Search Results (17)

The presence of mycotoxins in cheese is a significant concern due to their potential health risks. Mycotoxins can contaminate cheese through two main routes: indirectly via contaminated animal feed, and/or directly, because of mold growth on dairy products. It has been reported that cheese may contain metabolites of aflatoxin B1 such as aflatoxin M1 (AFM1), aflatoxicol (AFL), and, its precursor, sterigmatocystin (STC). This study presents a reliable method for the simultaneous determination of AFM1, AFL, and STC in cheeses made from ovine, goat, or buffalo milk. The method was developed using single liquid extraction, clean-up by an immunoaffinity column (IAC), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) determination. The method was subjected to initial validation according to EU regulations, which outline the required performance parameters and criteria of analytical methods for official food control. The limits of quantification (LOQs) of the method for AFM1, AFL, and STC are 2.0 ng/kg, 5.0 ng/kg, and 1.0 ng/kg, respectively. The method was applied in a study for the assessment of mycotoxin transfer from milk to cheeses and also their growth. Full article

The detection and quantification of mycotoxins in beer are critical for ensuring consumer safety and regulatory compliance. These contaminants, originating from barley and other grains, persist and potentially transform during the brewing process. This study presents an innovative analytical protocol using liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) for the simultaneous qualitative and quantitative analysis of nine mycotoxins, including aflatoxins (AFB1, AFB2, AFG1, AFG2), Ochratoxin A (OTA), Fumonisins (FB1, FB2), Deoxynivalenol (DON), and HT-2. The method leverages the efficiency of multi-mycotoxin immunoaffinity columns, providing streamlined sample preparation with high specificity and sensitivity. Validation was conducted using craft beers from Calabria, including freeze-dried samples to enhance analytical consistency and stability. The method’s accuracy was confirmed by using spiking samples with mycotoxins at concentrations compliant with the European Commission’s regulations (Recommendation 2024/1038/EU). The developed protocol delivers reliable results with minimized resource consumption, offering a robust tool for quality control and safety assessments in brewing. By addressing knowledge gaps in freeze-dried craft beer, this study contributes to advancing food safety standards in the brewing industry. Full article

This study evaluates the effects of fermented whey (FW) and pumpkin (P) on the excretion of aflatoxin B1 (AFB1) and ochratoxin A (OTA) in rats using immunoaffinity column cleanup and high-performance liquid chromatography–fluorescence detection (IAC-LC-FLD). The method achieved detection limits of 0.1 µg/kg for AFB1 and 0.3 µg/kg for OTA, with recovery rates ranging from 72–92% for AFB1 and 88–98% for OTA. A fecal analysis of 100 rats showed peak AFB1 concentrations of 418 µg/kg and OTA of 1729 µg/kg. In the toxin-exposed groups, OTA levels were higher than AFB1, with males in the OTA-only group showing significantly higher OTA (1729 ± 712 µg/kg) than females (933 ± 512 µg/kg). In the AFB1-only group, the fecal levels were 52 ± 61 µg/kg in males and 91 ± 77 µg/kg in females. The AFB1 + FW group showed notable AFB1 concentrations (211 ± 51 µg/kg in males, 230 ± 36 µg/kg in females). The FW + P combination further influenced excretion, with higher AFB1 and OTA levels. These findings suggest that FW and P modulate mycotoxin excretion and may play a role in mycotoxin detoxification, providing insight into dietary strategies to reduce mycotoxin exposure and its harmful effects. Full article

Aflatoxin B1 (AFB1) and ochratoxin A (OTA) are highly toxic mycotoxins present in food and feed, posing serious health risks to humans and animals. This study aimed to validate an efficient and cost-effective analytical method for quantifying AFB1 and OTA in rat urine using immunoaffinity column extraction and liquid chromatography with fluorescence detection (IAC-LC-FD). Additionally, the study evaluated the effect of incorporating fermented whey and pumpkin into the feed on the urinary excretion of these mycotoxins. The limits of detection and quantification were determined to be 0.1 µg/kg and 0.3 µg/kg, respectively, for both mycotoxins in feed, and 0.2 ng/mL and 0.6 ng/mL, respectively, in urine. The method demonstrated robust recovery rates ranging from 74% to 119% for both AFB1 and OTA in both matrices. In feed samples, the levels of AFB1 and OTA ranged from 4.3 to 5.2 µg/g and from 5.4 to 8.8 µg/g, respectively. This validated method was successfully applied to analyze 116 urine samples from rats collected during the fourth week of an in vivo trial. The results indicated that the addition of fermented whey and pumpkin to the feed influenced mycotoxin excretion in urine, with variations observed based on the sex of the rats, type of mycotoxin, and exposure dosage. Full article

