Analysis of Patulin in Apple Products Marketed in Belgium: Intra-Laboratory Validation Study and Occurrence.

Apple and apple derivatives (e.g., juices, puree) are the most important foodstuffs contaminated with patulin (PAT) in the human diet. To routinely monitor these foodstuffs and ensure that the PAT levels are below the maximum permitted levels, a method using liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) has been developed. Afterwards, the method was successfully validated, reaching quantification limits of 1.2 µg/L for apple juice and cider, and 2.1 µg/kg for puree. Recovery experiments were performed with samples fortified with PAT in the range of 25-75 µg/L for juice/cider and 25-75 µg/kg for puree. The results show overall average recovery rates of 85% (RSDr = 13.1%) and 86% (RSDr = 2.6%) with maximum extended uncertainty (Umax, k = 2) of 34 and 35% for apple juice/cider and puree, respectively. Next, the validated method was applied to 103 juices, 42 purees and 10 ciders purchased on the Belgian market in 2021. PAT was not found in the cider samples, but it was present in 54.4% of the tested apple juices (up to 191.1 µg/L) and 7.1% of the puree samples (up to 35.9 µg/kg). When comparing the results to the maximum levels set by Regulation EC n° 1881/2006 (i.e., 50 µg/L for juices and 25 µg/kg for puree for adults, and 10 µg/kg for infants and young children), exceedances were observed in five apple juices and one puree sample, for infants and young children. Using these data, a potential risk assessment for consumers can be suggested, and it is found that the quality of apple juices and purees sold in Belgium needs further regular surveillance.

Development of a reliable UHPLC-MS/MS method for simultaneous determination of zearalenone and zearalenone-14-glucoside in various feed products.

A reliable ultra-high-performance liquid chromatography-tandem mass spectrometry method (UHPLC-MS/MS) was developed for the simultaneous determination of two mycotoxins, that is, zearalenone (ZEN) and zearalenone-14-glucoside (ZEN-14G) in formula feed, concentrated feed, and premixed feed products. An improved sample pretreatment was achieved with the hydrophilic-lipophilic balance (HLB) cartridges efficiently removing the impurities and enriching the target analytes in different feeds. The critical parameters affecting the performance of the solid-phase extraction (SPE) procedure were carefully optimized, and 20% acetonitrile in water as the loading solution, 50% methanol in water as the washing solvent, and 5 ml of methanol as the elution solvent yielded the optimal purification efficiencies. The established method was thoroughly validated in terms of linearity (R 2 ≥ 0.999), sensitivity (limit of quantification in the range of 0.50-5.00 µg kg-1), recovery (89.35 ± 2.67% to 110.93 ± 1.56%), and precision (RSD, 3.00-14.20%), and it was then successfully applied to investigate a total of 60 feed samples. Among them, 50 samples were found to be contaminated with ZEN (an incidence of 83.3%) at levels ranging from 0.63 to 615.24 µg kg-1, whereas 22 samples were contaminated with ZEN-14G (an incidence of 36.7%) in the range of 0.89-15.31 µg kg-1. The developed method proved to be a specific and reliable tool for intensive monitoring of ZEN and ZEN-14G in complex feed matrices.

Organisation of Multi-Mycotoxin Proficiency Tests: Evaluation of the Performances of the Laboratories Using the Triple A Rating Approach.

In accordance with the International Standard Organization ISO 17043, two proficiency tests (PTs) for the simultaneous determination of aflatoxins (AFB1, AFB2, AFG1, AFG2); deoxynivalenol; fumonisins FB1, FB2, and B3; ochratoxin A, the T-2 toxin; and the HT-2 toxin were conducted in 2019 and 2020 using cornflakes and rusk flours that were prepared in house. The homogeneity and the stability of these materials were verified according to the criteria laid down in ISO 13528 using randomly selected samples. Most of the targeted toxins were found to be homogenously distributed in both materials with no significant changes during the timescale of the PTs. Next, the materials were distributed to approximately 25 participating laboratories from Europe, Canada, and the United States. The obtained datasets were computed using robust statistics. The outliers were checked and removed, and the toxin concentrations were assigned as the consensus value of the results of the participants at Horwitz ratios <1.2. The z scores were generated for all mycotoxins, and the results were pooled to calculate the relative sum of squared z scores (SZ2) indexes and were clustered according to the triple A rating. Overall, at least 80% of the participating laboratories achieved good and acceptable performances. The most frequent categories assigned to good performances (SZ2 ≤ 2) were AAA (51%) and BAA (13%). Clusters of BBA + CBA (6%) included laboratories reporting acceptable z scores <90% of the total z scores for less than 90% or 50% of the mycotoxins targeted in the 2 matrices. The triple A rating seems to be appropriate in evaluating the performances of laboratories involved in multi-mycotoxin analyses. Accredited and non-accredited analytical methods achieved good and acceptable performances.

