006/1881, with the exception of OTA in all cereals and AFB1 in yellow noodles. Extra target analysis of OTA in oat flakes and wheat noodles was performed by HPLC with fluorescence detection. High rates of contamination were observed in paddy rice (23% DON, 23% FB₁, 7% AFB₁, 2% AFG₁ and 2% AFG₂), white wheat noodles (33% DON and 5% OTA) and oat flakes (17% DON, 2% OTA and 2% AFB₁), whereas the rates of contamination were lower in polished rice (2% AFG₁ and 4% HT-2 toxin) and yellow noodles (5% DON). Low rates of co-occurrence of several mycotoxins were observed only for white wheat noodles (5%) and paddy rice (7%). White noodles were contaminated with DON and/or OTA, while combinations of AFG₁, AFB₁, DON and FB₁ were found in paddy rice. Yellow noodles were contaminated with DON only; oat flakes contained DON, OTA or AFB₁, and polished rice was contaminated with AFG₁ and HT-2 toxin.     

Mycotoxin detection in infant formula milks in Italy

After birth, infant formulas constitute an important or often sole food source for infants during the first months of life. In this study, a survey on the presence of aflatoxin M₁ (AFM1) and ochratoxin A (OTA) in the 14 leading brands of infant formulas marketed in Italy was conducted. Mycotoxins were determined by immunoaffinity column clean-up and high-performance liquid chromatography (HPLC) with fluorescence detection. AFM1 was found in two of 185 samples, but at levels below the European legislation limit of 25 ng l^?1. OTA was detected in 133 (72%) samples (range = 35.1–689.5 ng l^?1). It has been observed that OTA contamination was 80% in the ready-to-use preparations and 63% in the powdered samples. The Scientific Committee for Food (SCF) reviewed the toxicology on OTA and concluded that it would be prudent to reduce exposure to OTA ensuring that exposure is towards the lower end of the range of tolerable daily intakes of 1.2–14 ng kg^?1 body weight day^?1. OTA was also evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and a provisional tolerable weekly intake (PTWI) of 100 ng kg^?1 body weight was established. The OTA levels in pre-term ready-to-use infant formulas were sufficient to cause a higher OTA intake than the suggested TDI. The results point out the need to perform controls for prevention programmes especially when attempting to identify risk markers of the infant feed quality.     

Immunoassay based on monoclonal antibodies versus LC-MS: deoxynivalenol in wheat and flour in Southern Brazil

An indirect competitive enzyme-linked immunosorbent assay (ELISA) method using a monoclonal antibody for deoxynivalenol (DON) detection in wheat and flour was standardised and validated (detection limit?=?177.1?µg?kg^?1) and its performance was compared with LC-MS, quantification limit?=140?µg?kg^?1). DON recovery ranged from 88.7% to 122.6% for wheat grain and from 70.6% to 139.3% for flour. Among the 38 wheat samples evaluated, DON was detected in 29 samples (76.3%) by ic-ELISA (281.6–12?291.4?µg?kg^?1) and in 22 samples (57.9%) by LC-MS (155.3–9906.9?µg?kg^?1). The 0.93 correlation coefficient between ic-ELISA and LC-MS data in 19 positive DON wheat samples demonstrated the reliability and efficiency of ic-ELISA. Results indicated that standardised ic-ELISA was suitable for DON screening in wheat samples and the need for continuous monitoring of mycotoxin levels in foodstuffs.     

Concentrations and exposure estimates of deoxynivalenol in wheat products from Argentina

The aim of this work was to determine deoxynivalenol (DON) contamination in wheat-based food products in Argentina and to estimate DON exposure. The numbers of samples were determined according to a developed sampling plan. A total of 156 samples of different wheat products were randomly collected from food markets in Luján, Argentina, and analyzed for DON by gas chromatography. DON contamination ranged 7–271?ng?g^?1 for French bread, 5–149?ng?g^?1 for Vienna bread, 11–85?ng?g^?1 for crackers, 8–85?ng?g^?1 for pizza, and was 79?ng?g^?1 for noodles. The maximum contribution to DON intake was 7% of the PMTDI for French bread; the minimum was less than 1% for noodles. Assuming all groups had eaten all sampled foods and summing all groups’ intake contribution, the highest estimate DON exposure would only be <14% (for the 18–24-year old men group) of the DON daily dietary intake.     

