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Hellany, H.; Assaf, J.C.; El-Badan, D.; Khalil, M. Mycotoxins in Groundnuts and Tree Nuts. Encyclopedia. Available online: https://encyclopedia.pub/entry/52894 (accessed on 08 July 2024).
Hellany H, Assaf JC, El-Badan D, Khalil M. Mycotoxins in Groundnuts and Tree Nuts. Encyclopedia. Available at: https://encyclopedia.pub/entry/52894. Accessed July 08, 2024.
Hellany, Heba, Jean Claude Assaf, Dalia El-Badan, Mahmoud Khalil. "Mycotoxins in Groundnuts and Tree Nuts" Encyclopedia, https://encyclopedia.pub/entry/52894 (accessed July 08, 2024).
Hellany, H., Assaf, J.C., El-Badan, D., & Khalil, M. (2023, December 18). Mycotoxins in Groundnuts and Tree Nuts. In Encyclopedia. https://encyclopedia.pub/entry/52894
Hellany, Heba, et al. "Mycotoxins in Groundnuts and Tree Nuts." Encyclopedia. Web. 18 December, 2023.
Mycotoxins in Groundnuts and Tree Nuts
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This entry is adapted from the peer-reviewed paper 10.3390/pr11123428

Mycotoxins are toxic compounds produced as secondary metabolites by certain types of filamentous fungi under specific conditions. The contamination of nuts and nut-related products with mycotoxins is a significant global concern due to their severe consequences on human health, including carcinogenicity and immunosuppression. Aflatoxins, with a particular emphasis on aflatoxin B1, are the most common and toxic mycotoxins found in human food. Aflatoxin B1 (AFB1) is known to be highly toxic and carcinogenic.

nuts mycotoxins quantification

1. Introduction

Mycotoxins, a group of secondary metabolites produced by filamentous fungi, have attracted significant attention due to their various effects on human and animal health [1][2]. The term “mycotoxin” was used for the first time in the 1960s in the UK to characterize a toxin found in peanuts used for animal feed. It was linked to the sudden death of turkeys after consuming contaminated peanuts (Turkey-X disease) [1][3]. Mycotoxins are small molecules (molecular weight < 700) mainly produced by fungal genera such as Aspergillus, Fusarium, and Penicillium [2][4].
Over 400 mycotoxins have been identified, but only a few species pose food safety concerns as they have been reported to contaminate food, including nuts [2][5]. These include aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), aflatoxin M1 (AFM1), aflatoxin M2 (AFM2)), ochratoxin A (OTA), patulin, zearalenone (ZEA), fumonisins (including fumonisin 1 (FB1) and fumonisin B2 (FB2)), citrinin (CIT), fusarenon-X (FUS-X), diacetoxyscirpenol (DAS), trichothecenes (including T-2 toxin (T-2), HT-2 toxin (HT-2), nivalenol (NIV), and deoxynivalenol (DON) [2][5]. They can contaminate a variety of nuts [1][5]. Contamination can occur pre- or post-harvest, and many conditions enhance the production of mycotoxins by fungi, such as water activity, moisture, temperature, and storage conditions. Mycotoxins are usually thermostable, even at high temperatures (80–121 °C) during nut processing (roasting, drying) [6]. Approximately 25% of the world’s food crops, including nuts, are contaminated annually, resulting in significant agricultural and industrial losses [7][8].
Different species of fungi can coexist in the same nut product and produce various mycotoxins. These mycotoxins can act synergistically or have cumulative effects, making the overall toxicity more complex and posing additional risks to human and animal health. Additionally, nut products may contain masked mycotoxins. These modified derivatives are produced during the metabolism of mycotoxins by plants and animals through enzymatic processes and chemical reactions. They are referred to as “masked” because they cannot be detected by the analytical methods typically used for mycotoxin detection. However, they can be released into toxic-free forms during digestion and nut processing methods, which increases the risk to human and animal health [1][9].
Nuts are frequently consumed in the Mediterranean region due to their numerous benefits. They are rich in healthy fats (monounsaturated and polyunsaturated fats), proteins, fibers, vitamins (such as vitamin E and vitamin K), and minerals (like magnesium and potassium) [10]. The term “nut” is used to describe all types of nuts consumed by humans. Nuts are categorized into two main classes: tree nuts, which are one-seeded fruits growing on trees (including pistachios, hazelnuts, almonds, and cashew nuts), and groundnuts, which grow underground and belong to the Leguminosae family, such as peanuts [11][12].
The widespread presence of mycotoxins in nuts and their high toxicity has become a worldwide concern. Several national and international organizations, such as the US Food and Drug Administration (FDA), the World Health Organization (WHO), the Food and Agriculture Organization (FAO), and the European Food Safety Authority (EFSA), have addressed the problem of mycotoxins through various regulations, recommendations, and regulatory guidelines for major mycotoxins found in food and feedstuffs [1][2]. For instance, the EU limits the sum of aflatoxins (AFB1, AFB2, AFG1, AFG2) in pistachios, and almonds are set at 15 μg/kg, while the FDA sets the limits for total aflatoxins at 20 μg/kg [7]. The EU sets the maximum limits for OTA (Ochratoxin A) in nuts at 10 g/kg for dried nuts, and the maximum levels for fumonisins are 200–500 g/kg in foods, including nuts [7][13].

