Simultaneous Determination Of Deoxynivalenol Research Articles

Simultaneous Determination of Deoxynivalenol, Deoxynivalenol-3-Glucoside and Nivalenol in Wheat Grains by HPLC-PDA with Immunoaffinity Column Cleanup

Deoxynivalenol-3-glucoside (D3G) is a modified mycotoxin formed by the metabolism of plants through the conjugation of deoxynivalenol (DON) with glucose. Toxicology studies of D3G for human and animal health are still under investigation, and the development of practical and reliable methods for its direct determination, especially in cereal matrices, is of great importance. In the present study, a methodology for simultaneous determination of D3G, DON, and nivalenol (NIV) in wheat grains, using immunoaffinity column (IAC) cleanup, separation by C18 column and detection by ultraviolet (UV) absorption, was optimized and in-house validated. The results demonstrated adequate values of D3G recovery from IAC and spiked samples. Intraday precision, linearity, limit of detection and limit of quantification (LOQ) were also adequate for the determination of these mycotoxins. Range of applicability varied from 47.1 to 1000 μg/kg for D3G and from 31.3 to 1000 μg/kg for DON and NIV, with recovery ranging from 84.7 ± 7.2 % to 112.3 ± 8.1 %. A high incidence of D3G (41.2 %, all samples <LOQ) was verified in commercial samples of wheat grains and whole wheat-flour (n = 17). Also, the presence of D3G occurred simultaneously with DON in 100 % of the D3G-positive samples. DON levels varied from <LOQ to 325.8 μg/kg, and NIV was detected in only 29.5 % (from <LOQ to 140.6 μg/kg). To the best of our knowledge, this is the first method of simultaneous determination of NIV, D3G, and DON by high-performance liquid chromatography with photodiode array detector (HPLC-PDA) reported until now.

Quantitative determination of trichothecenes in breadsticks by gas chromatography-triple quadrupole tandem mass spectrometry

Breadsticks are pencil-sized sticks of dry bread widely consumed as a pre-meal appetiser. They are basically wheat-based snacks, which makes them a good matrix to evaluate mycotoxin contamination, since wheat is very susceptible to fungal attack. In this sense, the fast, selective and sensitive gas chromatography-triple quadrupole tandem mass spectrometry (GC-QqQ-MS/MS) method proposed here allows simultaneous determination of deoxynivalenol (DON), 3-acetyldeoxynivalenol, fusarenon-X, diacetoxyscirpenol, nivalenol, neosolaniol, HT-2 and T-2 toxin in breadsticks after QuEChERS extraction and clean-up. The performance of the method was assessed with respect to European Commission Regulations by studying the selectivity and specificity, limit of detection (LOD), limit of quantitation (LOQ), linearity, matrix effect, accuracy, precision and trueness. Satisfactory results in terms of validation parameters were obtained for all selected mycotoxins (recovery range of 70–110%, RSD < 10%, LOQ <40 µg kg–1). The trueness of the method was supported by using certified reference material (DON 1062 ± 110 µg kg–1). The method was successfully used to evaluate the occurrence of the studied Fusarium toxins in 61 breadstick samples. A total of 64% of the samples showed mycotoxin contamination, DON being the most frequently detected toxin. Nonetheless, mean levels obtained were far below the maximum levels permitted by European Union legislation. An additional goal was to carry out a risk-characterisation approach to DON by comparing probable daily intake and provisional maximum tolerable daily intake (PMTDI).