Production of Alternaria Toxins in Yellow Peach (Amygdalus persica) upon Artificial Inoculation with Alternaria alternate.

The yellow peach (Amygdalus persica), an important fruit in China, is highly susceptible to infection by Alternaria sp., leading to potential health risks and economic losses. In the current study, firstly, yellow peaches were artificially inoculated with Alternariaalternate. Then, the fruits were stored at 4 °C and 28 °C to simulate the current storage conditions that consumers use, and the Alternaria toxins (ATs) contents from different parts of the fruits were analyzed via ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The results showed that the growth of A. alternate and the ATs production were dramatically affected by the storage temperature. At 28 °C, the fungi grew rapidly and the lesion diameter reached about 4.0 cm within 15 days of inoculation, while, at 4 °C, the fungal growth was noticeably inhibited, with no significant change in the lesion diameter. To our surprise, high contents of ATs were produced under both storage conditions even though the fungal growth was suppressed. With an increase in the incubation time, the amounts of ATs showed a steady tendency to increase in most cases. Remarkably, alternariol monomethyl ether (AME), alternariol (AOH), and tenuazonic acid (TeA) were detected in the rotten tissue and also in the surrounding tissue, while a large amount of TeA could also be found in the healthy tissue. To the best of our knowledge, this is the first report regarding the production of ATs by the infection of Alternaria sp. in yellow peach fruits via artificial inoculation under regulated conditions, and, based on the evidence herein, it is recommended that ATs be included in monitoring and control programs of yellow peach management and food safety administration.

Citrinin Determination in Food and Food Supplements by LC-MS/MS: Development and Use of Reference Materials in an International Collaborative Study.

The development of incurred reference materials containing citrinin (CIT) and their successful application in a method validation study (MVS) in order to harmonize CIT determination in food and food supplements are demonstrated. CIT-contaminated materials made of red yeast rice (RYR), wheat flour, and Ginkgo biloba leaves (GBL), as well as food supplements made of red yeast rice (FS-RYR) and Ginkgo biloba leaves (FS-GBL), were manufactured in-house via fungal cultivation on collected raw materials. The homogeneity and stability from randomly selected containers were verified according to the ISO 13528. CIT was found to be homogenously distributed and stable in all contaminated materials, with no significant degradation during the timescale of the MVS when storage was performed up to +4 °C. Next, an MVS was organized with eighteen international laboratories using the provided standard operating procedure and 12 test materials, including three RYRs (blank, <50 µg/kg, <2000 µg/kg), two wheat flours (blank, <50 µg/kg), two GBL powders (blank, <50 µg/kg), three FS-RYRs (blank, <50 µg/kg, <2000 µg/kg), and two FS-GBLs (blank, <50 µg/kg). The results of seven CIT-incurred materials showed acceptable within-laboratory precision (RSDr) varying from 6.4% to 14.6% and between-laboratory precision (RSDR) varying from 10.2% to 37.3%. Evidenced by HorRat values < 2.0, the results of the collaborative trial demonstrated that the applied analytical method could be standardized. Furthermore, the appropriateness of producing CIT reference materials is an important step towards food and feed quality control systems and the organization of proficiency tests.

In Vivo Kinetics and Biotransformation of Aflatoxin B1 in Dairy Cows Based on the Establishment of a Reliable UHPLC-MS/MS Method.