2. Analytical Quantification of Mycotoxins in Nuts

The detection and quantification of mycotoxins in nut samples are critical to ensure food safety and prevent health hazards. Analytical techniques play an important role in identifying and quantifying mycotoxin levels in nuts. These techniques can be categorized into direct techniques like ELISA and TLC, as well as indirect techniques like HPLC and LC-MS/MS.

2.1. Indirect Techniques

2.1.1. High-Performance Liquid Chromatography (HPLC)

HPLC is widely used for mycotoxin detection in nuts due to its accuracy and sensitivity. It separates mycotoxins by passing samples through a chromatographic column using a high-pressure pump, utilizing the differences in their properties. Coupled with a UV or fluorescence detector, HPLC can be automated, offering increased throughput, precision, and accuracy compared to other methods like ELISA and TLC [1][14]. Moreover, it enables automation. However, it suffers from several drawbacks, including the high cost of equipment, the need for specialized expertise, and the requirement of derivatization for some mycotoxins [15][16]. Numerous researchers have extensively utilized HPLC to measure various mycotoxins found in nuts [17][18][19][20][21][22][23][24][25][26][27].

2.1.2. LC-MS

Since 1980, liquid chromatography–mass spectrometry (LC-MS) has become a widely used technique for mycotoxin analysis due to its many advantages [28]. It involves separating mycotoxins with reversed-phase LC, followed by ionization and mass analysis [28][29]. While thermospray (MS) was initially common for mycotoxin analysis, electrospray ionization (ESI) has largely replaced it due to higher sensitivity and selectivity, enabling the detection of lower analyte levels in complex matrices like nuts [28][30]. LC-MS is a highly sensitive method for mycotoxin detection in nuts, and it has the ability to detect multi-analytes simultaneously [28][31][32][33][34]. It can be used to analyze and quantify mycotoxins without the need for derivatization [4][31]. However, LC-MS is time-consuming with variable signal suppression/enhancement due to multiple steps involved [28][31]. Additionally, LS-MS instruments are usually expensive [28]. “Matrix effects” are a major challenge in mycotoxin analysis, impacting accuracy and precision. These effects arise from the co-elution of matrix components and their influence on analyte ionization efficiency, resulting in signal suppression/enhancement, particularly in complex matrices with diverse chemical properties [28][35][36]. Matrix effects appear especially when complex matrices are analyzed due to the presence of many interferences having different chemical properties [37]. LC-MS/MS has become a popular technique in mycotoxin analysis in nuts in recent years. Almost 80% of all published LC-MS studies on mycotoxins since 2012 have used LC-MS/MS [36].
Several methods aim to reduce matrix effects in mycotoxin analysis of nuts by LC-MS, with “dilute-and-shoot” being suitable if highly sensitive LC-MS is present. However, as many LC-MS instruments cannot detect aflatoxins below EU limits, a clean-up step might be necessary. The most effective solution could be using stable isotope dilution assay (SIDA) with a commercial C-aflatoxins internal standard for accurate quantification in food, including nuts [28][36].
Several assays were developed to increase the sensitivity of the LC-MS/MS technique and to reduce the matrix effects on mycotoxin analysis in nuts.
SIDA was used for the first time for the detection of aflatoxins in nuts by Cervino et al. [38], who utilized LC-MS/MS stable isotope dilution assay (SIDA) to detect aflatoxins in nuts. Deuterated aflatoxins B2 and G2 were synthesized using palladium nanoparticles and used for quantification. This technique effectively reduced matrix effects, yielding high recoveries (94–105%) for aflatoxins in almonds. SIDA proved to be a sensitive method for analyzing aflatoxins in various nuts, even below EU regulatory limits. Xavier et al. [39] developed an LC-MS technique for aflatoxins analysis in Brazil nuts using the“dilute-and-shoot” method and atmospheric pressure chemical ionization (APCI). Nut samples were diluted and injected into the LC-MS instrument after extraction. The technique demonstrated high sensitivity with low LOD (0.04–0.060 μg/kg) and LOQ (0.08–0.12 μg/kg) values, achieving accurate quantification of aflatoxins (recoveries: 92–100%). Despite the need for an expensive instrument and regular maintenance, the technique provided quick results (total run time was less than 5 min), increased confidence in the findings, and removed the necessity for a clean-up step. Huang et al. [31] developed a dilute-and-shoot method using an ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) to simultaneously detect six types of aflatoxins (B1, B2, G1, G2, M1, and M2) in peanuts and their derivative products. Peanut samples were extracted with 84% acetonitrile, diluted with an acetonitrile/water mixture (10:90, v/v), and then analyzed using the UHPLC-MS/MS instrument. The developed method demonstrated good recovery (74.7–86.8%) and excellent precision (RSD < 10.9%).
The sensitivity and selectivity of LC-MS/MS can be influenced by many factors, such as the choice of columns and the mobile phase. Huang et al. [31] improved LC-MS/MS sensitivity by using a UHPLC-MS/MS technique with a 1.8 µm column and a water/formic acid–acetonitrile/methanol mobile phase. Aflatoxin analysis in peanuts showed over 74.7% recovery, indicating accuracy. Their technique detected six types of aflatoxins in 75 peanut samples, including AFM1 and AFM2, for the first time.

