Ochratoxin A and 2′R-Ochratoxin A in Selected Foodstuffs and Dietary Risk Assessment

The aim of this study was to estimate the contamination of grain coffee, roasted coffee, instant coffee, and cocoa purchased in local markets with ochratoxin A (OTA) and its isomerization product 2′R-ochratoxin A (2′R-OTA), and to assess risk of dietary exposure to the mycotoxins. OTA and 2′R-OTA content was determined using the HPLC chromatography with immunoaffinity columns dedicated to OTA. OTA levels found in all the tested samples were below the maximum limits specified in the European Commission Regulation EC 1881/2006. Average OTA concentrations calculated for positive samples of grain coffee/roasted coffee/instant coffee/cocoa were 0.94/0.79/3.00/0.95 µg/kg, with the concentration ranges: 0.57–1.97/0.44–2.29/0.40–5.15/0.48–1.97 µg/kg, respectively. Average 2′R-OTA concentrations calculated for positive samples of roasted coffee/instant coffee were 0.90/1.48 µg/kg, with concentration ranges: 0.40–1.26/1.00–2.12 µg/kg, respectively. In turn, diastereomer was not found in any of the tested cocoa samples. Daily intake of both mycotoxins with coffee/cocoa would be below the TDI value even if the consumed coffee/cocoa were contaminated with OTA/2′R-OTA at the highest levels found in this study. Up to now only a few papers on both OTA and 2′R-OTA in roasted food products are available in the literature, and this is the first study in Poland.

Natural Occurrence of Ochratoxin A in Spices Marketed in the Czech Republic during 2019–2020

Spices are a popular ingredient in cuisine worldwide but can pose a health risk as they are prone to fungal infestation and mycotoxin contamination. The purpose of this study was to evaluate ochratoxin A (OTA) in 54 single-kind traditional and less traditional spices, each of which was purchased in six samples of different batches (324 samples in total) at the Czech market during 2019–2020. The HPLC-FLD method with pre-treatment by immunoaffinity columns was employed to determine OTA. The limits of detection and quantification were 0.03 ng g−1 and 0.10 ng g−1, respectively. A total of 101 (31%) samples of 19 spice kinds were positive at concentrations ranging from 0.11–38.46 ng g−1. Only turmeric was contaminated with an OTA level exceeding the European Union limits. However, most spices have no regulation, thus further extensive monitoring of various mycotoxins in various kinds of spices is necessary. Chilli and black pepper are the most studied spices for OTA contamination, however, many other kinds of spice can also be highly contaminated, but studies on them are less common, rare, or have not yet been performed. The uniqueness of this study lies in the wide range of spice types studied for the presence of OTA on the Czech market.

Validation of an analytical method based on QuEChERS and LC-MS/MS to quantify nine mycotoxins in plant-based milk

Plant-based beverages (popularly known as vegetable milk) have become increasingly important in recent years. However, the nonexistence of information on mycotoxin contamination is noticeable. We herein describe the development and validation of an analytical methodology that employs QuEChERS and LC-MS/MS for the simultaneous determination of nine mycotoxins (aflatoxins B1, B2, G1, and G2, fumonisins B1 and B2, ochratoxin A, zearalenone, and citreoviridin) in seven types of vegetable milk (peanut, oat, rice, cashew, maize, soybean, and coconut). The method provided the following quantification limits, recoveries at the lowest validated concentration and relative standard deviations under repeatability conditions at the lowest validated concentration, respectively: aflatoxin B1 (0.023 μg/l, 84.98 and 9.23%); aflatoxin B2 (0.024 μg/, 93.00 and 4.85%); aflatoxin G1 (0.057 μg/l, 98.85 and 5.53%); aflatoxin G2 (0.031 μg/l, 96.64 and 4.08%); fumonisin B1 (2.166 μg/l, 75.55 and 16.78%); fumonisin B2 (1.105 μg/l, 70.47 and 11.89%); ochratoxin A (0.104 μg/l, 72.05 and 5.12%); zearalenone (8.093 μg/l, 107.10 and 6.37%); citreoviridin (1.305 μg/l, 97.25 and 7.28%). The method uses small amounts of samples, solvents, and other inexpensive reagents with no need for laborious clean-up and pre-concentration steps. Its attractive characteristics (simplicity, low cost compared to procedures that use immunoaffinity columns, and full compatibility with routine analyses) make it potentially valuable. As a proof-of-principle, the validated methodology was applied to seven commercial samples of different compositions showing that some were contaminated with aflatoxins and ochratoxin A.