The in vivo kinetics of aflatoxin B1 (AFB1) and its carry-over as aflatoxin M1 (AFM1) in milk as well as the toxin loads in the tissue of dairy cows were assessed through a repetitive feeding trial of an AFB1-contaminated diet of 4 µg kg-1 body weight (b.w.) for 13 days. This was followed by a clearance period that ended with a single dose trial of an AFB1-contaminated diet of 40 µg kg-1 b.w. An ultra-high performance liquid chromatography tandem mass spectrometry method was developed and successfully validated by the determination of linearity (R 2 ≥ 0.990), sensitivity (lower limit of quantification, 0.1-0.2 ng ml-1), recovery (79.5-111.2%), and precision relative standard deviation (RSD) ≤14.7%) in plasma, milk, and various tissues. The repetitive ingestion of AFB1 indicated that the biotransformation of AFB1 to AFM1 occurred within 48 h, and the clearance period of AFM1 in milk was not more than 2 days. The carry-over rate of AFM1 in milk during the continuous ingestion experiment was in the range of 1.15-2.30% at a steady state. The in vivo kinetic results indicated that AFB1 reached a maximum concentration of 3.8 ± 0.9 ng ml-1 within 35.0 ± 10.2 min and was slowly eliminated from the plasma, with a half-life time (T1/2) of 931.1 ± 30.8 min. Meanwhile, AFM1 reached a plateau in plasma (0.5 ± 0.1 ng ml-1) at 4 h after the ingestion. AFB1 was found in the heart, spleen, lungs, and kidneys at concentrations of 1.6 ± 0.3, 4.1 ± 1.2, 3.3 ± 0.9 and 5.6 ± 1.4 µg kg-1, respectively. AFM1 was observed in the spleen and kidneys at concentrations of only 0.7 ± 0.2 and 0.8 ± 0.1 µg kg-1, respectively. In conclusion, the in vivo kinetics and biotransformation of AFB1 in dairy cows were determined using the developed UHPLC-MS/MS method, and the present findings could be helpful in assessing the health risks to consumers.

A reliable and accurate UHPLC-MS/MS method for screening of Aspergillus, Penicillium and Alternaria mycotoxins in orange, grape and apple juices.

An ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for simultaneous determination of 15 mycotoxins, including aflatoxins (B1, B2, G1, and G2), ochratoxins (A, B, and C), citrinin, patulin, and emerging Alternaria toxins (alternariol, alternariol monomethyl ether, altenuene, tentoxin, tenuazonic acid, and altenusin) in orange, grape and apple juices. Different extraction approaches, sorbents, chromatographic columns and mobile phases were investigated for establishment of an optimal QuEChERS procedure and UHPLC-MS/MS conditions. Recoveries were in the range of 74-110%, and the limits of detection (LODs) and limits of quantification (LOQs) ranged from 0.05 to 0.1 ng mL-1 and from 0.1 to 5.0 ng mL-1, respectively. Matrix effects were evaluated and matrix-matched calibration curves were used to compensate for matrix effects and achieve accurate quantification. The correlation coefficients (R2) of linearity were higher than 0.99 and the relative standard deviations (RSDs) of intra- and inter-day precision were under 13%. The method was subsequently applied to 22 fruit juice samples. The high frequencies (90.9%) of mycotoxins not only proved the reliability and sensitivity of the currently established method, but also demonstrated that fruit juices are susceptible to different mycotoxins, which need to be continuously monitored in the future.

Determination of ochratoxin A in edible pork offal: intra-laboratory validation study and estimation of the daily intake via kidney consumption in Belgium.

