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Determination of mycotoxins in plant-based beverages using QuEChERS and liquid chromatography–tandem mass spectrometry
Abstract
A method was developed for the simultaneous determination of 11 mycotoxins in plant-based beverage matrices, using a QuEChERS extraction followed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry detection (UHPLC–(ESI)MS/MS). This multi-mycotoxin method was applied to analyse plant-based beverages such as soy, oat and rice. QuEChERS extraction was applied obtaining suitable extraction recoveries between 80 and 91%, and good repeatability and reproducibility values. Method Quantification Limits were between 0.05 μg L⁻¹ (for aflatoxin G1 and aflatoxin B1) and 15 μg L⁻¹ (for deoxynivalenol and fumonisin B2). This is the first time that plant-based beverages have been analysed, and certain mycotoxins, such as deoxynivalenol, aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, ochratoxin A, T-2 toxin and zearalenone, were found in the analysed samples, and some of them quantified between 0.1 μg L⁻¹ and 19 μg L⁻¹.
... The most common mycotoxins are aflatoxins (AFs), trichothecenes, fumonisins (FB1, FB2), zearalenone (ZEA), and ochratoxin A (OTA) [7,8]. Raw agricultural products, including cereals, legumes, nuts, fruits, herbs, and other crops used in herbal beverages, can be contaminated with fungi, leading to the occurrence of mycotoxins in processed foods, such as plant-based beverages [9][10][11]. Mycotoxins exhibit acute and chronic toxicity, including genotoxicity, carcinogenicity, immunotoxicity, mutagenicity, nephrotoxicity, and teratogenicity [7,8,12]. ...
... Limited information is available in the scientific literature on the mycotoxin content of plant-based beverages [9][10][11]. ...
... The concentration of tentoxin ranged from 15 to 98 µg L −1 , the concentration of ENNB ranged from 10 to 109 µg L −1 , and the concentration of ENNB1 ranged from 6 to 60 µg L −1 in almond-based beverages. Conversely, in oat beverages, the highest concentrations were observed for tentoxin, ENNs, ZEA, and HT-2 toxin [9][10][11]. ...
Mycotoxins are toxic mold metabolites that can adversely affect human and animal health. More than 400 mycotoxins have been identified so far. Cereals and nuts are the predominant mycotoxin-contaminated foodstuffs. Plant-based drinks produced from cereals, nuts, and legumes have grown in popularity. The mycotoxins accumulated in these crops may transfer to these beverages. A liquid chromatography–tandem mass spectrometry method was developed and optimized for the assessment of 22 mycotoxins in commercially available plant-based drinks in Latvia and Lithuania. A total of 64% of the seventy-two analyzed beverages were positive for one to sixteen mycotoxins, with deoxynivalenol, beauvericin, and enniatins A, B, B1, T-2, and HT-2 toxins detected most frequently. The European Commission has not yet set guidelines for the maximum mycotoxin concentrations in plant-based beverages, nor has the European Food Safety Authority conducted a risk assessment. Therefore, acute exposure studies were provided for the Latvian population based on the assumed replacement of dairy milk with plant-based beverages to ascertain the safety of plant-based milk substitutes. Based on the observed levels of mycotoxin prevalence and contamination levels and assumed exposure, it can be concluded that tested plant-based beverages may be relatively safe. However, exposure to emerging mycotoxins should be considered.
... The amount of mycotoxin in food should be as low as possible due to the many adverse effects [1]. The effects of carcinogenesis, malformation, nephrotoxicity, and suppression of the immune system are some of the harmful effects of mycotoxins on human health [2][3][4][5]. Therefore, the identification and measurement of mycotoxins are essential to control these dangerous toxins. ...
... In the studies, it has been found that the recovery percentage of this method is similar to other extraction methods such as solid-liquid extraction and matrix solid phase dispersion [12]. QuEChERS can be used for both solid and liquid foods [4]. In this method, salts are added to solvents [2]. ...
... In the study of Miró-Abella et al., which was conducted on the plant-based beverages, the composition of PSA was not used. Howere, the authors stated that the samples were pretreated with formic acid [4]. PSA is recommended for juice samples due to the fact that they contain interfering factors such as sugar, esters, and pigments [31]. ...
... For this reason, rice, soy, oatmeal, almond and tigernut drinks can be another source of exposure to mycotoxins through the diet. Currently, in the bibliography, there are very few studies in this regard, and one was only found recently from 2017, in which a QuEChERS extraction method has been optimized for the determination in rice, soy and oat drinks, of only 11 mycotoxins (aflatoxins (AFB 1 , AFB 2 , AFG 1 and AFG 2 ), ochratoxin A (OTA) and 6 fusarotoxins (DON, ZEA, T-2, HT-2, FB 1 and FB 2 )) [8]. ...
... Plant-based beverage are complex matrices (proteins, carbohydrates, lipids and fiber) [9]; therefore, pre-treatments and cleanup steps to avoid the matrix effects that might interfere in quantification by analytical instruments are necessary [10]. Several authors developed methods in plant-based beverages by applying salting-out assisted liquid-liquid extraction in the case of Fusarium toxin determination [11,12]; dispersive liquid-liquid microextraction for aflatoxins [13]; or QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) extraction in the case of a multidetermination of mycotoxins [8] to avoid matrix effects. ...
... the number of samples containing these combinations is indicated in parentheses. Recently, works studied mycotoxin presence in oat, soy and rice-based beverage, as they are the most consumed globally [8,[11][12][13]30]. Miró-Abella et al. [8] reported the presence of mycotoxins and found DON, OTA, ZEN and T-2 toxin co-occurrence with AFB 1 , AFB 2 , AFG 1 and AFG 2 , and oat-based beverages were the most frequently contaminated. ...
This study developed and validated an analytical methodology for the determination of aflatoxins, enniatins, beauvericin, zearalenone, ochratoxin-A, alternariols, HT-2 and T-2 toxin in soy, oat, rice and almond beverages, based on solid phase extraction columns (SPE) and analyzed by liquid chromatography coupled to mass spectrometry in tandem. C18 SPE was successfully applied, obtaining recoveries that range from 72 ± 12% (ochratoxin-A) to 99 ± 4% (ENA1) at high level (L1) and 65 ± 8% (T-2) to 128 ± 9% (alternariol monomethyl ether) at low levels (L3). The methodology was validated according to Commission Decision 2002/657/EC, with limits of quantification ranging from 0.3 (AFs in oat beverages) to 18 ng/mL (HT-2 in rice beverage). The analysis of 56 beverage samples purchased from Valencia (Spain) showed at least one mycotoxin occurring in 95% of samples, including carcinogenic aflatoxins, and oat beverage was the most contaminated. This is a newest validated methodology for the quantification of sixty mycotoxins in oat, rice, almond and soy beverages.
... Mycotoxins are hazardous chemicals produced by Aspergillus, Fusarium Penicillium, and Claviceps genus. Mycotoxins can contaminate foods and feeds and agricultural products [1]. To date, there are more than four hundred mycotoxins with different toxicity, which have been identified in cereals, fruits, vegetables, and other agricultural commodities, resulting in potential adverse effects on human and animal health, and economic losses [2][3][4]. ...
... The mycotoxin levels in all feed samples almost complied with the EU regulation (≤1,000 mg/kg of 11 ergot alkaloids) [28]. There are several reports on the presence of EAs in the feed from different countries, with 86-100% of listed EAs detected in feed samples from Germany [1] and 83% of compound feeds containing EAs with an average concentration of 89 μg/kg and a maximum concentration of 1,231 μg/kg in the Netherlands [19]. The major detected EAs were ergosine, ergotamine, ergocristine, and ergocryptine. ...
... The mycotoxin levels in all feed samples almost complied with the EU regulation (≤1000 mg/kg of 11 ergot alkaloids) [28]. There are several reports on the presence of EAs in the feed from different countries, with 86-100% of listed EAs detected in feed samples from Germany [1] and 83% of compound feeds containing EAs with an average concentration of 89 µg/kg and a maximum concentration of 1231 µg/kg in the Netherlands [19]. The major detected EAs were ergosine, ergotamine, ergocristine, and ergocryptine. ...
Ergot alkaloids (EAs) are mycotoxins mainly produced by the fungus Claviceps purpurea. EAs are known to affect the nervous system and to be vasoconstrictors in humans and animals. This work presents recent advances in swine and dairy feeds regarding 11 major EAs, namely ergometrine, ergosine, ergotamine, ergocornine, ergocryptine, ergocristine, ergosinine, ergotaminine, ergocorninine, ergocryptinine, and ergocristinine. A reliable, sensitive, and accurate multiple mycotoxin method, based on extraction with a Mycosep 150 multifunctional column prior to analysis using UHPLC-MS/MS, was validated using samples of swine feed (100) and dairy feed (100) for the 11 targeted EAs. Based on the obtained validation results, this method showed good performance recovery and inter-day and intra-day precision that are in accordance with standard criteria to ensure reliable occurrence data on EA contaminants. More than 49% of the swine feed samples were contaminated with EAs, especially ergocryptine(-ine) (40%) and ergosine (-ine) and ergotamine (-ine) (37%). However, many of the 11 EAs were not detectable in any swine feed samples. In addition, there were contaminated (positive) dairy feed samples, especially for ergocryptine (-ine) (50%), ergosine (-ine) (48%), ergotamine (-ine), and ergocristine (-ine) (49%). The mycotoxin levels in the feed samples in this study almost complied with the European Union regulations.
... In addition to the well-known presence of AFs (AFB1, AFB2, AFG1, and AFG2) in crops, cereals, and nuts, several recent reports have shown trace amounts of AFs in beverages such as sugarcane juice, 9,10 alcoholic beverages (mainly beer), [11][12][13][14][15] and non-dairy beverages (mainly soy-based milk). [16][17][18][19] Some of the reports have been focused on total AF 18 or AFB1 (ref. 11-13, 15 and 19) screening by electrochemical 11,12 or optical 13,18,19 sensors, and immunoassays; 15 whereas, other proposals have been based on liquid chromatography (LC). ...
... 11-13, 15 and 19) screening by electrochemical 11,12 or optical 13,18,19 sensors, and immunoassays; 15 whereas, other proposals have been based on liquid chromatography (LC). 14,16,17 Although automated LC analysis of AFs in beer has been proposed, 14 methods for AF determination in plant-based milks by LC required a previous sample pretreatment stage such as dispersive liquid-liquid microextraction with a hollow ber supported liquid membrane, 16 and the Quick, Easy, Cheap, Effective, Rugged and Safe method (QuEChERS). 17 Benets from applying a previous sample pretreatment are AF enrichment (pre-concentration) in interference-free extracts (clean-up) which is quite important due to the presence of trace levels of AFs in foodstuffs. ...
... 14,16,17 Although automated LC analysis of AFs in beer has been proposed, 14 methods for AF determination in plant-based milks by LC required a previous sample pretreatment stage such as dispersive liquid-liquid microextraction with a hollow ber supported liquid membrane, 16 and the Quick, Easy, Cheap, Effective, Rugged and Safe method (QuEChERS). 17 Benets from applying a previous sample pretreatment are AF enrichment (pre-concentration) in interference-free extracts (clean-up) which is quite important due to the presence of trace levels of AFs in foodstuffs. ...