In recent years, there has been an intensification of weather variability worldwide as a result of climate change. Some regions have been affected by drought, while others have experienced more intense rainfall. The incidence and severity of moldy grain and mycotoxin contamination during the growing and harvesting seasons have increased as a result of these weather conditions. Additionally, torrential rains and wet conditions may cause delays in grain drying, leading to mold growth in the field. In July 2023, a wheat field in Lecco (Lombardy, Italy) was affected by torrential rains that led to the development of the Claviceps fungi. In the field, dark sclerotia were identified on some ears. Wheat ears, kernels, and sclerotia were collected and analyzed by LC-MS/MS at IZSLER, Food Chemical Department, in Bologna. The wheat ears, kernels, and sclerotia were analyzed for 12 ergot alkaloids (EAs) according to (EU) Regulation 2023/915 (ergocornine/ergocorninine; ergocristine/ergocristinine; ergocryptine/ergocryptinine; ergometrine/ergometrinine; ergosine/ergosinine; ergotamine/ergotaminine), after QuEChERS (Z-Sep/C18) purification. The analyzed sclerotia showed significant differences in total alkaloid content that vary between 0.01 and 0.5% (w/w), according to the results of the 2017 EFSA scientific report. EAs detected in sclerotia were up to 4951 mg/kg, in wheat ears up to 33 mg/kg, and in kernels were 1 mg/kg. Additional mycotoxins, including ochratoxin A, deoxynivalenol, zearalenone, fumonisins, T2-HT2 toxins, and aflatoxins, were investigated in wheat kernels after purification with immunoaffinity columns (IAC). The analysis revealed the presence of deoxynivalenol in wheat kernels at a concentration of 2251 µg/kg. It is expected that climate change will increase the frequency of extreme weather events. In order to mitigate the potential risks associated with mycotoxin-producing fungi and to ensure the protection of human health, it is suggested that official controls be implemented in the field. Full article

The prevalence of mycotoxins in the environment is associated with potential crop contamination, which results in an unavoidable increase in human exposure. Rice, being the second most consumed cereal worldwide, constitutes an important source of potential contamination by mycotoxins. Due to the increasing number of notifications reported, and the occurrence of mycotoxins at levels above the legislated limits, this work intends to compile the most relevant studies and review the main methods used in the detection and quantification of these compounds in rice. The aflatoxins and ochratoxin A are the predominant mycotoxins detected in rice grain and these data reveal the importance of adopting safety storage practices that prevent the growth of producing fungi from the Aspergillus genus along all the rice chain. Immunoaffinity columns (IAC) and QuECHERS are the preferred methods for extraction and purification and HPLC-MS/MS is preferred for quantification purposes. Further investigation is still required to establish the real exposition of these contaminants, as well as the consequences and possible synergistic effects due to the co-occurrence of mycotoxins and also for emergent and masked mycotoxins. Full article