2.1.3. LC Techniques–QqQ-MS/MS

Co-contamination of aflatoxins and other mycotoxins in nuts is common, leading to increased toxicity and amplified effects [40]. Monitoring and controlling their presence in food and feed is crucial due to potential synergistic or additive effects [41][42]. Liquid chromatography (LC) and gas chromatography–mass spectrometry (GC-MS) are widely used to detect the co-occurrence of mycotoxins [43]. LC-MS/MS, specifically using a triple quadrupole mass spectrometer (usually abbreviated as QqQ), allows simultaneous detection and quantitation of multiple mycotoxins, providing high specificity and sensitivity [28][29]. Multiple reaction monitoring (MRM) scans enable efficient analysis of complex mixtures, making it a powerful technique for mycotoxin analysis in nuts [44][45]. The development of LC-QqQ-MS-based multi-mycotoxin techniques in many studies has increased the ability to detect multiple mycotoxins in a single analysis, and they have seen significant advancements in terms of sensitivity and capability for quantitative analysis [28]. Cunha et al. [46] developed an effective LC-MS/MS technique for determining 16 mycotoxins in nuts using a modified QuEChERS procedure. The method showed good recovery (70–93%), repeatability (RSD ≤ 13%), and low LOQ values (1.5–5 μg/kg). The co-contamination was observed in 35% of the nut samples. Oyedele et al. [41] employed LC-QqQ-MS/MS to screen 84 groundnut samples from different agro-ecological zones in Nigeria, detecting 58 microbial metabolites (54 fungal, four bacterial) and quantifying 10 major mycotoxins. A similar LC-MS/MS multi-technique was developed by Spanjer et al. [47], in which 33 mycotoxins were analyzed in peanut and pistachio samples in a single 30-minute run time. The recoveries ranged between 80% and 110%, and the LOD values in peanuts were 0.15–10 μg/kg, while in pistachios, they were 0.5–200 μg/kg. Furthermore, a study conducted by Warth et al. [48] showed the power of the LC-MS/MS multi-toxin technique to detect 27 metabolites in groundnut samples. The LOD values ranged between 0.05 μg/kg and 250 μg/kg, and good recoveries were obtained (38–1155). In another study, Liao et al. [49] developed a similar LC-ESI-MS/MS technique for mycotoxin analysis in several nut samples (almonds, peanuts, pistachios). This innovative approach enabled the simultaneous detection of an impressive array of 26 mycotoxins. The results were good, with almond samples exhibiting a remarkable 87 ± 12% recovery rate, peanuts displaying an even more impressive 104 ± 16% recovery rate, and pistachios showing a 92 ± 18% recovery rate. Moreover, the technique had excellent sensitivity, as evidenced by the low LOQ values ranging from 0.2 to 12.4 μg/kg for almonds, 0.3 to 12.1 μg/kg for peanuts, and 0.3 to 12.8 μg/kg for pistachios.