Exposure of consumers to deoxynivalenol from consumption of white bread in Hungary

In view of the frequent occurrence of mycotoxins in cereals, a study was initiated to assess the exposure of the Hungarian adult population. Consumption data for 1360 individuals, based on a 3-day questionnaire, indicated that white bread accounted for the major intake of cereal-based products. Various cereal products were analysed for 16 mycotoxins by a LC/MS/MS multi-toxin method with LOD of 16?µg?kg^?1 and LOQ of 50?µg?kg^?1. Deoxynivalenol (DON) was most frequently detected, but no acetyl-deoxynivalenol was present in detectable concentrations. Consumer exposure was calculated with standard Monte Carlo probabilistic modelling and point estimates, taking into account bread consumption and DON contamination in independently taken wheat flour and wheat grain samples. Over 55% of cases the DON intake were below 15% of the provisional maximum tolerable daily intake (PMTDI) of 1?µg/(kg?bw)/day. However, in 5–15% of cases, the intake from bread consumption alone exceeded the PMTDI. Wheat grain data led to the higher percentage. Intakes estimated from both data sets were at or below the acute reference dose (ARfD) of 8?µg/(kg?bw)/day in 99.94–99.97% of cases.     

Occurrence and intake of deoxynivalenol in cereal-based products marketed in Korea during 2007–2008

The occurrence of deoxynivalenol (DON) was investigated in 514 cereal-based products (corn-based, n = 125; barley-based, n = 96; wheat-based, n = 94; rice-based, n = 199) marketed in Korea during 2007?2008, and estimates of DON intake were determined. Samples were analysed by high-performance liquid chromatography (HPLC) with ultraviolet light (UV) detection after immunoaffinity clean-up. The limits of detection (LOD) and limits of quantification (LOQ) were 2.2 and 5.6 µg kg^–1, respectively. Recoveries and repeatability expressed as coefficients of variation (CV) were 82.3–100% and 2.4–15.3% in beer, bread and dried corn. The incidences and mean levels of DON were 56% and 68.9 µg kg^?1 for corn-based products, 49% and 24.1 µg kg^?1 for wheat-based products, 43% and 7.5 µg kg^?1 for barley-based products, and 16% and 3.4 µg kg^?1 for rice-based products, respectively. The estimated daily intake of DON from the consumption of rice-based, wheat-based, barley-based and corn-based products were 0.0038 µg kg^?1 bw day^?1, 0.0032 µg kg^?1 bw day^?1, 0.0015 µg kg^?1 bw day^?1 and 0.0002 µg kg^?1 bw day^?1, respectively. These values represent 0.38%, 0.32%, 0.25% and 0.01% of the provisional maximum tolerable daily intake (PMTDI) of 1 µg kg^?1 bw day^?1. These results indicate that rice-based products are major contributors to DON exposure in Korea, even though the current exposure level is unlikely to cause adverse health effects.     

Co-occurrence of mycotoxins in commercial formula milk and cereal-based baby food on the Qatar market

The present study was conducted to explore the occurrence of mycotoxins in commercial baby foods in Doha-Qatar. LCMS/MS- and HPLC-based analysis of baby food (n = 67) for 12 mycotoxins confirmed the presence of aflatoxin M1 (AFM1, 33%), ochratoxin A (OTA, 31%), deoxynivalenol (DON, 27%), aflatoxin B1 (AFB1, 22%), fumonisin B2 (FB2, 10%), zearalenone (ZEN, 4%) and T-2 toxin (2%). Noodles exhibited the maximum contamination percentage, with 33% of the samples being contaminated above the EU maximum limits, for at least one mycotoxin. Among the multi-grain flake samples, up to 28% and for the milk and milk-based-cereal samples, 14% contained at least one mycotoxin above the EU maximum limits. From all cereal-based food samples, 22%, 5%, 2% and 2% were concurrently contaminated with 2, 3, 4 and 5 mycotoxins, respectively. The occurrence of toxicological important mycotoxins in Qatari market warrants the implementation of strict regulatory limits to protect human health.     

