Development of a corn flour certified reference material for the accurate determination of zearalenone.

A certified reference material (CRM, KRISS 108-01-002) for zearalenone in corn flour was developed to assure reliable and accurate measurements in testing laboratories. Commercially available corn flour underwent freeze-drying, pulverization, sieving, and homogenization. The final product was packed in amber bottles, approximately 14 g per unit, and preserved at -70 °C. 13C18-Zearalenone was used as an internal standard (IS) for the certification of zearalenone by isotope-dilution liquid chromatography-tandem mass spectrometry (ID-LC‒MS/MS) and for the analysis of α-zearalenol, ß-zearalenol, and zearalanone by LC‒MS/MS. The prepared CRM was sufficiently homogeneous, as the among-unit relative standard deviation for each mycotoxin ranged from 2.2 to 5.7 %. Additionally, the stability of the mycotoxins in the CRM was evaluated under different temperature conditions and scheduled test periods, including storage at -70°C, -20°C, and 4°C and room temperature for up to 12 months, 6 months, and 1 month, respectively. The content of each target mycotoxin in the CRM remained stable throughout the monitoring period at each temperature. Zearalenone content (153.6 ± 8.0 µg/kg) was assigned as the certified value. Meanwhile, the contents of α-zearalenol (1.30 ± 0.17 µg/kg), ß-zearalenol (4.75 ± 0.33 µg/kg), and zearalanone (2.09 ± 0.16 µg/kg) were provided as informative values.

Occurrence of zearalenone and its major metabolites in cereal flour from Korea.

The occurrence of zearalenone (ZEN) and its metabolites (α-zearalenol (α-ZEL), ß-zearalenol (ß-ZEL), α-zearalanol (α-ZAL), ß-zearalanol (ß-ZAL), and zearalanone (ZAN)) was investigated in 78 cereal flour from Korea using UHPLC-MS/MS. Among these mycotoxins, ZEN was the most abundant in the analyzed samples at an incidence rate of 41% and concentration range of 0.5-536 µg/kg. The highest contamination and incidence rate of ZEN were found in corn flour samples, while oat flour samples showed the lowest contamination and incidence rate of this mycotoxin. α-ZEL, ß-ZEL, and ZAN were detected only in corn flour samples but at lower frequencies of 23%, 17%, and 15%, respectively, while α-ZAL and ß-ZAL were not detected in any sample. To the best of our knowledge, this is the first investigation of the simultaneous occurrence of ZEN and its major metabolites in commercially available cereal flour from Korea. Among the tested samples, only four were contaminated with ZEN at levels exceeding the maximum regulatory level established in Korea. The co-occurrence of ZEN, α-ZEL, ß-ZEL, and ZAN was observed in 14% of all samples. Although ZEN metabolites were detected at relatively lower levels than ZEN, the relatively high co-occurrence rate of those mycotoxins is of significant concern from a food safety perspective, since they can synergistically contribute to the overall toxicity and estrogenic effects.

Type B Trichothecenes in Cereal Grains and Their Products: Recent Advances on Occurrence, Toxicology, Analysis and Post-Harvest Decontamination Strategies.

Type B trichothecenes (deoxynivalenol, nivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol) and deoxynivalenol-3-glucoside (DON-3G) are secondary toxic metabolites produced mainly by mycotoxigenic Fusarium fungi and have been recognized as natural contaminants in cereals and cereal-based foods. The latest studies have proven the various negative effects of type B trichothecenes on human health. Due to the widespread occurrence of Fusarium species, contamination by these mycotoxins has become an important aspect for public health and agro-food systems worldwide. Hence, their monitoring and surveillance in various foods have received a significant deal of attention in recent years. In this review, an up-to-date overview of the occurrence profile of major type B trichothecenes and DON-3G in cereal grains and their toxicological implications are outlined. Furthermore, current trends in analytical methodologies for their determination are overviewed. This review also covers the factors affecting the production of these mycotoxins, as well as the management strategies currently employed to mitigate their contamination in foods. Information presented in this review provides good insight into the progress that has been achieved in the last years for monitoring type B trichothecenes and DON-3G, and also would help the researchers in their further investigations on metabolic pathway analysis and toxicological studies of these Fusarium mycotoxins.

Accurate determination of four tetracycline residues in chicken meat by isotope dilution-liquid chromatography/tandem mass spectrometry.

An analytical method based on isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC‒MS/MS) was developed to accurately determine four representative tetracyclines (tetracycline, chlortetracycline, doxycycline, and oxytetracycline) in chicken meat. Tetracyclines are known to have a great tendency for epimerization and keto-enol tautomerism, which often provoke major challenges in their determination. Since this isomerization was found to be unavoidable during the whole chain of the current analysis, the total content (µg kg‒1) of individual tetracycline was quantified as a sum of each parent compound and its respective isomeric forms. Using this approach in combination with IDMS analysis, more consistent, accurate, and reproducible measurement results for the four tetracyclines in chicken meat were acquired. LC-MS/MS conditions and sample preparation processes were comprehensively optimized to minimize the chelating effect of tetracyclines and possible co-extracted interferences. Details of the sample preparation scheme, LC‒MS/MS detection, calculation equation, and method validation are described in this article. The method provided very good accuracy (97.7-102.6%) for all analytes across the concentration range of 10-200 µg kg‒1, with relative standard deviations for intra-day and inter-day precision of less than 4%. The limits of quantification were below 0.2 µg kg‒1, demonstrating the high sensitivity of the method. Furthermore, the measurement uncertainty was generally below 5.5%. Hence, the established method exhibits high-order metrological quality with superior performance over various existing methodologies. Moreover, this method can provide references for general food testing laboratories close to and far below the established maximum residue limits (100 µg kg‒1) for animal muscle tissues.