Mycotoxins Detection and Fungal Contamination in Black and Green Tea by HPLC-Based Method

The fungal contamination and total aflatoxins (AF) and ochratoxin A (OTA) of tea samples were examined. A total of 60 tea samples were extracted and treated with immunoaffinity columns. The amount of AF and OTA were determined by using high-performance liquid chromatography (HPLC) with a fluorescence detector (FD). Tea samples were cultured and the fungi were identified. The results showed that 24 (40%) samples were contaminated with AFs and none of the tea samples were above the acceptable limit of AFs (≥10 μg/kg). All of the samples were contaminated with OTA where only 3 black tea samples (6.6%) and 1 green tea sample (6.7%) were detected to have more than the standard limits of toxin (10 μg·kg−1). The mean concentration of OTA in the black tea was higher than green tea. Aspergillus niger was the predominant fungi isolated from black and green tea samples. Considering the high contamination of mycotoxins in tea samples, regular monitoring in the tea process for improving quality is recommended.

Simultaneous Determination of Deoxynivalenol, Its Modified Forms, Nivalenol and Fusarenone-X in Feedstuffs by the Liquid Chromatography–Tandem Mass Spectrometry Method

A liquid chromatography-tandem mass spectrometry method was developed for simultaneous determination of deoxynivalenol (DON), 3-acetyldeoxynivalenol (3Ac-DON), 15-acetyldeoxynivalenol (15Ac-DON), DON-3-glucoside (DON-3Glc) nivalenol and fusarenone-X in feedstuffs. Different techniques of sample preparation were tested: solid-liquid-extraction, QuEChERS, solid phase extraction with OASIS HLB columns or immunoaffinity columns and a Mycosep 225 Trich column. None of the six immunoaffinity columns tested showed cross-reactivity to all of the mycotoxins. Surprisingly, the results show that if the immunoaffinity columns bound 3Ac-DON, then they did not bind 15Ac-DON. The most efficient sample preparation was achieved with a Mycosep 225 Trich column clean-up. The chromatography was optimised to obtain full separation of all analytes (including 3Ac-DON and 15Ac-DON isomeric form). The validation results show the relative standard deviations for repeatability and reproducibility varied from 4% to 24%. The apparent recovery ranged between 92% and 97%, and the limit of quantification described a 1.30 to 50 µg/kg range. The method trueness was satisfactory, as assessed by a proficiency test and analysis of reference material. A total of 99 feed samples were analysed by the developed method, revealing the presence of DON and DON-3Glc in 85% and 86% of examined animal feeds, respectively at concentrations between 1.70 and 1709 µg/kg. The ratios DON-3Glc to DON in the surveyed feedstuffs were from a low of 3% to high of 59%.

Contamination of Wheat Cultivated in Various Regions of Poland during 2017 and 2018 Agricultural Seasons with Selected Trichothecenes and Their Modified Forms

Cross-interaction of antibodies within the immunoaffinity columns used in this study facilitated the simultaneous determination of nivalenol (NIV), deoxynivalenol (DON), their glucoside derivatives (NIV-3G, DON-3G), and 3-acetyl-deoxynivalenol (3-AcDON) in wheat grain harvested in various regions of Poland. In Poland, 2018 was a warm, dry agricultural season, and hence, was relatively less favourable for cereal cultivation than 2017. Data on the natural occurrence of NIV-3G in wheat grain are among the first published in the literature. DON was the most frequently found mycotoxin in the tested samples; the percentage occurrence of DON-positive samples was 92% in 2017 and 61% in 2018. Moreover, DON concentrations were generally higher in 2017 samples (5.2–1670.7 µg/kg) than those in 2018 samples (range 5.0–461.7 µg/kg). A similar pattern was found for DON-3G. However, no statistically significant differences between the samples from the two agricultural seasons were observed for the other three mycotoxins that were analysed, and their concentrations were generally considerably lower. DON was strongly correlated with DON-3G (correlation coefficient r = 0.9558), while NIV was strongly correlated with NIV-3G (r = 0.9442). The percentage occurrence of NIV-3G- and DON-3G-positive samples was 14% in 2017 and 49% in 2018. The NIV-3G/NIV ratio was 5.9–35.7%, while the DON-3G/DON ratio range was 3.2–53.6%. In 2018, wheat samples from Southern Poland exhibited statistically significantly higher levels of DON than those from Northern Poland. The dry and hot summer of 2018 not only reduced wheat yields, but also limited development of Fusarium spp. Therefore, grain harvested that year was generally contaminated with relatively low levels of mycotoxins. Lower levels of DON were also accompanied by lesser amounts of DON-derivatives.

A simple and high throughput parallel dual immunoaffinity liquid chromatography-mass spectrometry system for urine drug testing

A simple and rapid method for direct quantitation of drugs in human urine samples was developed using a system composed of an automatic column switch and two home-made capillary immunoaffinity columns (CIACs, 100 μm × 15 cm).