Additionally, ultra-high-performance liquid chromatography (UHPLC) is a highly efficient and accurate technique for mycotoxin analysis in food. Its advantages include high resolution and retention-time reproducibility, as well as a high peak capacity when used with MS, enabling the separation of numerous components in a single run [50]. UHPLC’s use of submicron columns (<2.0 μm) allows for rapid detection of mycotoxins at extremely low levels [28]. Moreover, it enables fast and efficient analysis of multiple samples due to its high throughput [50]. UHPLC-MS also minimizes sample-matrix effects, making it a popular choice for multiclass mycotoxin analysis [50][51]. UHPLC-MS/MS has become a popular analytical technique for the multiclass analysis of mycotoxins in nuts because of its several advantages [52]. Therefore, in many studies, the UHPLC-QqQ-MS/MS technique allows for the simultaneous determination of several mycotoxins in nut samples.
Arroyo-Manzanares et al. [52] developed a sensitive UHPLC-MS/MS technique to detect 14 mycotoxins in nuts using QuEChERS and DLLME extraction. This technique showed low LOD values (0.17–45.1 μg/kg) and low LOQ values (0.57–150 μg/kg) with high recoveries (60.7–104.3%), indicating reliability in nut sample analysis. Furthermore, a UPLC-QqQ-MS/MS technique for the simultaneous determination of several mycotoxins in nut samples (peanuts, pistachios, almonds) was described by Kafouris et al. [53]. The technique was rapid, sensitive, and validated for 11 mycotoxins. LOD values ranged from 0.15 to 7.5 μg/Kg for pistachios, 0.15 to 15.3 μg/kg for almonds, and 0.08 to 15 μg/kg for peanuts. It showed mean recoveries of 74.4% to 131.7% in spiked samples. In the case of Hidalgo-Ruiz et al. [54], a UHPLC-QqQ-MS/MS technique was developed and validated for detecting six mycotoxins in nuts. Peanuts were chosen as the representative matrix after evaluating different types. The LOQ ranged from 0.5 to 1 µg/kg. Recoveries were 80–120%, with precision values < 20% for intra- and inter-day. Another multi-target UHPLC-QqQ-MS/MS technique was developed by Varga et al. [55] for mycotoxin analysis in nuts. It utilized sub-2-µm particle columns, enhancing resolution and accuracy. It successfully analyzed 191 fungal metabolites in 53 nut samples, quantifying 65 mycotoxins and semi-quantifying 126 others. Good recoveries were obtained and ranged from 80 to 120%.

2.2. Direct Techniques

2.2.1. Thin-Layer Chromatography (TLC)

TLC was used for aflatoxin analysis, with a significant impact on purification and identification in 1960 [35][56]. High-performance thin-layer chromatography (HPTLC) extended TLC, providing enhanced separation ability, higher resolution, and sensitivity and improving compound analysis like aflatoxins, was replaced by HPLC [56]. It employed different stationary phases like silica gel, F254 fluorescent silica gel, or organic acid-impregnated silica gel, with silica gel being the most common [1]. Visualization is achieved through fluoro densitometry or visual procedures (using UV-Vis spectroscopy) [1][57]. While TLC is simple, cost-effective, and high-throughput [1][35][58], it has a poor separation of closely related compounds, a low accuracy in determining the amounts of each component, and it is less sensitive for detection of small amounts of a substance [35][59]. Proper sample preparation varies depending on mycotoxin properties and type [1][35]. TLC is still a widely applied method for both quantitative and semi-quantitative measurements of mycotoxins in nut samples [1]. The TLC technique was used by many researchers for the first screening of mycotoxin presence in nut samples [60][61][62][63].