Development of a certified reference material for the accurate determination of type B trichothecenes in corn.

A corn flour certified reference material (KRISS CRM 108-01-011) was developed to ensure accurate and reliable measurements of type B trichothecenes (deoxynivalenol, nivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol), and deoxynivalenol-3-glucoside. The material was freeze-dried, ground, sieved, and well-mixed. The final produced CRM was packaged at 14 g per unit and stored at -70 °C. The certification was performed using isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). For simultaneous characterization and homogeneity assessments, ten units were randomly selected and analyzed. The among-unit relative standard deviations were below 1 % for all mycotoxins, indicating excellent homogeneity of the CRM. The stability of the CRM was also assessed at various designated temperatures and test periods. The uncertainties of the certified values varied between 2.4 % and 6.2 %, thereby confirming their higher-order metrological quality to provide references for testing laboratories. In case of deoxynivalenol-3-glucoside, an information value was assigned due to the lack of its traceability to the SI units.

Development of isotope dilution-liquid chromatography/tandem mass spectrometry for the accurate determination of zearalenone and its metabolites in corn.

A method using isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC/MS/MS) was developed for the accurate determination of zearalenone (ZEN) and its five major metabolites in corn. 13C- or 2H-labeled analogues of the target mycotoxins were used as internal standards. As the immunoaffinity columns used demonstrated selectivity to a specific chiral isomer of a racemic mixture of zearalanone-d6, a clean-up cartridge without stereoselectivity (Mycosep 226 column) was selected for the same recovery of the analyte and its internal standard with adequate elimination of matrix interferences. The method demonstrated sufficient selectivity, sensitivity, accuracy and precision over a concentration range of 20-400 µg/kg. The limit of detections and limit of quantifications were 0.14-0.33 µg/kg and 0.45-1.11 µg/kg, respectively. The accuracy values were 96.7%-103.6%, with intra and inter-day precisions of less than 3% and 4%, respectively. The expanded measurement uncertainty was less than 7% (with a 95% confidence level).

Development of isotope dilution-liquid chromatography/tandem mass spectrometry as a candidate reference method for the accurate determination of patulin in apple products.

The incidence of patulin (PAT) in fruit products is a worldwide concern due to its acute and chronic toxic effects. Therefore, accurate and reliable PAT measurements are important for preventing consumer health risks. Our previous method, which was based on a common technique that uses ethyl acetate extraction and liquid chromatography-tandem mass spectrometry with isotope dilution (ID-LC-MS/MS), has shown great performance for the determination of PAT in apple products. However, prolonged extraction times and multistep clean-up processes were required to sufficiently eliminate the matrix interferences. Herein, a feasible alternative ID-LC-MS/MS method was successfully established, employing simplified and reliable sample preparation steps. The clean-up process was performed using molecularly imprinted polymer-solid-phase extraction (MIP-SPE) cartridges, which eliminated matrix interferences and facilitated the trace quantification. While the previous method used a multimode LC column for the retention of polar patulin, the current method used a UPLC HSS T3 column, which further improved the peak sharpness and reduced the run time. The method was validated by measuring fortified samples in the concentration range of 5‒100 µg/kg. The accuracy varied between 97.8 and 102.0%, with relative standard deviation for interday and intraday precision being below 3%. The measurement uncertainty was lower than 4% (at a 95% level of confidence). Therefore, this method demonstrated adequate metrological quality with greatly enhanced performance over various reported methods. Additional key benefits of this method are easy manipulation, short preparation time, and lower consumption of hazardous solvents. Finally, the method was successfully applied to commercially available apple-based products.

Development of isotope dilution-liquid chromatography/tandem mass spectrometry for the accurate determination of type-A trichothecenes in grains.

We report the development of a highly accurate method based on isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) for the simultaneous determination of four major type-A trichothecenes in cereal grains. Uniformly labeled [13C] analogues of the target analytes were used as internal standards. An expedient sample preparation procedure was established. After extraction with acetonitrile/water (84:16; v/v), further clean-up was performed using MycoSep 227 solid-phase extraction cartridges. Unlike the commonly used immunoaffinity columns having strong selectivity for only certain target analytes, the cartridges allowed the simultaneous recovery of all four mycotoxins and efficient elimination of co-extracted matrix interferences. The ID-LC-MS/MS method exhibited very good analytical performance in the concentration range of 10-200 µg/kg; accuracy ranged from 97 to 103% with intra-day and inter-day relative standard deviations of less than 5% and 4%, respectively. Measurement uncertainties were generally below 5%. The applicability of the method was assessed by measuring the target mycotoxins in several samples at sub-µg/kg levels.