Natural occurrence of type-B trichothecene mycotoxins in Korean cereal-based products

Type-B trichothecenes (deoxynivalenol (DON), nivalenol (NIV), fusarenone-X (FUS-X), 15-acetyldeoxynivalenol (15ADON), and 3-acetyldeoxynivalenol (3ADON)) were determined in 338 cereal-based products. Detection limit, quantification limit and mean recovery for five toxins were in the ranges 0.7–2.6?µg?kg^?1, 2.1–7.8?µg?kg^?1 and 73–110%, respectively. The range of occurrence and average level in samples were, respectively, 21–88% and 5.2–121.8?µg?kg^?1 for NIV, 10–96% and 1.7–109.5?µg?kg^?1 for DON, 2–39% and 0.4–3.6?µg?kg^?1 for FUS-X, 0–80% and 0–17.3?µg?kg^?1 for 15ADON, and 0–29% and 0–1.5?µg?kg^?1 for 3ADON. Regarding co-occurrence, 64% of samples had more than two type-B trichothecenes. The estimated daily intakes of NIV, DON, FUS-X, 15ADON, and 3ADON were 0.077, 0.048, 0.004, 0.006 and 0.002?µg?kg^?1?bw?day^?1, respectively. These results suggest that current exposure levels do not indicate the possibility of adverse effects, but consideration of the combined exposure of type-B trichothecenes may be required due to the high frequency of co-occurrence.     

Stability of the mycotoxin deoxynivalenol (DON) during the production of flour-based foods and wheat flake cereal

Deoxynivalenol (DON) is a mycotoxin found in cereal grains and cereal-based foods. DON concentrations in finished products are reduced under some processing conditions, but not others. DON concentrations in flour, wheat and selected foods made from them under commercially relevant conditions were compared by GC with electron capture detection. Average concentrations (n?=?9/item) in cookies, crackers and pretzels ranged from 61% (cookies) to 111% (pretzels) compared with flour (100%?=?0.46?µg?g^?1). Lesser amounts were found in donuts and bread: their respective DON concentrations were 44% and 30% that of flour. Mass balance estimates for DON (µg?g^?1 flour equivalents) ranged from 50% (bread?=?0.23?µg?g^?1 flour equivalents) to 120% (donuts), indicating that dilution by recipe ingredients contributed to DON reductions in bread and accounted for all of the apparent reduction in donuts. Mass balance estimates averaged 76% (crackers) to 107% (pretzels) for the other flour products. DON concentrations were higher in cereal flakes (0.55?µg?g^?1 in the finished product and 0.58?µg?g^?1 on a mass balance basis) than in wheat (0.40?µg?g^?1), suggesting that DON concentrations might increase during processing of wheat cereals under some conditions. In summary, DON concentrations of finished food products were reduced?≥50% only in bread and donuts. Reduction in bread resulted from a combination of DON ‘loss’ and dilution by recipe ingredients whereas the reduction in donuts was due entirely to dilution. These results are further evidence of DON stability during the preparation of popular flour or wheat-based products.     

Deoxynivalenol,zearalenone and T-2 in grain based swine feed in Hungary

Fusarium genera can produce trichothecenes like deoxynivalenol (DON), zearalenone (ZEN) and T-2 toxin, which can occur in feed cereal grains. Enzyme-linked immunosorbent assays (ELISA) tests of different Hungarian swine feedstuff proved that these mycotoxins were present. In this survey, 45 feed samples from 3 significant Hungarian swine feedstuff manufacturers were tested. ELISA methodology validation showed mean recovery rates in ranges from 85.3% to 98.1%, with intermediate precision of 86.9-96.9% and variation coefficients of 3.4–5.7% and 5.9–7.1%, respectively. The results showed that among Fusarium toxins, generally DON was present in the highest concentration, followed by T-2 and finally ZEN in all tested swine feeds. Each of the mycotoxins was found above the limit of detection in all swine feedstuffs. Boars feed’s DON (average ± standard deviation was 872 ± 139 µg kg^?1) and ZEN (172 ± 18 µg kg^?1) results of one of the manufacturers were above the guidance values. It indicates the necessity for efficient monitoring of DON, ZEN and T-2 mycotoxins in swine feeds.     