Critical evaluation of LC-MS-based methods for simultaneous determination of deoxynivalenol, ochratoxin A, zearalenone, aflatoxins, fumonisins and T-2/HT-2 toxins in maize

The results of a proficiency test for the LC-MS/(MS) determination of up to 11 mycotoxins (aflatoxins B1, B2, G1 and G2, fumonisins B1 and B2, ochratoxin A, deoxynivalenol, T-2 and HT-2 toxins and zearalenone) in maize were evaluated to identify possible strengths and weaknesses of various methodologies used by the 41 participating laboratories. The majority of laboratories (56%) used mixtures of acetonitrile:water for extraction. Other laboratories used methanol:water mixtures (17%) or performed two consecutive extractions with phosphate buffer solution (PBS) followed by methanol (15%). Few laboratories used mixtures of acetonitrile:water:methanol (7%), water:ethyl acetate (2.5%) or PBS alone (2.5%). The majority of laboratories (58%) used a clean-up step prior to chromatography. The remaining laboratories analysed crude extracts (37%) or used a mixed approach (5%). The amount of sample equivalent injected into LC-MS/(MS) ranged between 0.1-303 mg for purified extracts and 0.08-20 mg for directly analysed crude extracts. External (54%), matrix-matched (22%) or stable isotope-labelled internal standards calibration (24%) were used for toxin quantification. In general, extraction mixtures of water with acetonitrile, methanol or both provided good results for quantitative extraction of mycotoxins from maize. Laboratories using sample extract clean-up reported acceptable results for the majority of mycotoxins. Good results were also obtained by laboratories that analysed crude extracts although a high variability of results was observed for all tested mycotoxins. Matrix-matched calibration or isotope-labelled internal standards efficiently compensated matrix effects whereas external calibration gave reliable results by injecting ≤10 mg of matrix equivalent amounts. Unacceptable high recovery and high variability of fumonisin results were obtained by the majority of laboratories, which could not be explained and thus require further investigation. These findings provide the basis for the optimization and selection of methods to be used in future interlaboratory validation studies to derive their performance characteristics for simultaneous determination of mycotoxins in maize.

Simultaneous determination of deoxynivalenol, and 15- and 3-acetyldeoxynivalenol in cereals by HPLC-UV detection

Fusarium head blight is an important cereal crop disease, which not only causes yield losses but also mycotoxin contamination in wheat and other cereal grains. Developing an accurate, rapid and efficient assay is critical to minimise the risk ofFusarium mycotoxins for human and animal health. In this study, HPLC with UV detection was used to separate and quantify deoxynivalenol, 15-acetyldeoxynivalenol and 3-acetyldeoxynivalenol in cereals. Samples were extracted with water, and the extracting solution was precipitated by adding an equal volume of ethanol followed by solid-phase extraction. The analytes were separated on a reversed-phase C18 column by a mobile phase composed of acetonitrile and 1 mM H3PO4 with gradient elution. 15- and 3-acetyldeoxynivalenol showed effective baseline separation. All analytes were well-resolved from matrix co-extractives and detected at 224 nm. The results showed good linearity of calibration curves (R2 ranged from 0.997 to 0.999) and excellent precision for inter- and intra-day determinations. Average recovery rates for the tested matrices ranged from 71 to 92%. The limits of detection and quantification ranged from 16 to 25 ng/g and 48 to 60 ng/g, respectively. The results indicate that the feasibility and practicality of the presented LC-UV method are excellent and that the method is suitable for routine analysis of DON and its acetyl derivatives in cereal grains.

Simultaneous determination of deoxynivalenol, zearalenone, T‐2 and HT‐2 toxins in breakfast cereals and baby food by high‐performance liquid chromatography and tandem mass spectrometry

In this study, the simultaneous determination of deoxynivalenol (DON), zearalenone (ZEA), T-2 and HT-2 toxins in foodstuff was investigated. A new kind of multi-mycotoxin immunoaffinity columns (IACs) available on the market (DZT MS-PREP(®)) was tested. A sensitive, selective and accurate method by high-performance liquid chromatography and tandem mass spectrometry was developed, with electrospray ionization mass spectrometer operating in multiple reaction monitoring (MRM) mode, with negative-positive-negative ion switching. The method was used for the analysis of samples marked in Italy, in the frame of official monitoring plans. The advantages of combining IACs and LC-MS/MS technique are as follows: efficient removal of matrix interferences, simple chromatographic outline, high selectivity, low detection limits (DLs) and separation of a wide range of molecules with different physico-chemical properties in a single run. The method was studied on two different matrices, breakfast cereal and baby food, at contamination levels close to Regulation limits (EC) 1126/2007. The recoveries obtained (60-100%) fulfil the performance criteria required by Regulation (EC) 401/2006. The DL is 60 µg/kg for DON and 10 µg/kg for ZEA, T-2 and HT-2. Linearity range of the calibration curves is suitable for adult and baby food.