A selective molecularly imprinted polymer (MIP) adsorbent was synthesised and used in a batch micro-solid phase extraction format for isolating aflatoxins (AFB1, and AFB2) from non-dairy beverages before liquid chromatography-tandem mass spectrometry determination. MIP synthesis (precipitation polymerization in 3 : 1 acetonitrile/toluene as a porogen) was performed with 5,7-dimethoxycoumarin (DMC), methacrylic acid (MAA) and divinylbenzene-80 (DVB) as a dummy template, functional monomer and cross-linker, respectively (1 : 4 : 20 molar ratio). 2,2'-Azobisisobutyronitrile (AIBN) was used as a polymerization initiator. The adsorbent MIP (50 mg) was enclosed in a cone-shaped polypropylene membrane (porous membrane protected molecularly imprinted micro-solid phase extraction), and parameters such as sample pH, mechanical (orbital-horizontal) shaking, the extraction time (loading stage), the composition of the eluting solution, and the desorption time were optimised. The highest extraction yields were obtained by using 5 mL of non-dairy beverages (pH adjusted at 6.0), and mechanical shaking (150 rpm) for 15 min. Elution was performed with 5 mL of an acetonitrile/formic acid (97.5 : 2.5) mixture under ultrasound (325 W, 35 kHz) for 15 min. After eluate evaporation to dryness and re-dissolution in 150 μL of the mobile phase, the pre-concentration factor of the method was 33.3, which yields limits of detection within the 0.085-0.207 μg L-1 range. In addition, the current proposal was shown to be an accurate and precise method through relative standard deviation of intraday and inter-day assays below 18% and analytical recoveries in the range of 91-104%. However, the method was found to suffer from matrix effects.
... In the reports of the few studies carried out on the presence of mycotoxins in PBDs aflatoxins, deoxynivalenol (DON), zearalenone (ZEN) and ochratoxin A (OTA) were detected in various samples (Hamed, Abdel-Hamid, Gámiz-Gracia, García-Campaña, & Arroyo-Manzanares, 2019;Miró-Abella, et al., 2017). In addition, Miro-Abella et al. (2017) was able to detect aflatoxins, deoxynivalenol (DON), zearalenone (ZEN) and ochratoxin A (OTA) in some samples, highlighting that in all cases the toxins found in the drinks correlated well with literature data on the presence of these toxins in the respective raw materials. ...
... Two or more toxins were found in more than half of the products with detectable levels of mycotoxins. Overall, the results indicate a correlation between the toxins found in the drinks and the literature data on the presence of these toxins in the respective raw materials (Miró-Abella, et al., 2017), e.g. aflatoxins in almonds (European Food Safety Authority, 2007) and T2/HT2 toxins in oats (Pettersson, et al., 2011). ...
... QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) is a new enrichment method based on dispersive solid-phase extraction (d-SPE). It has been developed and widely used to extract multiple pesticides from tea [25] and mycotoxins from food and liquid samples [26]. Therefore, QuEChERS would be a suitable choice for extracting both pesticides and mycotoxins from Pu-erh tea, since it would help minimize the sample treatment procedure and prevent exposure to matrix effects. ...
... Currently, the most employed techniques for analyzing pesticides and mycotoxins are, respectively, gas chromatography-mass spectrometry (GC-MS) and liquid chromato graphy-tandem mass spectrometry (LC-MS/MS) [18,19,21,[23][24][25][26][27][28][29][30][31][32][33][34]. Depending on the chemical properties of the pesticides and mycotoxins, these methods employ different extraction solvents, comprehensive cleanup steps and longer analysis times in GC or LC, aiming to reduce matrix effects and improve method sensitivity. ...
Mycotoxins and pesticides are the most concerning chemical contaminants that can affect the quality of Pu-erh tea during its production and storage. This study presents a method that can simultaneously determine 31 pesticide residues and six mycotoxins in Pu-erh tea within 11 min using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) after QuEChERS extraction. The lower limit of quantification (LOQ) for all analytes ranged between 0.06 and 50 ppb. Recoveries for each pesticide and mycotoxin ranged between 62.0 and 130.3%, with intra- and inter-day precisions lower than 15%. Good linear relationships were obtained, with correlation coefficients of r2 > 0.991 for all analytes. The established method was applied to 31 Pu-erh tea samples, including raw and ripened Pu-erh tea with different storage times. As a result, pesticide residues were not detected in any of the collected samples, and the mycotoxins detected in the samples were well below the official maximum residue limits (MRLs). Notably, the levels of aflatoxin B1 (AFB1), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2) were lower than 1 ppb in the samples stored for more than 30 years.
... However, aflatoxins contaminated oats and other plant ingredients can remain in dairy products, and this phenomenon has been reported in recent studies (Miro-Abella et al., 2017;Pinto et al., 2021). Considering the co-occurrence of aflatoxins in milk and the multi-exposure toxicological effects for human, it is of a paramount importance to develop an analytical method for the quantitation of six aflatoxins in milk and plantbased milk (Bashiry et al., 2021;Iqbal et al., 2015;Min et al., 2020). ...
... However, the simultaneous quantitation of aflatoxin B 1 , B 2 , G 1 , G 2 , M 1 , and M 2 in milk and oat milk in one method is still limited (Zhang and Banerjee, 2020). Nowadays, the occurrence and the potential risk of aflatoxins in dairy products and plant-based milk have aroused a worldwide concern (Miro- Abella et al., 2017;Pinto et al., 2021). In the present study, a novel strategy of OSCC based on the multiple isotopologue reaction monitoring was proposed, where six MIRM channels for each aflatoxin and its isotopic labelled form were selected. ...
Preparing of calibration curves are critical steps for accurate quantitative LC-MS bioanalysis. Traditional multi-sample external calibration curve (MSCC) is labor-intensive and prone to error. In this study, a novel strategy of one sample multi-point calibration curve (OSCC) using multiple isotopologue reaction monitoring (MIRM) was proposed and validated using LC-MS for the quantitation of six aflatoxins in milk and oat-based milk samples. The developed MIRM-OSCC methodology is comprehensively validated and the results indicated that the established method exhibits good performance in selectivity, sensitivity, accuracy and precision. Furthermore, the OSCC could realize sample dilution by monitoring the MIRM channel with less intensity for samples beyond the upper limit of quantification, without the need of sample dilution, which improves the assay throughput. Considering the advantages of excluding the MSCC preparation and sample dilution in OSCC, this strategy can be widely applied in various fields such as drugs, food safety and environmental analysis.
... So far, there are only a few published studies, all with very limited sample size, which report investigations of PBMAs for mycotoxins. Although no study specific for the German market exists, data from other European countries clearly demonstrate that PBMAs may be contaminated by multiple mycotoxins belonging to different chemical groups including trichothecenes and aflatoxins Hamed et al. 2017Hamed et al. , 2019Juan et al. 2022;Miró-Abella et al. 2017). ...
... The levels measured for these samples corresponded well with contamination data for oats specifically (EFSA 2017;Curtui et al. 2009), and for cereals in general (Gottschalk et al. 2009). Furthermore, they are in good agreement with the results reported by Miró-Abella et al. (2017). Considering that the total amount of solids in these products typically ranged from 5 to 10%, the concentration of these toxins in the cereal ingredients should be about 10-20-fold higher, in a range roughly between 100 and 800 µg/kg for DON, and between 4 and 80 µg/kg for T-2/ HT-2. ...
Plant-based milk alternatives (PBMAs) are a potential source of mycotoxin uptake. To ensure food safety, simple and rapid testing methods of PBMAs for mycotoxins are therefore required. This study investigated the applicability of enzyme immunoassay (EIA) methods for direct testing of PBMAs without sample extraction. Mycotoxin analyses included aflatoxin B 1 (AFB 1 ), sterigmatocystin (STC), ochratoxin A (OTA), deoxynivalenol (DON), and T-2/HT-2-toxin (T-2/HT-2). It was found that the PBMA matrix negatively affected the EIA to varying degrees, thus affecting the reliability of the results. A dilution of PBMAs of at least 1:8 was necessary to overcome matrix interference. This resulted in calculated detection limits of 0.4 µg/L (AFB 1 ), 2 µg/L (STC), 0.08 µg/L (OTA), 16 µg/L (DON), and 0.4 µg/L (T-2/HT-2). After analysis of 54 PBMA products from German retail stores, positive results in at least one test system were obtained for 23 samples. However, most positive results were near the calculated detection limit. Control analyses of selected samples by LC–MS/MS for AFB 1 , STC, and OTA qualitatively confirmed the presence of trace amounts of STC in some samples, but quantitative agreement was poor. It was concluded that the high diversity of ingredients used in PBMAs led to a highly variable degree of sample matrix interference even in a 1:8 dilution. Since the use of higher dilutions conflicts with the need to achieve low detection limits, the application of EIA for routine mycotoxin analysis in PBMA for mycotoxins requires further study on the development of a feasible sample preparation method.
... F. sporotrichioides, F. poae, and F. acumninatum, and F. equiseti have also been observed to generate T-2 and HT-2 toxins [52,53]. T-2 and HT-2 toxins have been detected in barley, oat, maize, wheat, rice, beer, and plant-based milks, especially in oat-and soy-based milks and beverages [28,49,54,55]. T-2 and HT-2 toxins are linked to hematotoxicity, myelotoxicity, and growth retardation [56]. ...
... Fourteen mycotoxins (AFB1, AFB2, AFG1, AFG2, AFM1, T-2 toxin, HT-2 toxin, OTA, PAT, DON, ZEN, and FB1, FB2, FB3) were identified and quantified in wines using two solid-phase extractions and ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) within 13 min [103]. Miró-Abella et al. [28] documented a procedure for the simultaneous identification of 11 mycotoxins in plantbased beverage matrices such as soy, oat, and rice using QuEChERS extraction followed by ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-(ESI)MS/MS). ...
Mycotoxins are secondary metabolites of filamentous fungi that contaminate food products such as fruits, vegetables, cereals, beverages, and other agricultural commodities. Their occurrence in the food chain, especially in beverages, can pose a serious risk to human health, due to their toxicity, even at low concentrations. Mycotoxins, such as aflatoxins (AFs), ochratoxin A (OTA), patulin (PAT), fumonisins (FBs), trichothecenes (TCs), zearalenone (ZEN), and the alternaria toxins including alternariol, altenuene, and alternariol methyl ether have largely been identified in fruits and their derived products, such as beverages and drinks. The presence of mycotoxins in beverages is of high concern in some cases due to their levels being higher than the limits set by regulations. This review aims to summarize the toxicity of the major mycotoxins that occur in beverages, the methods available for their detection and quantification, and the strategies for their control. In addition, some novel techniques for controlling mycotoxins in the postharvest stage are highlighted.