Pork-derived products can contribute to the overall ochratoxin A (OTA) intake via carry-over from contaminated feed or via mould spoilage of meat products (salami, dry-cured ham, sausage). An analytical method using liquid chromatography coupled with mass spectrometry (LC-MS/MS) was developed and validated in accordance with the specifications laid down by European Commission. It offered quantification limits of 0.2 for kidney, liver and 0.4 µg/kg for black sausage. Spiking experiments of blank samples at 5-10 µg/kg showed recoveries ranging from 88 to 101%, 89 to 97% and 80 to 85% for kidney, liver and black sausage, respectively. The respective intra-laboratory repeatabilities ranged between 9.8-11.1%, 9.4-14.4% and 9.7-14.2%, and extended measurement uncertainties MU(k = 2) were 33%, 35% and 43% for kidney, liver and black sausage. Next, the validated method was applied to kidney (110), liver (20) and black sausage (20) samples collected in Belgium in the period 2012-2019. Neither liver nor black sausage samples were contaminated with OTA. Kidney samples (37.3%) were OTA contaminated at the mean level of 0.22 ± 0.25 µg/kg (up to 1.91 µg/kg). These data combined with the offal consumption in the Belgian population revealed average daily OTA exposures ranged from 0.167 and 0.319 ng/kg bw for 3 age groups (3-9, 10-17 and 18-64 years). Taking into account, the OTA non-neoplastic and neoplastic effects, risk characterization assessed via the margin of exposure for reference endpoints revealed no potential health risk for the consumers. As the presence of low OTA content in foods together with other mycotoxins or derivatives may interactively potentiate its toxicity, monitoring of OTA and its metabolites in meat and meat by-products is advised.

Dietary exposure assessment and risk characterization of citrinin and ochratoxin A in Belgium.

Exposure to mycotoxins is a worldwide problem. To ensure public health, it is imperative to characterize the risks related to these toxins. The present study aims to conduct a dietary exposure assessment of citrinin (CIT) and ochratoxin A (OTA) in the Belgian population using consumption data of a variety of foodstuffs. A total of 367 food samples from different food categories were collected in Belgian supermarkets and analysed for CIT and OTA using a validated liquid chromatography-tandem mass spectrometry method. Daily CIT and OTA exposure to the Belgian population was calculated based on the analytical results and food consumption data in three age categories (3-9, 10-17 and 18-64 years), obtained from a national food consumption survey. Furthermore, a risk characterization was performed for CIT, in which no intake values exceeded the tolerable daily intake (TDI) of 200 ng kg-1 bw day-1, indicating no health risk. However, a CIT intake level of 187 ng kg-1 bw day-1 was detected for children in the age category of 3-9 years in the worst case scenario for rice, indicating that rice consumption could contain a potential health hazard for young children. For OTA, a potential health risk was detected in several food categories (biscuits, croissants, rice, flour, meat imitates, herbs and spices) in the higher percentiles (P99) or at maximum found concentrations when calculating the margin of exposure (MoE) for neoplastic effects. An attempt to perform a cumulative health risk assessment for both toxins was done. Although a high number of uncertainties is involved, combined margin of exposure (MoET) values indicated a potential health risk related to the combined exposure to CIT and OTA. For the first time, our study demonstrated the potential health risks of CIT and OTA after individual and combined exposure, in particular related to rice consumption. Moreover, further research is recommended concerning multiple mycotoxin exposure in young children.

A Study of Carry-Over and Histopathological Effects after Chronic Dietary Intake of Citrinin in Pigs, Broiler Chickens and Laying Hens.

Citrinin (CIT) is a polyketide mycotoxin occurring in a variety of food and feedstuff, among which cereal grains are the most important contaminated source. Pigs and poultry are important livestock animals frequently exposed to mycotoxins, including CIT. Concerns are rising related to the toxic, and especially the potential nephrotoxic, properties of CIT. The purpose of this study was to clarify the histopathological effects on kidneys, liver, jejunum and duodenum of pigs, broiler chickens and laying hens receiving CIT contaminated feed. During 3 weeks, pigs (n = 16) were exposed to feed containing 1 mg CIT/kg feed or to control feed (n = 4), while 2 groups of broiler chickens and laying hens (n = 8 per group) received 0.1 mg CIT/kg feed (lower dose group) and 3 or 3.5 mg CIT/kg feed (higher dose group), respectively, or control feed (n = 4). CIT concentrations were quantified in plasma, kidneys, liver, muscle and eggs using a validated ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method. Kidneys, liver, duodenum and jejunum were evaluated histologically using light microscopy, while the kidneys were further examined using transmission electron microscopy (TEM). Histopathology did not reveal major abnormalities at the given contamination levels. However, a significant increase of swollen and degenerated mitochondria in renal cortical cells from all test groups were observed (p < 0.05). These observations could be related to oxidative stress, which is the major mechanism of CIT toxicity. Residues of CIT were detected in all collected tissues, except for muscle and egg white from layers in the lowest dose group, and egg white from layers in the highest dose group. CIT concentrations in plasma ranged between 0.1 (laying hens in lower dose group) and 20.8 ng/mL (pigs). In tissues, CIT concentrations ranged from 0.6 (muscle) to 20.3 µg/kg (liver) in pigs, while concentrations in chickens ranged from 0.1 (muscle) to 70.2 µg/kg (liver). Carry-over ratios from feed to edible tissues were between 0.1 and 2% in pigs, and between 0.1 and 6.9% in chickens, suggesting a low contribution of pig and poultry tissue-derived products towards the total dietary CIT intake for humans.