A novel and efficient immunoaffinity column (IAC) based on bispecific monoclonal antibody (BsMAb) recognizing aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) was prepared and applied in simultaneous extraction of AFB₁ and OTA from food samples and detection of AFB₁/OTA combined with ic-ELISA (indirect competitive ELISA). Two deficient cell lines, hypoxanthine guanine phosphoribosyl-transferase (HGPRT) deficient anti-AFB₁ hybridoma cell line and thymidine kinase (TK) deficient anti-OTA hybridoma cell line, were fused to generate a hybrid-hybridoma producing BsMAb against AFB₁ and OTA. The subtype of the BsMAb was IgG₁ via mouse antibody isotyping kit test. The purity and molecular weight of BsMAb were confirmed by SDS-PAGE method. The cross-reaction rate with AFB₂ was 37%, with AFG₁ 15%, with AFM₁ 48%, with AFM₂ 10%, and with OTB 36%. Negligible cross-reaction was observed with other tested compounds. The affinity constant (Ka) was determined by ELISA. The Ka (AFB₁) and Ka (OTA) was 2.43 × 10⁸ L/mol and 1.57 × 10⁸ L/mol, respectively. Then the anti-AFB₁/OTA BsMAb was coupled with CNBr-Sepharose, and an AFB₁/OTA IAC was prepared. The coupling time and elution conditions of IAC were optimized. The coupling time was 1 h with 90% coupling rate, the eluent was methanol–water (60:40, v:v, pH 2.3) containing 1 mol/L NaCl, and the eluent volume was 4 mL. The column capacities of AFB₁ and OTA were 165.0 ng and 171.3 ng, respectively. After seven times of repeated use, the preservation rates of column capacity for AFB₁ and OTA were 69.3% and 68.0%, respectively. The ic-ELISA for AFB₁ and OTA were applied combined with IAC. The IC₅₀ (50% inhibiting concentration) of AFB₁ was 0.027 ng/mL, the limit of detection (LOD) was 0.004 ng/mL (0.032 µg/kg), and the linear range was 0.006 ng/mL~0.119 ng/mL. The IC₅₀ of OTA was 0.878 ng/mL, the LOD was 0.126 ng/mL (1.008 µg/kg), and the linear range was 0.259 ng/mL~6.178 ng/mL. Under optimum conditions, corn and wheat samples were pretreated with AFB₁-OTA IAC. The recovery rates of AFB₁ and OTA were 95.4%~105.0% with ic-ELISA, and the correlations between the detection results and LC-MS were above 0.9. The developed IAC combined with ic-ELISA is reliable and could be applied to the detection of AFB₁ and OTA in grains. Full article

Carotenoids are naturally occurring fat-soluble pigments found in many organisms. Because of their extensively conjugated carbon–carbon double bond system, carotenoids are potent antioxidants. Although the most abundant carotenoid and best singlet oxygen quencher found in red tomatoes is lycopene, carotenoid profiles may vary between genotypes. The objective of this work was to perform carotenoid profile indentification using HPLC-DAD-APCI-MS in ten wild cherry tomato accessions and one cultivated tomato. A mixture of hexane/acetone/ethanol (50:25:25) and 0.1% BHT was used for carotenoid extraction. For separation, a C30 column at 30 °C with a gradient consisting of methanol, methyl-tert-butyl ether, and water was used for their analysis. Ten major carotenoids were quantified within cherry tomato samples. All accessions present different profiles and quantities of carotenoids. Wild red tomatoes had more lycopene content that commercial tomato, whereas yellow tomatoes present no lycopene. From a functional viewpoint, higher concentrations of carotenoids that could play an antioxidant activity were measured from accessions IAC401, IAC426, LA1480, IAC391, and LA2692. This trait means that these germplasms may be targets for commercial activities. To the best of our knowledge, this is the first time that HPLC-DAD-APCI-MS has been used to analyze these accessions of wild cherry tomatoes that are both functionally promising and suitable for projects with social implementation at a local scale. Full article

As a class of mycotoxins with regulatory and public health significance, aflatoxins (e.g., aflatoxin B₁, B₂, G₁ and G₂) have attracted unparalleled attention from government, academia and industry due to their chronic and acute toxicity. Aflatoxins are secondary metabolites of various Aspergillus species, which are ubiquitous in the environment and can grow on a variety of crops whereby accumulation is impacted by climate influences. Consumption of foods and feeds contaminated by aflatoxins are hazardous to human and animal health, hence the detection and quantification of aflatoxins in foods and feeds is a priority from the viewpoint of food safety. Since the first purification and identification of aflatoxins from feeds in the 1960s, there have been continuous efforts to develop sensitive and rapid methods for the determination of aflatoxins. This review aims to provide a comprehensive overview on advances in aflatoxins analysis and highlights the importance of sample pretreatments, homogenization and various cleanup strategies used in the determination of aflatoxins. The use of liquid-liquid extraction (LLE), supercritical fluid extraction (SFE), solid phase extraction (SPE) and immunoaffinity column clean-up (IAC) and dilute and shoot for enhancing extraction efficiency and clean-up are discussed. Furthermore, the analytical techniques such as gas chromatography (GC), liquid chromatography (LC), mass spectrometry (MS), capillary electrophoresis (CE) and thin-layer chromatography (TLC) are compared in terms of identification, quantitation and throughput. Lastly, with the emergence of new techniques, the review culminates with prospects of promising technologies for aflatoxin analysis in the foreseeable future. Full article