... The extraction efficiency was determined by finding the recovery of AFB1 in the extract. As shown in Fig. 4, it is clear that pure acetonitrile is the best choice for the extraction of the target compounds from the two miscellaneous grain samples, which is comparable to the method of E Miró-Abella, E [29] and Yogendrarajah, P [30] . ...
... These solvents were selected as the organic phase because they influenced the chromatographic separation quality and facilitated analyte ionization (Cortese et al., 2020). The mobile phase was used to achieve optimal conditions and regulate the ionization extent of AFs in ESI + mode since AFs are reported to ionize better in positive mode (Miró-Abella et al., 2017;Leite et al., 2023). Moreover, including buffers in the mobile phase and the organic phase enhances ionization efficiency and improves the peak shapes (Luo et al., 2023). ...
This study identified and monitored the levels of aflatoxins (B1 and B2) produced by Aspergillus flavus isolate VKMN22 (OP355447) in maize samples sourced from a local shop in Johannesburg, South Africa. Maize samples underwent controlled incubation after initial rinsing, and isolates were identified through morphological and molecular methods. In another experiment, autoclaved maize grains were intentionally re-inoculated with the identified fungal isolate using spore suspension (106 spore/mL), after which 1 g of the contaminated maize sample was inoculated on PDA media and cultured for seven days. The aflatoxin concentrations in the A. flavus contaminated maize inoculated on culture media was monitored over seven weeks and then measured using liquid chromatography–mass spectroscopy (LC-MS). Results confirmed the successful isolation of A. flavus strain VKMN22 with accession number OP355447, which consistently produced higher levels of AFB1 compared to AFB2. AF concentrations increased from week one to five, then declined in week six and seven. AFB1 levels ranged from 594.3 to 9295.33 µg/kg (week 1–5) and then reduced from 5719.67 to 2005 µg/kg in week six and seven), while AFB2 levels ranged from 4.92 to 901.67 µg/kg (weeks 1–5) and then degraded to 184 µg/kg in week six then 55.33 µg/kg (weeks 6–7). Levene's tests confirmed significantly higher mean concentrations of AFB1 compared to AFB2 (p ≤ 0.005). The study emphasizes the importance of consistent biomonitoring for a dynamic understanding of AF contamination, informing accurate prevention and control strategies in agricultural commodities thereby safeguarding food safety.
... The QuEChERS method has been desired by researchers for its ability to be quick, easy, cheap and effective. This technique has been studied for the extraction of mycotoxins from plants (Miró-Abella et al., 2017), polyaromatic hydrocarbons in food (Słowik-Borowiec et al., 2022), pesticides and pharmaceuticals in fertilizers (Dong et al., 2023). ...
sitosterol and solasodine are major bioactive ingredients in Hypoxis hemerocallidea ( H. hemerocallidea) with significant pharmacological properties. As a result, developing a simple and efficient extraction method for simultaneous extraction of both analytes is critical. The purpose of this study was to identify and separate β -sitosterol and solasodine from ethanolic extracts of H. hemerocallidea using a modified QuEChERS method and subsequent analysis via UPLC triple quadrupole mass spectrometry. Response surface methodology was carried out, which included numerical parameters such as ultrasonication time, centrifugation time, and ultrasonication power. The categorical factors included the type of salt used to facilitate extraction, which was (NH 4 ) 2 SO 4 and Na 2 SO 4 . Fitting the response surface model to the experimental data produced a quadratic model with a good fit ( R ² = 0.9966 for solasodine and R ² = 0.9857 for β -sitosterol). The optimum conditions for extraction of β-sitosterol and solasodine were an ultrasonication time of 30 min, ultrasonication power of 300 W and centrifugation time of 12 min. The generally higher concentrations of analytes obtained for (NH 4 ) 2 SO 4 indicated that it had a superior salting-out ability compared to Na 2 SO 4. In conclusion, for the first time, β -sitosterol and solasodine were simultaneously extracted using modified QuEChERS with good yields through the salting-out action of (NH 4 ) 2 SO 4 in the presence of environmentally friendly solvents, ethanol and water. This modified QuEChERS technique can potentially be applied on a large scale as a sustainable and quick method for enrichment of therapeutic compounds from natural products.
... This technique affords enrichment of the analytes by their adsorption to the stationary phase; therefore, it is important to choose an appropriate SPE column to improve the selectivity [119]. There are numerous SPE cartridges with varying chemistries available on the market and used in the analysis of HT-2 and T-2 toxins in foodstuffs, which include: the study of wheat and wheat products using Oasis HLB cartridges, the occurrence of type-A trichothecenes in oats and oat products using MycoSep columns, the analysis of cereals using Strata-XL cartridges, the analysis of cereals including oats using Oasis HLB cartridges and the determination of 12 type-A and -B trichothecenes in cereals and cereal-based food using a Bond Elute Mycotoxin column [129][130][131][132][133]. One thing to note from the aforementioned analyses is that none were specific to HT-2 and T-2 only; instead, they are multi-methods analysing a range of mycotoxins including the Fusarium-produced type-A trichothecenes. ...
One of the major classes of mycotoxins posing serious hazards to humans and animals and potentially causing severe economic impact to the cereal industry are the trichothecenes, produced by many fungal genera. As such, indicative limits for the sum of T-2 and HT-2 were introduced in the European Union in 2013 and discussions are ongoing as to the establishment of maximum levels. This review provides a concise assessment of the existing understanding concerning the toxicological effects of T-2 and HT-2 in humans and animals, their biosynthetic pathways, occurrence, impact of climate change on their production and an evaluation of the analytical methods applied to their detection. This study highlights that the ecology of F. sporotrichioides and F. langsethiae as well as the influence of interacting environmental factors on their growth and activation of biosynthetic genes are still not fully understood. Predictive models of Fusarium growth and subsequent mycotoxin production would be beneficial in predicting the risk of contamination and thus aid early mitigation. With the likelihood of regulatory maximum limits being introduced, increased surveillance using rapid, on-site tests in addition to confirmatory methods will be required. allowing the industry to be proactive rather than reactive.
... QuEChERS is a straightforward, quick, and economical methodology that necessitates minimal amounts of solvent in comparison to other approaches. Over the last few years, this methodology has been used for the analysis of type B trichothecenes and other multiple mycotoxins in various food matrices, such as cereal grain, cereal products, plant-based beverages, spices, coffee, and livestock products (meat, milk, and eggs) [210][211][212][213][214][215][216]. For instance, Zhou et al. [216] proposed a facile and sensitive method based on modified QuEChERS that can quantify 10 mycotoxins, including DON, NIV, 3-ADON, 15-ADON, and FUS-X in wheat flour. ...
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. Key Contribution: Contamination of food with type B trichothecenes has gained growing concern worldwide due to their adverse health effects and substantial economic losses. This review briefly highlights the occurrence profile and toxicological effects of these mycotoxins. Current trends in analytical methodologies for their determination as well as management strategies to control their contamination in foods are also overviewed.
... Sample pretreatment reduces the interference of impurities and improves the sensitivity of detection by extracting, enriching and purifying the target substance (Gonzalez-Jartin et al. 2019). Currently, sample pretreatment methods including one-step extraction (Zhou, Liu, Yan, et al. 2021) and dispersed solid phase extraction QuEChERS (quick, easy, cheap, effective, rugged, and safe) (Miro-Abella et al. 2017) are routinely used. Due to its simplicity and potential for application, QuEChERS has become increasingly popular as an extraction method in food detection (Lago et al. 2021). ...
Mycotoxin contamination has become a challenge in the field of food safety testing, given the increasing emphasis on food safety in recent years. Mycotoxins are widely distributed, in heavily polluted areas. Food contamination with these toxins is difficult to prevent and control. Mycotoxins, as are small-molecule toxic metabolites produced by several species belonging to the genera Aspergillus, Fusarium, and Penicillium growing in food. They are considered teratogenic, carcinogenic, and mutagenic to humans and animals. Food systems are often simultaneously contaminated with multiple mycotoxins. Due to the additive or synergistic toxicological effects caused by the co-existence of multiple mycotoxins, their individual detection requires reliable, accurate, and high-throughput techniques. Currently available, methods for the detection of multiple mycotoxins are mainly based on chromatography, spectroscopy (colorimetry, fluorescence, and surface-enhanced Raman scattering), and electrochemistry. This review provides a comprehensive overview of advances in the multiple detection methods of mycotoxins during the recent 5 years. The principles and features of these techniques are described. The practical applications and challenges associated with assays for multiple detection methods of mycotoxins are summarized. The potential for future development and application is discussed in an effort, to provide standards of references for further research.
... Miro-Abella et al. [23] used QuEChERS method followed by liquid chromatography-tandem mass spectrometry to determine 11 mycotoxins in plant-based beverages which were reported to yield 80-91% recoveries with better repeatability and reproducibility values. Limit of quantification was between 0.05 μg/l (for AFGI and AFBI) and 15 μg/l for decoxynivalenol and fumonisin B2. ...
Mycotoxins are secondary metabolites present in foods which can cause adverse effects on humans and animals. Therefore, developing a simple, effective, sensitive and validated analytical method to monitor mycotoxins is essential. Sample preparation is an important step in the analysis of mycotoxins and other contaminants from complex food matrices. Food industries in developed and developing countries have faced serious challenges with contamination of mycotoxins especially aflatoxin in food and feed products. Thus, corn and cereal-based foods are mostly affected right from pre and postharvest periods. Owing to the complexity and structural nature of myco-toxins in foods and feeds there is an urgent need for simple, effective and environmentally friendly methods of sample preparation for the detection and quantification of aflatoxins in food samples. The paper reviews the application of the Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method for the analysis of aflatoxins in foods.
... Soy products don'tfavor the growth of mycotoxins, however, there is a chance for contamination of soy, products with the mycotoxin (Nesheim and Wood, 1995) 182 . Some studies using QuEChERS method confirm the presence of mycotoxins in beverages of soy, oat, and rice (Miro-Abella et al, 2017) 183 185 . ...
... Soy products don'tfavor the growth of mycotoxins, however, there is a chance for contamination of soy, products with the mycotoxin (Nesheim and Wood, 1995) 182 . Some studies using QuEChERS method confirm the presence of mycotoxins in beverages of soy, oat, and rice (Miro-Abella et al, 2017) 183 185 . ...
Demand for food and increasing with the population increasing thus may impart health benefits beyond basic nutrition. Beverages are a major part of the food industry and are composed of alcoholic and non-alcoholic drinks. Beverages were consumed to deliver high concentrations of functional ingredients. Vegetables, fruits, grains,
legumes, grains and seeds were the major functional foods that provide health benefits. Recently, the number of functional foods that have a potential benefit on health has hugely grown and scientific evidence is supporting the role of functional foods in the prevention and treatment of several diseases. Due to the increased consumption of
beverages, it has become very important to ensure the proper assessment of the antioxidant properties of beverages. The present study deals with the preparation of raw materials, traditional processing, composition, and ethnomedicinal importance of each food to encourage entrepreneurs to develop large-scale production to meet the
growing market demand for functional foods.