Simultaneous Determination of Deoxynivalenol and Nivalenol in Traditional Chinese Medicine by SPE and LC

A liquid chromatography method for simultaneous determination of deoxynivalenol (DON) and nivalenol (NIV) in traditional Chinese medicine (TCM) is described for the first time. Different clean-up columns and extraction solvents have been optimized. The DON purification column was chosen as the best. The detection limits for DON and NIV were 62.5 and 50.0 μg kg−1, respectively. Recoveries from different TCMs spiked with DON and NIV at levels ranging from 0.5 to 10 mg kg−1 were 82.8–99.9% and 80.8–100.3%, respectively. None of the 30 commercially available TCM samples analyzed, were found to contain any detectable amounts of DON and NIV.

Simultaneous determination of deoxynivalenol, zearalenone, and their major masked metabolites in cereal-based food by LC–MS–MS

Cereals and cereal-based food have often been found to be contaminated with the mycotoxins deoxynivalenol (DON) and zearalenone (ZON), after infection of the grain with the pathogenic fungus Fusarium. Both the pathogen and the infected plants can chemically modify DON and ZON, including acetylation, glucosidation, and sulfation. Analytical strategies for detection and quantification of DON and ZON are well known and established but often fail to recognize the respective metabolites, which are, therefore, also referred to as "masked" mycotoxins. However, several masked forms are also known to be harmful to mammals. Failure to detect these could lead to significant underestimation of the toxic potential of a particular sample. To monitor the levels of DON and ZON metabolites in cereals and cereal-based food, we have developed a LC-MS-MS method capable of simultaneous determination of DON, ZON, and eight of their masked metabolites, namely deoxynivalenol-3-glucoside (D3G), 3-acetyl-deoxynivalenol (3ADON), zearalenone-4-glucoside (Z4G), alpha-zearalenol (alpha-ZOL), beta-zearalenol (beta-ZOL), alpha-zearalenol-4-glucoside (alpha-ZG), beta-zearalenol-4-glucoside (beta-ZG), and zearalenone-4-sulfate (Z4S). The suitability of several cleanup strategies including C(18)-SPE, primary and secondary amines (PSA), MycoSep push-through columns, and immunoaffinity columns was evaluated. The final method used no sample cleanup and was successfully validated for four cereal-based food matrices, namely cornflour, porridge, beer, and pasta, showing good recoveries and precision for all analytes.

OPTIMIZATION OF ATMOSPHERIC PRESSURE CHEMICAL IONIZATION INTERFACE PARAMETERS FOR THE SIMULTANEOUS DETERMINATION OF DEOXYNIVALENOL AND ZEARALENONE USING HPLC/MS

In this paper, the influence of several atmospheric pressure chemical ionization (APCI) parameters were investigated in flow-injection for the simultaneous determination of deoxynivalenol (DON) and zearalenone (ZON) using liquid chromatography (LC) coupled to a mass spectrometer (MS). During the optimization procedure of the APCI interface, it was revealed that vaporiser temperature and capillary temperature had a strong influence on the MS signal of DON and ZON in the positive and in the negative ionization mode. In the positive mode a higher absolute signal of both compounds was obtained, while in the negative mode a higher selectivity was achieved. Thereafter, tube lens offset, corona discharged current, and capillary voltage were investigated in the negative mode; it was shown that only the tube lens offset had a large influence on the MS signal of the analytes. Finally, the optimized conditions were confirmed injecting and separating a standard mixture on a LC column prior to MS detection.