Domoic acid (DA) is a neurotoxin associated with amnesic shellfish poisoning (ASP). Though LC coupled to tandem mass spectrometry (LC-MS/MS) has become the preferred method for DA determination, traditional sample pretreatment is still labor-intensive. In this study, a simple, efficient and selective method for LC-MS/MS analysis of DA in shellfish was established by optimizing clean-up procedures on a self-assembly immunoaffinity column (IAC). Shellfish was extracted with 75% methanol twice and diluted with phosphate buffered saline (PBS, 1:2). The mixture was purified on IAC as follows: preconditioned with PBS, loaded with sample, washed by 50% MeOH, and eluted with MeOH containing 2% ammonium hydroxide. Concentrated analyte was monitored by multiple reaction monitoring (MRM) using electrospray (ESI) positive ion mode throughout the LC gradient elution. Based on the post-extraction addition method, matrix effects for various shellfish matrices were found to be less than 8%. The developed method was fully validated by choosing mussel as the representative matrix. The method had a limit of detection (LOD) of 0.02 µg·g⁻¹, showed excellent linear correlation in the range of 0.05–40 µg·g⁻¹, and obtained ideal recoveries (91–94%), intra-day RSDs (6–8%) and inter-day RSDs (3–6%). The method was successfully applied to DA determination in 59 shellfish samples, with a detection rate of 10% and contaminated content of 0.1–14.9 µg·g⁻¹. Full article

Immunoaffinity columns (IACs) are most popularly used for mycotoxin clean-up in complex matrices prior to chromatographic analysis. But, their high cost has limited their wide application and the regeneration of IACs for multiple instances of reuse is important. This study aimed to investigate the feasibility of regeneration and reuse of IACs for purification of ochratoxin A (OTA) in spiked raw malt and dried ginger samples followed by high performance liquid chromatography-fluorescence detection. After each use, the IACs were filled with phosphate buffer saline (PBS) as the preservation solution and stored at 8 °C overnight for regeneration and reuse until the recovery rate was <70%. The results showed that matrix type, preparation procedure, and pH value of sample extraction exhibited major effects on the reuse of IACs for OTA clean-up. While, after modifying the sample preparation procedure using water as the diluent and the solution at a pH of 7 to 8, the IACs could be used eight and three times for the spiked raw malt and dried ginger samples with OTA after regeneration. Regarding the traditional procedure recommended in Chinese Pharmacopoeia (2015 edition), the IACs could be used for three and two times for the spiked raw malt and dried ginger samples with OTA, respectively. Therefore, the corresponding experimental cost could be reduced to one-eighth and one-third of the original cost. This is the first study on the regeneration and reuse of IACs for OTA clean-up in complex Chinese herbal medicines, providing a green and economical tool for a large number of samples analysis with low cost. Full article

This study investigated the distribution of twelve mycotoxins (aflatoxins B₁, B₂, G₁, and G₂; ochratoxin A; fumonisins B₁ and B₂; deoxynivalenol; nivalenol; zearalenone; T-2 toxin; and HT-2 toxin) in corn and corn by-products (corn bran, cornstarch, corn gluten, corn gluten feed, corn germ, light steep water, and corn steep liquor) produced by wet-milling in Korea. Fifty-two samples were collected from three factories producing cornstarch and other corn by-products. The samples were pretreated on an immunoaffinity column (IAC), and then the levels of the 12 mycotoxins were analyzed simultaneously by liquid chromatography-coupled triple-quadrupole mass spectrometry (LC-MS/MS). Fusarium mycotoxins were mainly found in raw corn and corn gluten feed samples. Other mycotoxins—such as aflatoxins, ochratoxin A, and HT-2 toxin—were detected in tiny amounts below the limit of quantification (LOQ) in cornstarch, corn germ, and corn bran. Ochratoxin A and nivalenol were mainly carried over into cornstarch. Aflatoxin B₁, deoxynivalenol, T-2 toxin, HT-2 toxin, and the fumonisins were concentrated in corn gluten feed. Zearalenone was evenly distributed in all corn by-products except cornstarch during the milling process. Full article