... AFs have been also determined in nuts using solid-phase microextraction (SPME) (Nonaka et al. 2009), dispersive liquid-liquid micro-extraction (DLLME) (Arroyo-Manzanares et al. 2013). The QuEChERS method, a combination of extraction and clean-up steps which was first used for the analysis of pesticides in fruits and vegetables (Anastassiades et al. 2003) has increasingly applied to mycotoxins due to its ease of use and suitability for the extraction from complex matrices (Desmarchelier et al. 2010;Yogendrarajah et al. 2013;Miró-Abella et al. 2017;Hidalgo-Ruiz et al. 2019). There are a number of different variations to the QuEChERS method in use worldwide, but all consist of the same basic ordered steps. ...
The present study describes a simple and rapid method for the determination of aflatoxins in almonds using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Aflatoxins were extracted using a modified QuEChERS method with little sample preparation, excluding the use of laborious purification procedures. Extracts were frozen overnight to separate the majority of lipids. The method was successfully validated for almonds. Linearity was demonstrated in the range 0.125–20 µg/kg. Limits of quantification (LOQ) ranged from 0.34 to 0.5 μg/kg. Matrix effect was not significant for the aflatoxins. Satisfactory recoveries were obtained at spike levels below 1 μg/kg and between 1 and 10 μg/kg. Relative standard deviations (RSDs) of repeatability and reproducibility were below 15%. The method was successfully tested with two proficiency tests in almond powder and peanut paste, with acceptable z-scores (−2 ≤ z ≤ 2). Only one of 11 local almond samples contained detectable aflatoxins, at concentrations below the maximum permitted level.
... It was observed that all analytes exhibited acceptable results using MeOH as compared to MeCN, with no significant differences. These results were consistent with those of other studies [28,29,[35][36][37][38][39], which suggested that MeOH was the optimal mobile phase in multi-mycotoxin analysis using LC-MS/MS system. Furthermore, MeOH is less expensive and more environmentally friendly in view of laboratory waste disposal, and hence have been preferred by many [40]. ...
Rice bran, a by-product of the rice milling process, has emerged as a functional food and being used in formulation of healthy food and drinks. However, rice bran is often contaminated with numerous mycotoxins. In this study, a method to simultaneous detection of aflatoxins (AFB1, AFB2, AFG1, and AFG2), ochratoxin A (OTA), deoxynivalenol (DON), fumonisins (FB1 and FB2), sterigmatocystin (STG), T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS) and zearalenone (ZEA) in rice bran was developed, optimized and validated using dispersive liquid–liquid microextraction (DLLME) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In DLLME, using a solvent mixture of methanol/water (80:20, v/v) as the dispersive solvent and chloroform as the extraction solvent with the addition of 5% salt improved the extraction recoveries (63–120%). The developed method was further optimized using the response surface methodology (RSM) combined with Box–Behnken Design (BBD). Under the optimized experimental conditions, good linearity was obtained with a correlation coefficient (r2) ≥ 0.990 and a limit of detection (LOD) between 0.5 to 50 ng g−1. The recoveries ranged from 70.2% to 99.4% with an RSD below 1.28%. The proposed method was successfully applied to analyze multi-mycotoxin in 24 rice bran samples.
Climate change and changing consumer demand are the main factors driving the protein transition. This shift toward more sustainable protein sources as alternatives to animal proteins is also reflected in the rapid upscaling of meat and dairy food analogues. Such changes could challenge food safety, as new food sources could result in new and unexpected food safety risks for consumers. This review analyzed the current knowledge on chemical and microbiological contamination of emerging alternative protein sources of plant origin, including soil‐based (faba bean, mung bean, lentils, black gram, cowpea, quinoa, hemp, and leaf proteins) and aquatic‐based (microalgae and duckweeds) proteins. Moreover, findings on commercial analogues from known alternative protein sources were included. Overall, the main focus of the investigations is on the European context. The review aimed to enable foresight approaches to food safety concerning the protein transition. The results indicated the occurrence of multiple chemical and microbiological hazards either in the raw materials that are the protein sources and eventually in the analogues. Moreover, current European legislation on maximum limits does not address most of the “contaminant‐food” pairs identified, and no legislative framework has been developed for analogues. Results of this study provide stakeholders with a more comprehensive understanding of the chemical and microbiological safety of alternative protein sources and derived analogues to enable a holistic and safe approach to the protein transition.
Food-based dietary guidelines recommend the reduction of animal-derived ingredients such meat and dairy products and a transition toward more sustainable diets. The ongoing shift toward plant-based diets is governed by their health benefits and environmental-friendly character. However, this transition comes with potential health effects such as increased exposure to natural toxins and possible allergic reactions. This chapter presents an overview on the need of a regulatory framework taking into account legislations for natural toxins in plant-based foods and updated labeling related to potential allergens. The economic impact and how the regulatory framework will support the growth and success of the plant-based food industry are also examined.
Newly designed micro-solid phase extraction cartridges are now available, reflecting the increasing shift towards laboratory automation, especially in the clean-up step for the analysis of pesticide residues in food and feed. In the present study, the introduction of different sorbents on the newly designed PAL µSPE CTC cartridges was investigated for the removal of matrix interferents and the recovery of pesticides. Eight cartridges containing different sorbent combinations and different amounts were used including EMR-lipid (not activated), Z-sep, chitin, C18, PSA, and GCB. The evaluation of co-extractive removal for each cartridge showed that the optimal choice for removing fatty acids was the cartridges containing PSA and Z-sep as clean-up sorbents. However, the presence of C18 and EMR-lipid was still required for the removal of sterols and tocopherols. Two grams of sample, fish feed (FF) and rapeseed cake (RSC) were extracted using QuEChERS citrate buffer, followed by a freeze-out step. The recoveries and repeatability of QuEChERS using µ-SPE clean-up were evaluated for 216 pesticide residues (112 compounds analyzed by GC-MS/MS and 143 compounds by LC-MS/MS, from which 39 compounds were analyzed using both techniques). The best results, with recovery between 70 and 120% and RSD <20%, were achieved when FF samples were cleaned-up with 15 mg EMR-lipid and 20 mg MgSO4. This was achieved for 94% of GC-amenable compounds and 86% of LC-amenable compounds. In the case of RSC, the best results were seen when samples were cleaned-up with the cartridge containing only 20 mg Z-sep and 20 mg MgSO4. This was achieved for 88% of GC-amenable compounds and 90% of LC-amenable compounds. Although these cartridges yielded optimal results in terms of recovery, their use could require more instrument maintenance, especially for GC-MS/MS, due to the lower removal of co-extractives.
Aflatoxins (AFs) including AFB1, AFB2, AFG1 and AFG2 are widely found in agriculture products, and AFB1 is considered one of the most toxic and harmful mycotoxins. Herein, a highly sensitive (at the pg mL−1 level) and group-specific enzyme-linked immunosorbent assay (ELISA) for the detection of AFB1 in agricultural and aquiculture products was developed. The AFB1 derivative containing a carboxylic group was synthesized and covalently linked to bovine serum albumin (BSA). The AFB1-BSA conjugate was used as an immunogen to immunize mice. A high-quality monoclonal antibody (mAb) against AFB1 was produced by hybridoma technology, and the mAb-based ELISA for AFB1 was established. IC50 and limit of detection (LOD) of the ELISA for AFB1 were 90 pg mL−1 and 18 pg mL−1, respectively. The cross-reactivities (CRs) of the assay with AFB2, AFG1, and AFG2 were 23.6%, 42.5%, and 1.9%, respectively, revealing some degree of group specificity. Corn flour, wheat flour, and crab roe samples spiked with different contents of AFB1 were subjected to ELISA procedures. The recoveries and relative standard deviation (RSD) of the ELISA for AFB1 in spiked samples were 78.3–116.6% and 1.49–13.21% (n = 3), respectively. Wheat flour samples spiked with the mixed AF (AFB1, AFB2, AFG1, AFG2) standard solution were measured by ELISA and LC-MS/MS simultaneously. It was demonstrated that the proposed ELISA can be used as a screening method for evaluation of AFs (AFB1, AFB2, AFG1, AFG2) in wheat flour samples.
As bebidas são alimentos de significativa importância econômica, nutricional e afetiva. São uma classe ampla de produtos industrializados, com composições complexas e processos produtivos variados. O perfil da indústria de bebidas envolve indústrias de pequeno, médio e grande porte, e fatores como a complexidade das amostras, a frequência com que é alvo de fraudes e adulterações, a facilidade da ocorrência de contaminações, e os rígidos parâmetros de identidade e qualidade requerem que estas
indústrias empreguem métodos analíticos para controle de qualidade e garantia da segurança alimentar dos consumidores. As indústrias de bebidas são um dos segmentos da indústria alimentícia que mais possuem demandas de métodos analíticos que sejam viáveis economicamente e com facilidade operacional, para atender a diversidade de perfis de produção e econômicos das indústrias do segmento, e para a análise de compostos químicos de interesse. Neste capítulo serão apresentados a classificação das bebidas e a influência nos parâmetros analíticos para o controle de qualidade de cada
bebida, as inovações em métodos analíticos recomendados por protocolos oficiais e métodos inovadores para a análise de bebidas e como eles podem ser aplicados, seja na determinação de contaminantes e congêneres, no desenvolvimento de novos produtos e processos e na verificação de autenticidade e rastreabilidade de bebidas.
Realizing the simultaneous speedy detection of multiple mycotoxins in contaminated food and feed is of great practical importance in the domain of food manufacturing and security. Herein, a fluorescent aptamer sensor based on self-assembled DNA double-crossover was developed and used for effective simultaneous quantitative detection of aflatoxins M1 and B1 by fluorescence resonance energy transfer (FRET). Fluorescent dye-modified aflatoxin M1 and B1 aptamers are selected as recognition elements and signal probes, and DNA double crosses are consistently locked by the aflatoxin aptamers, which results in a "turn-off" of the fluorescent signal. In the presence of AFM1 and AFB1, the aptamer sequences are more inclined to form Apt-AFM1 and Apt-AFB1 complexes, and the fluorescent probes are released from the DNA double-crossing platform, leading to an enhanced fluorescent signal (Cy3: 568 nm; Cy5: 660 nm). Under the optimal conditions, the signal response of the constructed fluorescent aptamer sensor showed good linearity with the logarithm of AFM1 and AFB1 concentrations, with detection limits of 6.24 pg/mL and 9.0 pg/mL, and a wide linear range of 0.01-200 ng/mL and 0.01-150 ng/mL, respectively. In addition, the effect of potential interfering substances in real samples was analyzed, and the aptasensor presented a good interference immunity. Moreover, by modifying and designing aptamer probes, the sensor can be applied to high-throughput simultaneous screening of other analytes, providing a new approach for the development of fluorescent aptamer sensors.