2.2.2. Enzyme-Linked Immunosorbent Assay ELISA

ELISA is widely used for detecting and quantifying mycotoxins [7][64]. This immunological technique relies on specific interactions between the mycotoxin and its corresponding antibodies coated onto a solid surface. If the mycotoxin is present in the nut sample, it binds to the antibodies. A secondary antibody, linked to an enzyme, is added, producing a signal upon reacting with a substrate [11][14]. ELISA can be qualitative or quantitative; visual color intensity provides semi-quantitative results, while quantitative results require careful calibration and are more time-consuming [64][65][66]. Direct competitive ELISA is a common technique to detect and quantify mycotoxins in nuts, relying on labeled and unlabeled antigens competing for binding to specific antibodies. The signal intensity is inversely proportional to the mycotoxin concentration [7][64][66][67]. This sensitive, inexpensive, and rapid technique serves as a primary screening tool for detecting contaminated nut samples [7][31][66][67][68][69]. However, ELISA has limitations like cross-reactivity and matrix effects, causing false results. Validated mycotoxin ELISA kits exist, but they are specific to certain mycotoxins, contamination levels, and food matrices, limiting their universal applicability. Additionally, ELISA lacks the ability to detect multiple mycotoxins simultaneously in nut samples, requiring separate tests, which can be time-consuming, and ELISA kits are usually one-time use [7][31][66][67][70]. Commercial ELISA kits are available for various mycotoxins, including aflatoxins AFs, ZEA, OTA, DON, T2/HT2, and FBs [1][14]. ELISA kit has become an indispensable tool for researchers studying the prevalence of mycotoxins in nuts [71][72][73][74].
Efforts to develop cost-effective monoclonal antibodies against aflatoxins for accessible immunochemical analysis were made. Therefore, several monoclonal antibodies were reported by Li et al. [69][75] and Oplatowska-Stachowiak et al. [76] and used for an accurate detection of aflatoxins in nut samples. Common steps included selecting the target antigen, immunizing mice, screening hybridomas, and purifying antibodies. The best monoclonal antibody was chosen based on sensitivity and cross-reactivity for ELISA development. The validated class-specific monoclonal antibody-based ELISA successfully detected specific aflatoxins in contaminated nut samples. Oplatowska-Stachowiak et al. [76] produced seven unique monoclonal antibodies with high sensitivity and cross-reactivity for aflatoxin detection. Among them, two antibodies (1 NP-D and 1 NP-C) exhibited impressive IC50 values for aflatoxin B1. The developed ELISA test demonstrated low LOD values for AFB1 and total AFs (0.4 and 0.3 μg/kg, respectively), and acceptable recoveries were high (97.1–107.5%). Researchers attempted to develop monoclonal antibodies with improved cross-reactivity for G-group aflatoxins. Li et al. [75] created three promising antibodies (2G6, 3A4, and 4G4) with good cross-reactivity to AFG1 and AFG2. Among them, 2G6 showed the highest sensitivity and specificity (IC50 of 17.18 ng/mL, 100% CR with AFG1, and 87% CR with AFG2). They utilized 2G6 to design a competitive indirect ELISA (CI-ELISA) with optimized parameters, achieving a LOD of 0.06 ng/mL and demonstrating high accuracy in detecting G-group aflatoxins in peanut samples, with recoveries of 94–103%. Li et al. [69] conducted a study using three class-specific monoclonal antibodies (8E11, 8F6, and 10C9) to detect aflatoxins. Among them, 10C9 displayed the most similar sensitivity for five aflatoxins and the highest cross-reactivity (CR = 65.2) to AFG2. With the 10C9 antibody, they developed an ELISA for peanut samples, achieving recoveries of 85.5% to 102%. The developed ELISA method also demonstrated low LOD values (0.06–0.09 ng/mL).