Development and validation of an LC-MS/MS method for the simultaneous determination of deoxynivalenol,zearalenone, T-2-toxin and some masked metabolites in different cereals and cereal-derived food

An LC-MS/MS method was developed and validated for the simultaneous determination of deoxynivalenol, zearalenone, T-2-toxin, HT-2-toxin and metabolites, including 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, deoxynivalenol-3-glucoside, α-zearalenol, β-zearalenol, zearalenone-4-glucoside, α-zearalenol-4-glucoside, β-zearalenol-4-glucoside and zearalenone-4-sulfate in maize, wheat, oats, cornflakes and bread. Extraction was performed with acetonitrile/water/acetic acid (79/20/1, v/v/v) followed by a hexane defatting step. After filtration, the extract was evaporated and the residue was redissolved in mobile phase for injection. The mobile phase, which consisted of a mixture of methanol and water with 10?mM ammonium acetate, was adjusted to pH 3 with glacial acetic acid. A sample clean-up procedure was not included because of the low recoveries of free and masked mycotoxins and their differences in polarity. The method allowed the simultaneous determination of 13 Fusarium mycotoxins in a one-step chromatographic run using a Waters Acquity UPLC system coupled to a Quattro Premier XE mass spectrometer. The method was validated for several parameters such as linearity, apparent recovery, limit of detection, limit of quantification, precision, expanded measurement uncertainty and specificity. The limits of detection varied from 5 to 13?ng?g^?1; those for the limit of quantification from 10 to 26?ng?g^?1. The results of the performance characteristics of the developed LC-MS/MS method were in good agreement with the criteria mentioned in Commission Regulation (EC) No. 401/2006. Thirty samples of a variety of food and feed matrices were sampled and analysed between July 2010 and January 2011.     

Quantitative determination of mycotoxins in urine by LC-MS/MS

Naturally occurring mycotoxins are responsible for a wide array of adverse health effects. The measurement of urinary mycotoxin levels is a useful means of assessing an individual's exposure, but the development of sensitive and accurate analytical methods for detecting mycotoxins and their metabolites in urine samples is challenging. Urinary mycotoxins are present in low pg ml^–1 concentrations, and the chromatographic identification of their metabolites can be obscured by other endogenous metabolites. We developed an analytical method focused on the selection of two appropriate multiple-reaction monitoring transition for unambiguous identification and quantification of carcinogenic aflatoxin M₁ (AFM₁), ochratoxin A (OTA) and fumonisin B₁, B₂ (FB₁, FB₂) in urine samples from a small volunteer group in a pilot study. AFM₁, OTA, FB₁ and FB₂ were concentrated selectively, interfering substances were removed using an immunoaffinity column (IAC), and mycotoxins were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in combination with a stable-isotope standard-dilution assay (SIDA). The method was sensitive enough to measure mycotoxins and their metabolites at pg ml^–1 levels in urine. The combination of LC-MS/MS and SIDA was critical to distinguishing pseudo-OTα interference from genuine OTα. Twelve urine samples contained OTA ranging from 0.013 to 0.093 ng ml^?1 (mean = 0.031 ng ml^?1). AFM₁ were detected in one sample at a 0.002 ng ml^?1 level, while FB₁ and FB₂ were undetectable in all 12 samples. None of the samples in this pilot study contained a detectable level of OTα, despite the presence of OTA, and this may suggest the need for further epidemiological investigation of OTA exposure in the Korean population.