Mycotoxins pollution is a global concern, and can pose a serious threat to human health. People and livestock eating contaminated food will encounter acute and chronic poisoning symptoms, such as carcinogenicity, acute hepatitis, and a weakened immune system. In order to prevent or reduce the exposure of human beings and livestock to mycotoxins, it is necessary to screen mycotoxins in different foods efficiently, sensitively, and selectively. Proper sample preparation is very important for the separation, purification, and enrichment of mycotoxins from complex matrices. This review provides a comprehensive summary of mycotoxins pretreatment methods since 2017, including traditionally used methods, solid-phase extraction (SPE)-based methods, liquid-liquid extraction (LLE)-based methods, matrix solid phase dispersion (MSPD), QuEChERS, and so on. The novel materials and cutting-edge technologies are systematically and comprehensively summarized. Moreover, we discuss and compare the pros and cons of different pretreatment methods and suggest a prospect.
Fumonisins are mycotoxins present worldwide. They are mainly found in corn and its derived foods; however, they also have an important presence in other grains, fruits, and vegetables. Their consumption in excessive amounts can affect animal and human health. The most abundant of these is fumonisin B1, associated with a range of toxicological effects in animals, including equine leukoencephalomalacia, porcine pulmonary edema, and rodent carcinogenicity. In humans this mycotoxin has been shown to increase rates of esophageal cancer. The International Agency for Research on Cancer has classified FB1 within the 2B group, considering it a possible human carcinogen. Thus, analytical methods that identify/quantify fumonisins become a necessity to ensure adequate control of food and crops. An analytic method needs to be sensitive, selective, and robust to provide reliable data that can aid in monitoring risk assessment, quality control, and research. Recently, colorimetric methods which use immunologic and molecular approaches based on dyes, enzymes and aptamers have gained attention; some of these using nanomaterials. However, these methods are still in development. Currently, chromatographic methods remain the most confident and robust analytic tool, especially for quantification purposes. There is a great deal of information reported in the literature regarding these methods; despite this, there has not been a compilation of the methods for fumonisin analysis to facilitate its consult since 2005. Being the most common method for fumonisin detection worldwide, the present review focuses on the compilation of liquid chromatography methods published between 2006 and 2022 organized by matrix, analytes, instrument, and method conditions, using diverse detectors including MS, fluorescence, and an evaporative light scattering detector. Additionally, These techniques have been applied to diverse matrices, namely food and beverages, including grains, milk, meat, beer, wine; as well as biological samples such as urine, plasma, serum, and tissues. Other aspects pertaining to legislation, extraction, cleanup (selective pressurized liquid extraction, strong anion-exchange, immunoaffinity chromatography, and QuEChERS), derivatization procedures, limit of detection and quantification of fumonisins are also included. This review had compiled and organized 88 chromatographic methods for fumonisins analysis, and the analysts can consult all the procedures with detail.
Aflatoxins are secondary metabolites of fungi, particularly Aspergillus flavus and Aspergillus parasiticus, that known to be the most dangerous mycotoxin. Among the more than 20 types of aflatoxin identified, four types of AFB1, AFB2, AFG1, AFG2, and two AFM1 and AFM2 (hydroxylated metabolites of AFB1 and AFB2 in animals and livestock products, respectively) have been identified as major contributors to pathogenicity. The carcinogenic role of these toxins recognized in liver target tissue. Human exposure to mycotoxins occurs directly through the intake of contaminated agricultural products or indirectly through the consumption of products of animal origin prepared or obtained from animals that were fed with contaminated material. For detoxification and reduce threats to public health in the context of communities, and the economic damage caused by the aflatoxins in food products of animal and plants, different techniques (physical, chemical and biological) has been studied. All of these methods, by modifying and destroying the toxin molecular structure, inhibit its transfer to the digestive system, and reduce the accessibility of toxins to the target tissue and eliminate it. The current review performed based on search in database Scopus, Elsevier, PubMed, Google scholar, Science Direct, Iranmedex, Magiran, SID and Irandoc of biological substance (bacteria, fungi, and algae) reviews the biological degradation of AFs by fungal microorganisms and converting it into non-toxic, or less toxic products, and providing appropriate solutions.
Aflatoxins (AFs) as a category of polar mycotoxins possess a significant threat to human health. However, constructing of sorbents with suitable polarity for the preconcentration of AFs remains poorly developed due to the difficulty in tuning the polarity of the sorbents optionally. Herein, a series of novel hypercrosslinked polymers (OP-HCPs) with adjustable polarity were designed and synthesized by varying selection of alkylphenol ethoxylates monomers and benzyl halides crosslinkers for the preconcentration of AFs. By integration of magnetism into OP-HCP, a novel magnetic sorbent (M-OP10-DCX) was prepared to readily pre-concentrate of AFs from rice and maize samples prior to high performance liquid chromatography-fluorescence detection (HPLC-FD). Benefiting from the unique polarity interaction between M-OP10-DCX and AFs, the M-OP10-DCX displayed excellent adsorption ability for the AFs. Good linearity in the range of 5.0-4000 pg g⁻¹ (R² > 0.9983), low limits of detection (signal-to-noise ratio of 3) of 1.5-50 pg g⁻¹ and high enrichment factors of 105-205 were achieved. The recoveries of the method ranged from 82.8% to 115% with the relative standard deviation less than 8.0%. This work provided a new strategy for the rational design of HCPs with tunable polarity as valuable tools for specific adsorption of different kinds of compounds.
Coffee is an agricultural product highly susceptible to mycotoxin contamination. The present study evaluates the natural presence of 17 mycotoxins in different types of coffee sampled in Tunisia (n = 100), by using liquid chromatography-tandem mass spectrometry methods (LC-MS/MS). The results showed that all the analysed samples were contaminated by at least one mycotoxin. Tentoxin (TENT) was registered in 95 samples with maximum of 300 μg/Kg while aflatoxin B1 (AFB1) and ochratoxin A (OTA) in 7 and 18 samples, respectively. Most of OTA positive samples exceeded the OTA maximum regulatory limit (MRL). The highest levels of OTA, aflatoxin G1 (AFG1), and alternariol monomethyl ether (AME) levels were detected in Arabica samples reaching up to 122.6; 186.9 and 388.6 μg/Kg, respectively.82 samples were contaminated by at least two mycotoxins and up to nine different mycotoxins co-occurred simultaneously in two samples. The dietary exposure of the Tunisian population was also assessed, and the probable daily intake (PDI) ranged between 0.27 – 1.48 and 2.79–5.28 ng/Kg bw/day for OTA and zearalenone (ZEN), respectively. It was also found that, at the worst scenario (Mp), consumers may be reaching up to 2.1% and 8.7% of the tolerable daily intakes (TDIs) of ZEN and OTA, respectively. However, no toxicological risk arises to most of the analysed mycotoxins from the regular consumption of the evaluated coffee.
In the current work, for the first time, a vitamin–based metal–organic framework constructed from cobalt ions and vitamin B3 has been used as a sorbent in dispersive micro–solid–phase extraction. The proposed method was used to extract and enrich aflatoxins (B1, B2, G1, and G2) from soy milk samples before their quantification by high performance liquid chromatography–tandem mass spectrometry. In this work, first the metal–organic framework was synthesized and characterized using techniques such as X–ray diffraction, Fourier transform infrared spectrophotometry, scanning electron microscopy, nitrogen adsorption/desorption, and energy–dispersive X–ray spectroscopy. Then it was used as an efficient sorbent in the proposed dispersive micro–solid–phase extraction. For this purpose, after precipitating the proteins of soy milk sample with the aid of trichloroacetic acid, the supernatant phase was taken, mixed with the synthesized sorbent, and vortexed. After centrifuging, the analytes loaded on the adsorbent were eluted with methanol to transfer them into an organic phase which was compatible with the subsequently employed separation system. The adsorption capacity of the synthesized MOF for aflatoxins B1, B2, G1, and G2 were 0.77, 0.83, 0.70, and 0.54 mg g–1, respectively. Under the best experimental situations, satisfactory outcomes including acceptable extraction recoveries (64–75%), low limits of detection (11.3–48.2 ng L–1) and quantification (42.8–161.6 ng L–1), and good repeatability (relative standard deviations equal or less than 4.0 and 4.7% for intra– and inter– day precisions, respectively) were obtained. In addition, green synthesis of the metal–organic framework (using vitamin B3 as a linker, water as the reaction solvent, and mild conditions) and usage low amount or volume of the adsorbent and organic solvents during the extraction process were the other beneficial aspects of this work which caused the suggested analytical method to be environmentally friendly.
Aflatoxin B1 (AFB1) is one of the most common mycotoxins that threatens human health. As single-stranded oligonucleotides with high affinity and specificity, aptamers have incomparable effect on the targeted detection of AFB1. Herein, after 11 rounds of selection and analysis using a modified affinity chromatography-based SELEX strategy, the truncated 37 nt aptamer AF11-2 was successfully obtained. The aptamer shows good detection performance for AFB1, and can sensitively detect AFB1 in the range of 100-1000 nmol/L, with a detection limit of 42 nmol/L. In the detection of pretreated edible peanut oil samples, AF11-2 aptamer also showed a high recovery rate and good stability for AFB1, and achieved satisfactory results. In addition, AF11-2 aptamer can significantly enhance the fluorescence ability of AFB1, which is not available in traditional Afla17-2-3 aptamer. After molecular docking analysis, it was found that AF11-2 and Afla17-2-3 had different nucleotide binding sites for AFB1. Afla17-2-3 binds to the carbonyl O of AFB1, while AF11-2 binds to the pyrrolic O of AFB1, which may be the main reason that AF11-2 can enhance the fluorescence of AFB1.
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 B 1 , B 2 , G 1 , and G 2 , fumonisins B 1 and B 2 , 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 B 1 (0.023 μg/l, 84.98 and 9.23%); aflatoxin B 2 (0.024 μg/, 93.00 and 4.85%); aflatoxin G 1 (0.057 μg/l, 98.85 and 5.53%); aflatoxin G 2 (0.031 μg/l, 96.64 and 4.08%); fumonisin B 1 (2.166 μg/l, 75.55 and 16.78%); fumonisin B 2 (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.
One of the main objectives of routine laboratories is the development of simple and reliable methods as well as meeting fit-for-purpose criteria for regulatory surveillance. In this study, the accuracy profiles and the evaluation of the distribution of results in the case of aflatoxins in almonds have been performed using ultraperformance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). The method consists of designing the experiment and using certified reference material (CRM) to evaluate the bias, to calculate the combined uncertainty, and to construct the control charts. Good sensitivity (limit of quantifications (LOQs) 0.34–0.5 μg/kg) and recovery (between 82 and 107%) were achieved. The proposed method was successfully tested with a proficiency test in almond powder with acceptable z scores (−2 ≤ z ≤ 2). The results provided direct evidence for the proper functioning and stability of the whole analytical protocol, allowing acceptable combined uncertainty.
Mycotoxin contamination is a current issue affecting several crops and processed products worldwide. Among the diverse mycotoxin group, fumonisin B1 (FB1) has become a relevant compound because of its adverse effects in the food chain. Conventional analytical methods previously proposed to quantify FB1 comprise LC-MS, HPLC-FLD and ELISA, while novel approaches integrate different sensing platforms and fluorescently labelled agents in combination with antibodies. Nevertheless, such methods could be expensive, time-consuming and require experience. Aptamers (ssDNA) are promising alternatives to overcome some of the drawbacks of conventional analytical methods, their high affinity through specific aptamer-target binding has been exploited in various designs attaining favorable limits of detection (LOD). So far, two aptamers specific to FB1 have been reported, and their modified and shortened sequences have been explored for a successful target quantification. In this critical review spanning the last eight years, we have conducted a systematic comparison based on principal component analysis of the aptamer-based techniques for FB1, compared with chromatographic, immunological and other analytical methods. We have also conducted an in-silico prediction of the folded structure of both aptamers under their reported conditions. The potential of aptasensors for the future development of highly sensitive FB1 testing methods is emphasized.