Comprehensive toxicokinetic analysis reveals major interspecies differences in absorption, distribution and elimination of citrinin in pigs and broiler chickens.

A comprehensive toxicokinetic analysis of citrinin (CIT) revealed interspecies differences for all toxicokinetic parameters and in absolute oral bioavailability. Oral bioavailability for CIT was complete for broilers (113-131%), while ranging from 37 to 44% in pigs. CIT was more rapidly absorbed in pigs (Tmax = 0.92 h) compared to broiler chickens (Tmax = 7.33 h). The elimination of CIT was slower in pigs (T1/2el = 26.81 h after intravenous (IV) administration) compared to chickens (T1/2el = 1.97 h after IV administration), due to the striking difference in clearance (Cliv=9.87 mL/h/kg for pigs versus Cliv = 863.09 mL/h/kg for broilers). Also, the volume of distribution differed significantly between pigs (Vd = 0.30 L/kg after IV administration) and chickens (Vd = 2.46 L/kg after IV administration). However, plasma protein binding did not differ statistically significant (91-98%). It is imperative to further investigate biotransformation and elimination pathways in different species, including humans.

Development of a QuEChERS-Based UHPLC-MS/MS Method for Simultaneous Determination of Six Alternaria Toxins in Grapes.

A simple and reliable analytical method for the simultaneous determination of alternariol (AOH), altenuene (ALT), tentoxin (TEN), altenusin (ALS), tenuazonic acid (TeA), and alternariol monomethyl ether (AME) in grapes was developed by ultra-high-performance liquid chromatography⁻tandem mass spectrometry (UHPLC-MS/MS). A modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) procedure with the extraction by acetonitrile and purification by sodium chloride (0.5 g) and anhydrous magnesium sulfate (0.5 g) was established to recover the six Alternaria toxins. After validation by determining the linearity (R² > 0.99), recovery (77.8⁻101.6%), sensitivity (limit of detection in the range of 0.03⁻0.21 µg kg-1, and limit of quantification in the range of 0.09⁻0.48 µg kg-1), and precision (relative standard deviation (RSD) ≤ 12.9%), the analytical method was successfully applied to reveal the contamination state of Alternaria toxins in grapes. Among 56 grape samples, 40 (incidence of 71.4%) were contaminated with Alternaria toxins. TEN was the most frequently found mycotoxin (37.5%), with a concentration range of 0.10⁻1.64 µg kg-1, followed by TeA (28.6%) and AOH (26.8%). ALT (10.7%), AME (3.6%), and ALS (5.4%) were also detected in some samples. To the best of our knowledge, this is the first report about the Alternaria toxins contamination in grapes in China.

Development and validation of an LC-MS/MS method for the simultaneous determination of citrinin and ochratoxin a in a variety of feed and foodstuffs.