2.2.3. Lateral Flow Device (LFD)

Lateral flow tests are widely used for the rapid detection of mycotoxin prevalence in nut samples. These tests contain specific antibodies and mycotoxin–carrier protein conjugates to identify target mycotoxins [65][77][78]. The procedure involves applying a liquid sample to the test area. If the mycotoxin is present, a red color appears solely on the control line, indicating a positive result. In the absence of mycotoxins, both the test line and the control line turn red, showing a negative result. Lateral flow tests are rapid and simple techniques for mycotoxin screening in nut samples, providing visual results for the presence or absence of the target mycotoxin [65][78]. They are semi-quantitative, but accuracy can be improved with a lateral flow reader to measure color intensity [65]. Gold nanoparticles (AuNPs) can enhance sensing applications through metal enlargement, increasing optical or electrochemical signals by depositing silver or gold on their surface [79]. Colloidal gold, referring to suspended AuNPs, is widely used in mycotoxin detection due to its ease of preparation and visible signal production. Commonly, 40 nm particles are utilized in immunochromatographic strip tests for mycotoxin detection in nut samples [65][77][78][79].
Several studies have used the LFD that contains gold nanoparticles for the simultaneous detection of multiple mycotoxins in nuts. Li et al. [80] developed a multi-component immunochromatographic assay (ICA) for the simultaneous detection of three mycotoxins (AFB1, OTA, ZEA) in peanut samples. The assay utilized competitive immunoreactions between specific antibody–colloidal gold nanoparticle conjugate probes and mycotoxins or mycotoxin antigens. The ICA strip conditions were optimized, and visible results were obtained in 20 min. The visual detection limits of the ICA for AFB1, OTA, and ZEA were 0.25 ng/mL, 0.5 ng/mL, and 1 ng/mL, respectively. The prevalence of multiple mycotoxins in peanut samples was investigated using the developed ICA. In a study conducted by Chen et al. [81], a multiplex lateral flow immunoassay (LFA) was capable of detecting AFB1, ZEA, and OTA mycotoxins in peanut samples within 15 min. The optimized LFA employed 32 nm AuNPs, specific antibody amounts, and pH levels, resulting in visual detection limits of 10 µg/kg for AFB1, 50 µg/kg for ZEA, and 15 µg/kg for OTA in spiked samples. The quantitative analysis of peanut samples revealed LOD values of 0.13 µg/kg for AFB1, 0.46 µg/kg for ZEA, and 0.24 µg/kg for OTA, with good recovery rates ranging from 86.2 to 114.5%. In another study conducted by Zhang et al. [82], they developed an ultrasensitive immunochromatographic (IC) assay for detecting total aflatoxins in peanuts. They employed a competitive format using a monoclonal antibody (1C11) labeled with nanogold particles to enhance sensitivity. The optimized conditions resulted in lower visual detection limits (VDLs) compared to other studies: 0.03, 0.06, 0.12, and 0.25 ng/mL for AFB1, AFB2, AFG1, and AFG2, respectively. The validated IC assay was then used to analyze several peanut samples for aflatoxin detection.
While gold nanoparticle labels have been widely used in immunochromatographic assays due to their unique optical properties, they can be susceptible to interference from complex sample matrices, such as those found in food, which may lead to false positives or false negatives [83]. To address this issue, researchers have developed fluorescent lateral flow immunoassays based on lanthanide Eu3+ chelate labeling, which offers several advantages over gold nanoparticle labels. Wang et al. [83] developed fluorescent lateral flow immunoassays using lanthanide Eu3+ chelate labeling to overcome the limitations of gold nanoparticle labels in complex sample matrices, such as nuts. The optimized technique allowed simultaneous and quantitative detection of AFB1, ZEA, and CTN in peanut samples within 15 min. The limit of detection (LOD) for AFB1 and ZEA in peanut samples was 0.18 µg/kg and 0.57 µg/kg, respectively, with good recoveries (91.60–95.87% for AFB1 and 85.72–91.67% for ZEA).