The Fusarium fungi produce toxic substances called mycotoxins, which can cause disease and harmful effects in grains, livestock, and humans. Deoxynivalenol (DON), also known as vomitoxin, is one of the Fusarium mycotoxins that is known to cause vomiting in livestock. This study shows the occurrence of deoxynivalenol in feedstuffs (compound feed and feed ingredients) between 2009 and 2016 in South Korea. A total of 653 domestic samples were collected at five time points, including 494 compound feed samples and 159 feed ingredient samples. DON contamination levels were analyzed using high-performance liquid chromatography (HPLC) with pretreatment using an immunoaffinity column (IAC). The limit of detection (LOD) and the limit of quantification (LOQ) were estimated at 1–10 µg/kg and 3–35 µg/kg, respectively. Two compound feeds (two gestating sow feed samples) out of 160 pig feed samples exceeded the European Commission (EC) guidance value, while no feed ingredient samples exceeded the EC or South Korean guidance values. There were statistically significant differences in the mean contamination levels of compound feed and feed ingredients that indicated a decreasing trend over time. Full article

Antibody based techniques are widely used for the detection of aflatoxins which are potent toxins with a high rate of occurrence in many crops. We developed a murine monoclonal antibody of immunoglobulin A (IgA) isotype with a strong binding affinity to aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2) and aflatoxin M1 (AFM1). The antibody was effectively used in immunoaffinity column (IAC) and ELISA kit development. The performance of the IACs was compatible with AOAC performance standards for affinity columns (Test Method: AOAC 991.31). The total binding capacity of the IACs containing our antibody was 111 ng, 70 ng, 114 ng and 73 ng for AFB1, AFB2, and AFG1 andAFG2, respectively. Furthermore, the recovery rates of 5 ng of each AF derivative loaded to the IACs were determined as 104.9%, 82.4%, 85.5% and 70.7% for AFB1, AFB2, AFG1 and AFG2, respectively. As for the ELISA kit developed using non-oriented, purified IgA antibody, we observed a detection range of 2–50 µg/L with 40 min total test time. The monoclonal antibody developed in this research is hitherto the first presentation of quadruple antigen binding IgA monoclonal antibodies in mycotoxin analysis and also the first study of their utilization in ELISA and IACs. IgA antibodies are valuable alternatives for immunoassay development, in terms of both sensitivity and ease of preparation, since they do not require any orientation effort. Full article

Mycotoxins are secondary metabolites produced by several fungi contaminating crops. In several countries, the maximum permitted levels of mycotoxins are found in foodstuffs and feedstuffs. The common strategy of mycotoxin analysis involves extraction, clean-up and quantification by chromatography. In this paper, we analyzed the reasons of underestimation of ochratoxin A (OTA) content in wine, and overestimation of OTA in wheat, depending on the pH of the clean-up step and the simultaneous presence of citrinin (CIT). We demonstrated that the increase of pH by adding polyethylene glycol (PEG) to wine led to an underestimation of OTA by conversion of OTA into open ring ochratoxin A OP-OA. In comparing three methods of extraction and clean-up for the determination of OTA and CIT in wheat—(i) an inter-laboratory validated method for OTA in cereals using immunoaffinity column clean-up (IAC) and extraction by acetonitrile/water; (ii) a validated method using IAC and extraction with 1% bicarbonate Na; and (iii) an in-house validated method based on acid liquid/liquid extraction—we observed an overestimation of OTA after immunoaffinity clean-up when CIT is also present in the sample, whereas an underestimation was observed when OTA was alone. Under neutral and alkaline conditions, CIT was partially recognized by OTA antibodies. Full article