An ultra-performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-ESI+/--MS/MS) method for the simultaneous analysis of citrinin (CIT) and ochratoxin A (OTA) in feed (chicken and pig) and food (cereal-based products, fruit, vegetable juices, nuts, seeds, herbs, spices, vegetarian and soy products, alcoholic beverages, baby food products and food supplements) was developed. The mycotoxins were extracted from these matrices using a QuEChERS-based extraction method without any further clean-up step. The samples were 5-fold concentrated. Final extracts were analyzed using a UPLC-MS/MS system and chromatographic separation was achieved by applying a gradient elution for a total run time of 10 min. Mycotoxins were quantified using an internal calibration via analyte/13C-labeled internal standard ratio. The developed method was validated according to the criteria described in Commission Regulation No. 401/2006/EC and Commission Decision No. 2002/657/EC. Specificity, linearity, apparent recovery, limit of detection and quantification, intraday and interday precision, measurement uncertainty, matrix effect, and extraction efficiency were the parameters studied. Finally, 90 Belgian chicken and pig feed samples were analyzed, revealing the simultaneous presence of CIT (

In vitro glucuronidation of ochratoxin a by rat liver microsomes.

Ochratoxin A (OTA), one of the most toxic mycotoxins, can contaminate a wide range of food and feedstuff. To date, the data on its conjugates via glucuronidation request clarification and consolidation. In the present study, the combined approaches of ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), UHPLC-Orbitrap-high resolution mass spectrometry (HRMS) and liquid chromatography-multiple stage mass spectrometry (LC-MS(n)) were utilized to investigate the metabolic profile of OTA in rat liver microsomes. Three conjugated products of OTA corresponding to amino-, phenol- and acyl-glucuronides were identified, and the related structures were confirmed by hydrolysis with ß-glucuronidase. Moreover, OTA methyl ester, OTα and OTα-glucuronide were also found in the reaction solution. Based on these results, an in vitro metabolic pathway of OTA has been proposed for the first time.

Deoxynivalenol loads in matched pair wheat samples in Belgium: comparison of ELISA VERATOX kit against liquid chromatography.

A comparison of matched pairs deoxynivalenol (DON) loads in wheat samples via VERATOX for DON 5/5 performed by two laboratories against two liquid chromatographic methods (LC-MS/MS and HPLC-UV) used by two other laboratories was carried out using biometrical and sum of ranking differences (SRD) procedures. The Lin's Concordance correlation coefficients, the average discrepancies, the limits of agreement and the SRD between ELISA and reference values showed good overall agreement between VERATOX for DON 5/5 and reference methods for the two datasets. The VERATOX kits are valuable for quantitative screening and even for an initial exposure assessment in situations when there are practical or economical reasons not to use sophisticated methods such as HPLC or GC methods (with or without MS). However, networking of laboratories using this rapid method and laboratories with reference analytical methods should be encouraged.

Cross-reactivity of antibodies in some commercial deoxynivalenol test kits against some fusariotoxins.

Cross-reactivity of antibodies in AGRAQUANT, DON EIA, VERATOX, ROSA LF-DONQ, and MYCONTROLDON designed for deoxynivalenol (DON) determination in food and feedstuffs was evaluated against nivalenol, 3-acetylDON, 15-acetylDON, de-epoxy metabolite 1 of DON, DON-3ß-glucoside, T2-toxin, HT2-toxin, fusarenone X, diacetoxyscirpenol, verrucarol, and zearalenone. Cross-reactivity measurements were run in water using the 50% reduction of absorbance of the blank for ELISA kits or through direct DON determination upon using the standards of mycotoxins via ROSA LF-DONQ or MYCONTROLDON. For the tested toxin concentrations, all DON kits have low cross-reactivity toward diacetoxyscirpenol, T2-toxin, HT2-toxin, verrucarol, and zearalenone and moderate cross-reactivity toward 15-AcetylDON and fusarenone X. AGRAQUANT, DON EIA, and VERATOX kits showed high cross-reactivity in various ranking orders against DON-3-Glc, DOM-1, and 3AcDON. DON EIA showed also high cross-reactivity against nivalenol and fusarenone X. These mycotoxins could coexist in food or feedstuffs, and analytical results can be wrongly interpreted. Cross-reactivity does not allow checking the compliance with the legal norms, but it does allow an overall risk assessment for the consumers. Updating regularly the cross-reactivity evaluation of the produced batches is recommended for 3-acetylDON, nivalenol, DON-3-Glc, de-epoxy metabolite 1, and fusarenone X.