According to current demands and future perspectives in food safety, this study reports a fast and fully automated analytical method for the simultaneous analysis of the mycotoxins with high toxicity and wide spread, aflatoxins (AFs) and ochratoxin A (OTA) in dried fruits, a high-risk foodstuff. The method is based on pressurized liquid extraction (PLE), with aqueous methanol (30 %) at 110 °C, of the slurried dried fruit and online solid-phase extraction (online SPE) cleanup of the PLE extracts with a C18 cartridge. The purified sample was directly analysed by ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for sensitive and selective determination of AFs and OTA. The proposed analytical procedure was validated for different dried fruits (vine fruit, fig and apricot), providing method detection and quantification limits much lower than the AFs and OTA maximum levels imposed by EU regulation in dried fruit for direct human consumption. Also, recoveries (83-103 %) and repeatability (RSD < 8, n = 3) meet the performance criteria required by EU regulation for the determination of the levels of mycotoxins in foodstuffs. The main advantage of the proposed method is full automation of the whole analytical procedure that reduces the time and cost of the analysis, sample manipulation and solvent consumption, enabling high-throughput analysis and highly accurate and precise results.
Physicochemical and acid gelation properties of UHT-treated commercial soy, oat, quinoa, rice and lactose-free bovine milks were studied. The separation profiles were determined using a LUMiSizer dispersion analyser. Soy, rice and quinoa milks formed both cream and sediment layers, while oat milk sedimented but did not cream. Bovine milk was very stable to separation while all plant milks separated at varying rates; rice and oat milks being the most unstable products. Particle sizes in plant-based milk substitutes, expressed as volume mean diameters (d4.3), ranged from 0.55μm (soy) to 2.08μm (quinoa) while the average size in bovine milk was 0.52μm. Particles of plant-based milk substitutes were significantly more polydisperse compared to those of bovine milk. Upon acidification with glucono-δ-lactone (GDL), bovine, soy and quinoa milks formed structured gels with maximum storage moduli of 262, 187 and 105Pa, respectively while oat and rice milks did not gel. In addition to soy products currently on the market, quinoa may have potential in dairy-type food applications.
A sensitive and reliable multi-mycotoxin method was developed for the simultaneous determination of 16 toxicological important mycotoxins, such as aflatoxins B1, B2, G1, and G2; enniatins A, A1, B, and B1; beauvericin; ochratoxin A; fumonisin B1, B2, and B3; diacetoxyscirprenol; HT-2; and T-2 toxin in dried fruits using liquid chromatography combined with electrospray ionization-triple quadrupole tandem-mass spectrometry. Mycotoxins have been extracted from the samples using a modified quick, easy, cheap, effective, rugged, and safe procedure. The method was based on a single extraction with acidified acetonitrile, followed by partitioning with salts, avoiding any further clean-up step. Limits of detections ranged from 0.08 to 15 μg kg−1 and limits of quantification ranged from 0.2 to 45 μg kg−1, which were below the legal limit set by the European Union for the legislated mycotoxines. The recoveries in spiked samples ranged from 60 to 135 % except for beauvericin using matrix-matched calibration curves for quantification, with good inter- and intraday repeatability (respective relative standard deviation ≤20 and 9 %). The developed method was applied to 15 commercial dried fruits: raisins, figs, apricots, plums, and dates purchased in local markets from Spain. Among the mycotoxins studied, enniantins and aflatoxins were the most predominant mycotoxins.
Disease outbreaks due to the consumption of contaminated food and feedstuff are a recurring problem worldwide. The major factor contributing to contamination are microorganisms, especially fungi, which produce low-molecular-weight compounds as secondary metabolites, with confirmed toxic properties referred to as mycotoxins. Several mycotoxins reported to date are cosmopolitan in distribution and incur severe health-associated risks (including cancer and neurological disorders). Hence, creating awareness among consumers, as well as developing new methods for detection and inactivation is of great importance for food safety. In this review, the focus is on the occurrence of various types of mycotoxins in food and feed associated with risks to humans and livestock, as well as legislation put forth by various authorities, and on presently practiced detoxification methods. Brief descriptions on recent developments in mycotoxin detection methodology are also inlcuded. This review is meant to be informative not only for health-conscious consumers but also for experts in the field to pave the way for future research to fill the existing gaps in our knowledge with regard to mycotoxins and food safety. © 2010 Institute of Food Technologists®.
Deoxynivalenol (DON) is one of several mycotoxins produced by certain Fusarium species that frequently infect corn, wheat, oats, barley, rice, and other grains in the field or during storage. The exposure risk to human is directly through foods of plant origin (cereal grains) or indirectly through foods of animal origin (kidney, liver, milk, eggs). It has been detected in buckwheat, popcorn, sorgum, triticale, and other food products including flour, bread, breakfast cereals, noodles, infant foods, pancakes, malt and beer. DON affects animal and human health causing acute temporary nausea, vomiting, diarrhea, abdominal pain, headache, dizziness, and fever. This review briefly summarizes toxicities of this mycotoxin as well as effects on reproduction and their antagonistic and synergic actions.
A simple, fast, and inexpensive method for the determination of pesticide residues in fruits and vegetables is introduced. The procedure involves initial single-phase extraction of 10 g sample with 10 mL acetonitrile, followed by liquid-liquid partitioning formed by addition of 4 g anhydrous MgSO4 plus 1 g NaCl. Removal of residual water and cleanup are performed simultaneously by using a rapid procedure called dispersive solid-phase extraction (dispersive-SPE), in which 150 mg anhydrous MgSO4 and 25 mg primary secondary amine (PSA) sorbent are simply mixed with 1 mL acetonitrile extract. The dispersive-SPE with PSA effectively removes many polar matrix components, such as organic acids, certain polar pigments, and sugars, to some extent from the food extracts. Gas chromatography/mass spectrometry (GC/MS) is then used for quantitative and confirmatory analysis of GC-amenable pesticides. Recoveries between 85 and 101% (mostly > 95%) and repeatabilities typically < 5% have been achieved for a wide range of fortified pesticides, including very polar and basic compounds such as methamidophos, acephate, omethoate, imazalil, and thiabendazole. Using this method, a single chemist can prepare a batch of 6 previously chopped samples in < 30 min with approximately 1 dollar (U.S.) of materials per sample.
The European Food Safety Authority (EFSA) was asked to deliver a scientific statement on the increase of risk for public health related to a possible temporary derogation from the maximum level (tML) of deoxynivalenol (DON), fumonisins (FUMO) and zearalenone (ZON) in maize and maize products. EFSA relied on occurrence data reflecting levels of these mycotoxins in the 2013 maize harvest. Depending on the mycotoxin, mean levels estimated considering tMLs were increased by a factor comprised between 7.6 and 27 % in maize and maize milling fractions, and up to 99 % in some processed maize-based products, compared to levels estimated considering current ML. Chronic exposure levels estimated across the population groups – representing different age classes and countries – were increased by a factor up to 17 % for fumonisins, 20 % for deoxynivalenol and 83 % for zearalenone. The tolerable daily intake (TDI) for zearalenone set by EFSA at 0.25 µg/kg body weight (b.w.) per day, and the group provisional maximum tolerable daily intake (PMTDI) of 1 and 2 µg/kg b.w. per day set by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) for deoxynivalenol and fumonisins, were used as chronic health based guidance values (HBGV). For all three mycotoxins these values were exceeded in at least one age group when considering the current ML, reflecting a health concern. When considering the tMLs, percentages of consumers in the age groups with the highest exposures exceeding chronic HBGVs increased from 9.2 to 9.8 % for zearalenone, from 47 to 48 % for fumonisins and from 52 to 54 % for deoxynivalenol. Acute exposure scenarios resulted in exceedance of the group acute reference dose (ARfD) of 8 µg/kg b.w. established by JECFA for DON, with up to 1.2 % of the consumption days above the group ARfD with the current ML, and up to 8.1 % with the tML. This assessment, mainly based on French data, may lack representativeness for the European situation.
This study determined and compared drivers of liking for unflavored soymilk with different U.S. consumer groups. A highly trained panel documented appearance, mouthfeel and flavor attributes of 26 commercial soymilks. Twelve representative soymilks were then selected for evaluation by consumers from 3 age/cultural categories (n = 75 each category; Caucasian/African American females aged 18 to 30 y; Asian females aged 18 to 30 y; Caucasian/African American females aged 40 to 64 y). Consumers evaluated overall liking and liking and intensity of specific attributes. Results were evaluated by analysis of variance, followed by internal and external preference mapping. Age had no effect on overall liking, while ethnicity did (Caucasian/African American compared with Asian; P < 0.05). Caucasians/African Americans differentiated soymilks more than Asians and assigned a wider range of liking scores than Asians (2.1 to 7.2 compared with 4.0 to 6.1). Three consumer clusters were identified. Sweet taste with vanilla/vanillin and sweet aromatic flavors and higher viscosity were preferred by most consumers and differences between consumer clusters were primarily in drivers of dislike. Drivers of dislike were not identified for Cluster 1 consumers while Clusters 2 and 3 consumers (n = 84, n = 80) disliked beany, green/grassy and meaty/brothy flavors and astringency. Cluster 3 (n = 80) consumers scored all soymilks higher in liking (P < 0.05) than Cluster 2 consumers, and were willing to overlook disliked attributes with the addition of sweet taste, whereas the Cluster 2 consumers were not. These findings can be utilized to produce soymilks with attributes that are well liked by target consumers and to tailor attributes for segments of the population that have not yet been accommodated.
A multi-class methodology was developed to determine pesticides and mycotoxins in food supplements. The extraction was performed using acetonitrile acidified with formic acid (1%, v/v). Different clean-up sorbents were tested, and the best results were obtained using C18 and zirconium oxide for green tea and royal jelly, respectively. The compounds were determined using ultra high performance liquid chromatography (UHPLC) coupled to Exactive-Orbitrap high resolution mass spectrometry (HRMS). The recovery rates obtained were between 70% and 120% for most of the compounds studied with a relative standard deviation <25%, at three different concentration levels. The calculated limits of quantification (LOQ) were <10 μg/kg. The method was applied to green tea (10) and royal jelly (8) samples. Nine (eight of green tea and one of royal jelly) samples were found to be positive for pesticides at concentrations ranging from 10.6 (cinosulfuron) to 47.9 μg/kg (paclobutrazol). The aflatoxin B1 (5.4 μg/kg) was also found in one of the green tea samples.
Nowadays the interest and consumption of pseudocereals is increasing due to their nutritional properties. Like cereals and oilseeds, pseudocereal seeds are susceptible to fungal growth and mycotoxin contamination; however these matrices have received little attention in literature. A sensitive, simple and rapid method for the determination of fifteen mycotoxins (aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, ochratoxin A, fumonisin B1, fumonisin B2, nivalenol, deoxynivalenol, fusarenon-X, T-2 and HT-2 toxin, citrinin, sterigmatocystin and zearalenone) in pseudocereals (buckwheat, quinoa and amaranth) has been developed and validated by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS), using a QuEChERS-based sample treatment. This study also includes cereals such as spelt and white, brown and red rice. Matrix-matched calibration curves were established and limits of quantification were below the maximum contents established by EU regulation in cereals. The precision (repeatability and intermediate precision) was lower than 12% in all cases and recoveries were between 60.0% and 103.5%, fulfilling the current legislation. Finally, the content of aflatoxin B1 found in a red rice sample was confirmed by comparison of the result obtained by using immunoaffinity columns for extraction and clean-up. © 2013 Elsevier Ltd.
An automated, size-exclusion solid phase extraction (SPE)-UPLC-MS/MS protocol without pre-treatment of samples was developed to screen for four mycotoxins (OTA, ZEN, AFB1, and AFM1) in liquid milk and milk powder. Firstly, a mixed macropore-silica gel cartridge was established as a size-exclusion SPE column. The proposed methodology could be a candidate in green analytical chemistry because it saves on manpower and organic solvent. Permanent post-column infusion of mycotoxin standards was used to quantify matrix effects throughout the chromatographic run. Matrix-matched calibration could effectively compensate for matrix effects, which may be caused by liquid milk or milk powder matrix. Recovery of the four mycotoxins in fortified liquid milk was in the range 89-120% and RSD 2-9%. The LOD for the four mycotoxins in liquid milk and milk powder were 0.05-2ngL(-1) and 0.25-10ngkg(-1), respectively. The LOQ for the four mycotoxins in liquid milk and milk powder were 0.1-5ngL(-1) and 0.5-25ngkg(-1), respectively.
Copyright © 2014 Elsevier Ltd. All rights reserved.
Trichothecene mycotoxins, with T-2 and HT-2 toxins being the main representatives of the type A subgroup, are naturally and worldwide occurring contaminants frequently found in grain-based food and feed. Due to the high consumption of these products and the potential health risk associated herewith, concerns about the safety and quality of food and feed have increased over the last decades at both governmental and consumer levels. Since it is not possible to avoid their occurrence, tremendous efforts have been performed to identify and monitor mycotoxins in food and feed to make their consumption safe. However, suitable certified reference materials (CRMs) intended for quality assurance and quality control purposes are still lacking for many mycotoxin-matrix combinations. Therefore, in the framework of a European Reference Material (ERM®) project, the first CRM for T-2 and HT-2 toxin in ground oat flakes (ERM®-BC720) was developed according to the requirements of ISO Guide 35. The whole process of ERM®-BC720 development, including sample preparation, homogeneity and stability studies and value assignment, is presented. The assignment of the certified mass fractions was based upon an in-house study using high-performance liquid chromatography isotope-dilution tandem mass spectrometry. Simultaneously, an interlaboratory comparison study involving 24 expert laboratories was conducted in order to support the in-house certification study. The certified values and their corresponding expanded uncertainties (k = 2) for both T-2 and HT-2 toxin in ERM®-BC720, traceable to the international system of units, are (82 ± 4) μg kg(-1) and (81 ± 4) μg kg(-1), respectively.
An improved method for the quantitative determination of aflatoxins (B1, B2, G1, G2), ochratoxin A, fumonisins (B1, B2), zearalenone, deoxynivalenol, nivalenol, T-2 and HT-2 toxins in cereals and derived products, at levels comparable with EU maximum permitted levels, was developed. The effective co-extraction of the mycotoxins under investigation was achieved in 4min by a double extraction approach, using water followed by methanol. Clean up of the extract was performed by a new multi-toxin immunoaffinity column. Analytical performance characteristics were evaluated through single laboratory validation. Raw wheat and maize, corn flakes and maize snacks were chosen as representative matrices for method validation. The validation assay was carried out at 50, 100 and 150% of EU maximum permitted levels for each mycotoxin. Statistical analysis of the results (ANOVA) provided the within laboratory reproducibility and the error contributions from repeatability, between day effects, and influences from different matrix composition. Recoveries generally higher than 70% were obtained for all tested mycotoxins with relative standard deviation (within laboratory reproducibility) lesser than 37%. Limits of quantification (calculated as the lowest amount of each analyte which could be determined with a precision of 10%) ranged from 1μg/kg to 30μg/kg. The trueness of generated data was assessed by analysis of reference materials. The proposed method was proven to be suitable to assess, with a single analysis, compliance of the selected cereal based foods with the EU maximum permitted or recommended levels for all regulated mycotoxins.
A simple and efficient QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) sample preparation method was modified to provide good analytical results for 14 mycotoxins in rice. The method involved mixing sample with acidified aqueous acetonitrile, followed by salt-out liquid partitioning using MgSO4, NaCl, and citrate buffer salts. The extract was cleaned-up by dispersive solid-phase extraction with MgSO4, PSA, C18, and alumina-neutral. The analysis was performed using ultra-high performance liquid chromatography coupled to triple-quadrupole tandem mass spectrometry (UHPLC–MS/MS). Throughout the validation experiments, 70–98% overall recoveries were achieved with RSDs ⩽7% for most analytes at concentrations 10–100 μg kg−1. Limit of detections were 0.5–15 μg kg−1. Inter-laboratory precision was performed by proficiency testing, |z| ⩽2 was considered satisfactory. We compared our modified QuEChERS method against sample preparation using an immunoaffinity column; the recovery and specificity were comparable for the two methods, but the QuEChERS approach was more time- and cost-effective.
Soybean sauce, a traditional fermented food in China, has different levels of aflatoxin B1 pollution. Two kinds of direct and indirect immunomagnetic bead methods for the pretreatment of aflatoxin B1 were evaluated in this work. A method was established to detect aflatoxin B1 in soybean sauce using an immunomagnetic bead system for pretreatment and ELISA for quantification. The pretreatment method of immunomagnetic beads performed better compared with the conventional extraction and immunoaffinity column method. ELISA exhibited a good linear relationship at an aflatoxin B1 concentration of 0.05–0.3 μg/kg (r2 = 0.9842). The average recoveries across spike levels varied from 0.5 to 7 μg/kg were 83.6–104% with a relative standard deviation between 4.2% and 11.7%. With the advantages of rapid detection, easy operation, simple equipment, sensitivity, accuracy, and high recovery; this method can be well applied in the trace determination of aflatoxin B1 in soybean sauce samples.
In the present study, the occurrence of eighteen mycotoxins, nine trichothecenes (deoxynivalenol, 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, nivalenol, neosolaniol, diacetoxyscirpenol, fusarenon-X, T-2 toxin and HT-2 toxin), three zearalenones (zearalenone, α-zearalenol and β-zearalenol), and six emergent mycotoxins, beauvericin and five enniatins (A, A1, B, B1 and B4), was monitored in different Italian organic cereals and cereal products by using a liquid chromatography coupled to triple quadrupole mass spectrometry method. A total of 93 organic cereal samples (wheat, barley, rye and oat) were collected from Italy. Limits of quantification ranged from 5 to 15μg/kg. 80% of analyzed samples contained mycotoxins. The occurrence was 33%, 6.5%, 2%, 27%, 7%, 10% and 43% for deoxynivalenol, HT-2, T-2, nivalenol, zearalenone, beauvericin and enniatins, respectively. The major mycotoxin found was enniatin B4; it was detected in 40% of all analyzed samples and its levels ranged from 5.7 to 284.2μg/kg. Risk assessment was evaluated by EDI calculations which were lower than TDI for all legislated Fusarium mycotoxins.
Nine strains of lactic acid bacteria were tested for fermentation characteristics in an oat- based, non-dairy milk substitute, Mill MilkTM (MM medium). Viable counts, aroma formation, lactic acid production and viscosity were the parameters investigated. In general, bacterial strains grown at 30°C yielded a better flavour and higher viable counts than those incubated at 37°C. Strains were selected for their capacity to produce exopolysaccharides in a semi-defined medium. Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 was chosen for further fermentation experiments, using yoghurt as a control. Production of exopolysaccharide and shear thinning properties were measured using a viscosimetric method. A combination of temperature stress and an increased carbon/nitrogen ratio was shown to affect the production of exopolysaccharide. Increased viscosity was found after incubation at a low temperature using glucose as a supplementary carbon source. The production of exopolysaccharides was also favoured by prolonged incubation times. The use of physical factors such as time and temperature in combination with chemical factors, i.e. media and carbon/nitrogen levels, were crucial in the course of improving exopolysaccharide production during fermentation in the MM medium. Copyright 2000 Academic Press.
Mycotoxins are typically present in grain and are also concentrated in distillers dried grains with solubles (DDGS), common feed ingredients for food animals. The diversity of mycotoxins and feed matrices has made the routine detection and quantification of mycotoxins in feed both complex and prohibitively expensive.
Ultra-performance liquid chromatography/electrospray ionization triple quadrupole detection (UPLC/ESI-TQD) (tandem mass spectrometry, MS/MS) with (13) C-labeled isotopic dilution was used to analyze internal standard isotopologues of three mycotoxin molecules, as well as 29 other structurally differing mycotoxin molecules from four common feed matrices: corn, wheat, barley, or DDGS. Mycotoxins were extracted via a single-step procedure using a mixture of acetonitrile/water/formic acid. Labeled isotopologues were used as a surrogate to account for extraction quality and as internal standards for the evaluation of the feed matrix signal suppression/enhancement (SSE) contributed by each mycotoxin and by each matrix. The SSE was corrected by matrix-matched calibration with blank certified reference feed material.
The limits of detection for individual mycotoxins in buffer ranged from 0.01 to 206.7 µg/mL but could increase by up to four times depending on the matrix effect. The accuracy and precision were enhanced by the use of isotopically labeled standards. The recoveries were somewhat negatively affected by the SSE contributed by each matrix. Each mycotoxin was successfully detected and assigned to one of four SSE categories: high (-66%), intermediate (-48%), low (-19%) signal suppression and signal enhancement (> +300%).
An improved LC/MS method was validated, which offers a practical and economical means for large-scale detection and quantification of multiple mycotoxins in common animal-feed matrices, including DDGS. Copyright © 2012 John Wiley & Sons, Ltd.
More than 50 countries have enacted or proposed regulations for the control of aflatoxins in foods and/or feeds, and at least
15 of these countries also have regulations for permitted levels of contamination by other mycotoxins. Since 1965, the U.S.
Food and Drug Administration has used action levels to control aflatoxins in its compliance programs. Cooperative programs
with the U.S. Department of Agriculture, state agencies, and industry also have been used to keep exposure to aflatoxins as
low as practical. Soybeans support the growth of many mold species, which can produce toxins such as aflatoxins, trichothecenes
(such as T-2), and cytochalasins. The natural occurrence of these toxins in soybeans has not been a problem. Limited surveys
of soybeans and soy-based infant formulas have not revealed significant contamination. The sequence of events that leads to
consideration of a mycotoxin for control programs and other regulatory activity includes determination of a toxic response,
isolation and identification of the toxin, development of a sampling plan and method of analysis, and determination of incidence
and levels of contamination of the susceptible commodity. The quality of soybeans can vary widely, depending on environmental,
agronomic, and storage conditions. products susceptible to contamination from improper storage are subject to regulatory action
on a case-by-case basis. The government-industry cooperative programs have been successful in limiting human exposure to aflatoxins.
Mold metabolites that can elicit deleterious effects on other organisms are classified as mycotoxins. Human exposure to mycotoxins occurs mostly through the intake of contaminated agricultural products or residues due to carry over or metabolite products in foods of animal origin such as milk and eggs, but can also occur by dermal contact and inhalation. Mycotoxins contained in moldy foods, but also in damp interiors, can cause diseases in humans and animals. Nephropathy, various types of cancer, alimentary toxic aleukia, hepatic diseases, various hemorrhagic syndromes, and immune and neurological disorders are the most common diseases that can be related to mycotoxicosis. The absence or presence of mold infestation and its propagation are seldom correlated with mycotoxin presence. Mycotoxins must be determined directly, and suitable analytical methods are necessary. Hundreds of mycotoxins have been recognized, but only for a few of them, and in a restricted number of utilities, a maximum acceptable level has been regulated by law. However, mycotoxins seldom develop alone; more often various types and/or classes form in the same substrate. The co-occurrence might render the individual mycotoxin tolerance dose irrelevant, and therefore the mere presence of multiple mycotoxins should be considered a risk factor. The advantage of chromatography/mass spectrometry (MS) is that many compounds can be determined and confirmed in one analysis. This review illustrates the state-of-the-art of mycotoxin MS-based analytical methods for multiclass, multianalyte determination in all the matrices in which they appear. A chapter is devoted to the history of the long-standing coexistence and interaction among humans, domestic animals and mycotoxicosis, and the history of the discovery of mycotoxins. Quality assurance, although this topic relates to analytical chemistry in general, has been also examined for mycotoxin analysis as a preliminary to the systematic literature excursus. Sample handling is a crucial step to devise a multiclass analytical method; so when possible, it has been treated separately for a better comparison before tackling the instrumental part of the whole analytical method. This structure has resulted sometimes in unavoidable redundancies, because it was also important to underline the interconnection. Most reviews do not deal with all the possible mycotoxin sources, including the environmental ones. The focus of this review is the analytical methods based on MS for multimycotoxin class determination. Because the final purpose to devise multimycotoxin analysis should be the assessment of the danger to health of exposition to multitoxicants of natural origin (and possibly also the interaction with anthropogenic contaminants), therefore also the analytical methods for environmental relevant mycotoxins have been thoroughly reviewed. Finally, because the best way to shed light on actual risk assessment could be the individuation of exposure biomarkers, the review covers also the scarce literature on biological fluids.
Mycotoxins are natural contaminants produced by a range of fungal species. Their common occurrence in food and feed poses a threat to the health of humans and animals. This threat is caused either by the direct contamination of agricultural commodities or by a "carry-over" of mycotoxins and their metabolites into animal tissues, milk, and eggs after feeding of contaminated hay or corn. As a consequence of their diverse chemical structures and varying physical properties, mycotoxins exhibit a wide range of biological effects. Individual mycotoxins can be genotoxic, mutagenic, carcinogenic, teratogenic, and oestrogenic. To protect consumer health and to reduce economic losses, surveillance and control of mycotoxins in food and feed has become a major objective for producers, regulatory authorities and researchers worldwide. However, the variety of chemical structures makes it impossible to use one single technique for mycotoxin analysis. Hence, a vast number of analytical methods has been developed and validated. The heterogeneity of food matrices combined with the demand for a fast, simultaneous and accurate determination of multiple mycotoxins creates enormous challenges for routine analysis. The most crucial issues will be discussed in this review. These are (1) the collection of representative samples, (2) the performance of classical and emerging analytical methods based on chromatographic or immunochemical techniques, (3) the validation of official methods for enforcement, and (4) the limitations and future prospects of the current methods.
A rapid multianalyte-multiclass method with little sample manipulation has been developed for the simultaneous determination of eleven mycotoxins in different food commodities by using ultra-high-pressure liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC/MS/MS). Toxins were extracted from the samples with acetonitrile/water (80:20, v/v) 0.1% HCOOH and, after a two-fold dilution with water, directly injected into the system. Thanks to the fast high-resolution separation of UHPLC, the eleven mycotoxins were separated by gradient elution in only 4 min. The method has been validated in three food matrices (maize kernels, dry pasta (wheat), and eight-multicereal babyfood (wheat, maize, rice, oat, barley, rye, sorghum, millet)) at four different concentration levels. Satisfactory recoveries were obtained (70-110%) and precision (expressed as relative standard deviation) was typically below 15% with very few exceptions. Quantification of samples was carried out with matrix-matched standards calibration. The lowest concentration successfully validated in sample was as low as 0.5 microg/kg for aflatoxins and ochratoxin A in babyfood, and 20 microg/kg for the rest of the selected mycotoxins in all matrices tested. Deoxynivalenol could be only validated at 200 microg/kg, due the poor sensitivity for this mycotoxin analysis. With only two exceptions (HT-2 and deoxynivalenol), the limits of detection (LODs), estimated for a signal-to-noise ratio of 3 from the chromatograms of samples spiked at the lowest level validated, varied between 0.1 and 1 microg/kg in the three food matrices tested. The method was applied to the analysis of different kinds of samples. Positive findings were confirmed by acquiring two transitions (Q quantification, q confirmation) and evaluating the Q/q ratio.
The contamination levels of 16 different Fusarium- and Aspergillus-mycotoxins were chemically determined from randomly selected organic and conventional grain-based products purchased from Finnish and Italian markets. The cytotoxicity of the samples was analyzed with an in vitro test using feline fetal lung cells. Overall, the concentrations of the mycotoxins studied were low in all of the samples. Enniatins B and B1 as well as deoxynivalenol were the most predominant mycotoxins in the samples, being present in 97%, 97%, and 90% of the samples, respectively. The geographical origin or the agricultural practice had no influence on the mycotoxin concentrations of the samples. The babyfoods included in the samples had significantly lower concentrations of mycotoxins than the other products with a mean total mycotoxin content of 47 microg/kg compared with 99 microg/kg for the other kinds of food. All the samples evoked toxicity in the in vitro test, but no correlation between cytotoxicity and the mycotoxin concentrations was observed.
Mycotoxins are toxic secondary metabolites produced by filamentous fungi that are growing on agricultural commodities. Their frequent presence in food and their severe toxic, carcinogenic and estrogenic properties have been recognised as potential threat to human health. A reliable risk assessment of mycotoxin contamination for humans and animals relies basically on their unambiguous identification and accurate quantification in food and feedstuff. While most screening methods for mycotoxins are based on immunoassays, unambiguous analyte confirmation can be easily achieved with mass spectrometric methods, like gas chromatography/mass spectrometry (GC/MS) or liquid chromatography/mass spectrometry (LC/MS). Due to the introduction of atmospheric pressure ionisation (API) techniques in the late 80s, LC/MS has become a routine technique also in food analysis, overcoming the traditional drawbacks of GC/MS regarding volatility and thermal stability. During the last few years, this technical and instrumental progress had also an increasing impact on the expanding field of mycotoxin analysis. The aim of the present review is to give an overview on the application of LC-(API)MS in the analysis of frequently occurring and highly toxic mycotoxins, such as trichothecenes, ochratoxins, zearalenone, fumonisins, aflatoxins, enniatins, moniliformin and several other mycotoxins. This includes also the investigation of some of their metabolites and degradation products. Suitable sample pre-treatment procedures, their applicability for high sample through-put and their influence on matrix effects will be discussed. The review covers literature published until July 2006.
Mycotoxins likely have existed for as long as crops have been grown but recognition of the true chemical nature of such entities of fungal metabolism was not known until recent times. Conjecturally, there is historical evidence of their presence back as far as the time reported in the Dead Sea Scrolls. Evidence of their periodic, historical occurrence exists until the recognition of aflatoxins in the early 1960s. At that time mycotoxins were considered as a storage phenomenon whereby grains becoming moldy during storage allowed for the production of these secondary metabolites proven to be toxic when consumed by man and other animals. Subsequently, aflatoxins and mycotoxins of several kinds were found to be formed during development of crop plants in the field. The determination of which of the many known mycotoxins are significant can be based upon their frequency of occurrence and/or the severity of the disease that they produce, especially if they are known to be carcinogenic. Among the mycotoxins fitting into this major group would be the aflatoxins, deoxynivalenol, fumonisins, zearalenone, T-2 toxin, ochratoxin and certain ergot alkaloids. The diseases (mycotoxicoses) caused by these mycotoxins are quite varied and involve a wide range of susceptible animal species including humans. Most of these diseases occur after consumption of mycotoxin contaminated grain or products made from such grains but other routes of exposure exist. The diagnosis of mycotoxicoses may prove to be difficult because of the similarity of signs of disease to those caused by other agents. Therefore, diagnosis of a mycotoxicoses is dependent upon adequate testing for mycotoxins involving sampling, sample preparation and analysis.
The mycotoxins that commonly occur in cereal grains and other products are not completely destroyed during food processing operations and can contaminate finished processed foods. The mycotoxins most commonly associated with cereal grains are aflatoxins, ochratoxin A, fumonisins, deoxynivalenol and zearalenone. The various food processes that may have effects on mycotoxins include sorting, trimming, cleaning, milling, brewing, cooking, baking, frying, roasting, canning, flaking, alkaline cooking, nixtamalization, and extrusion. Most of the food processes have variable effects on mycotoxins, with those that utilize the highest temperatures having greatest effects. In general the processes reduce mycotoxin concentrations significantly, but do not eliminate them completely. However, roasting and extrusion processing show promise for lowering mycotoxin concentrations, though very high temperatures are needed to bring about much of a reduction in mycotoxin concentrations. Extrusion processing at temperatures greater than 150 degrees C are needed to give good reduction of zearalenone, moderate reduction of alfatoxins, variable to low reduction of deoxynivalenol and good reduction of fumonisins. The greatest reductions of fumonisins occur at extrusion temperatures of 160 degrees C or higher and in the presence of glucose. Extrusion of fumonisin contaminated corn grits with 10% added glucose resulted in 75-85% reduction in Fumonisin B(1) levels. Some fumonisin degredation products are formed during extrusion, including small amounts of hydrolyzed Fumonisin B(1) and N-(Carboxymethyl) - Fumonisin B(1) and somewhat higher amounts of N-(1-deoxy-d-fructos-1-yl) Fumonisin B(1) in extruded grits containing added glucose. Feeding trial toxicity tests in rats with extruded fumonisin contaminated corn grits show some reduction in toxicity of grits extruded with glucose.
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