2.3. Combination of Direct and Indirect Techniques

To achieve a comprehensive mycotoxin analysis in nuts, researchers and analysts often employ a hybrid approach that combines both direct and indirect methods. Initially, they use rapid screening techniques, like ELISA or TLC, to identify the presence or absence of specific mycotoxins. If contamination is detected during the screening, further confirmation and quantification can be performed using HPLC or LC-MS/MS to obtain more detailed information about the mycotoxin levels in the sample.
In TLC, the comparison of the migration distance ratio (Rf values) between nut samples and standards can provide preliminary or presumptive evidence of the presence of mycotoxins. However, this evidence is not conclusive, and further confirmation tests are necessary [28]. In many studies, TLC has been used for the detection of initial positive results. Subsequently, HPLC was employed for further confirmation and quantification of mycotoxins in nut samples. In a study conducted by Kujbida et al. [61], they investigated the aflatoxin occurrence in peanuts and cashew nuts using two chromatographic techniques (TLC and HPLC). TLC screening detected the contaminated samples, while HPLC was used for the quantification of four aflatoxins in positive samples. In many studies, TLC has been reported for assessing mycotoxigenic fungi in nuts, while HPLC was used for further confirmation and quantification of mycotoxins. A study by Amar et al. [17] aimed to detect aflatoxigenic fungi and aflatoxins in various nut samples (almonds, pistachios, hazelnuts, peanuts, walnuts) from the Algerian market. TLC was used for rapid screening of positive isolated fungi, and HPLC was employed for aflatoxin quantification in the nut samples, leading to good recoveries (72.6–91.8%). A similar approach has been reported in a study conducted by Ait Mimoune et al. [84] for mycotoxin analysis (Aflatoxins, CPA, OTA) in nut samples from various markets. TLC was used for qualitative analysis to detect aflatoxigenic strains in positive samples, followed by HPLC for quantification of mycotoxin levels. In a study conducted by Ozay et al. [63] in Turkey, hazelnut samples were analyzed for aflatoxin detection. The detection of aflatoxin-producing fungi involved qualitative tests using TLC plates, while quantitative analysis was conducted through HPLC. In another study in India conducted by Sharma et al. [19], TLC and HPLC were used to determine the presence and levels of AFs, OTA, and PAT in Chilgoza pine nuts. TLC was employed for the qualitative estimation of mycotoxins, while HPLC was used for their quantification. Overall, TLC is considered as a qualitative or semi-quantitative technique where positive results can be assessed visually without an accurate quantification of mycotoxin levels in nut samples.
The possibility of obtaining false-positive results in ELISA tests exists due to the cross-reactivity of antibodies. To address this issue, it is recommended to confirm the results using a suitable chromatographic technique, such as HPLC [7][70][77]. Therefore, several studies have used the ELISA test for rapid screening of mycotoxins in nut samples, and the results were then confirmed by chromatographic techniques such as HPLC and LC-MS. Chun et al. [73] conducted a study analyzing 85 nuts and nut products for aflatoxins, utilizing both ELISA and HPLC techniques. After ELISA screening, 31 samples were identified as positive for aflatoxins. Subsequently, HPLC quantification of total aflatoxins in these thirty-one samples revealed that nine of them were contaminated, and the results obtained were further confirmed by LC-MS. Two similar studies were reported by Shadbad et al. [74] and Leong et al. [71], in which nuts and nut products were first screened by ELISA to detect the contaminated samples. Subsequently, HPLC was employed for further confirmation and quantification of aflatoxins in the identified positive samples. Asis et al. [85] compared ELISA and HPLC techniques for AFB1 determination in peanut samples. The ELISA test demonstrated high sensitivity with an LOD of 0.5 µg/kg and 107% average recovery for peanut samples contaminated with only AFB1 due to its two-antibody system and lack of cross-reactivity with the peanut matrix. The results showed a strong correlation between the two techniques, with a correlation coefficient (r) of 0.977 and a p-value less than 0.0001 (p < 0.0001). In another study, Razzazi-Fazeli et al. [86] validated positive ELISA samples of peanut products using HPLC. The study confirmed thirty contaminated peanut samples detected by ELISA, then confirmed through HPLC. Comparing ELISA and HPLC, a stronger correlation was observed at lower levels of total aflatoxins (0–120 µg/kg) and AFB1 (0–80 µg/kg), with correlation coefficients (r) of 0.9244 and 0.8805, respectively. However, this correlation was not observed at higher levels of total aflatoxins or AFB1. In addition, researchers developing a class-specific monoclonal antibody ELISA for mycotoxin analysis in nuts aimed to validate its accuracy against more precise techniques like HPLC and UHPLC-MS/MS. Li et al. [69] compared the recoveries of the developed ELISA, using AFG2 as a competitor, with those of HPLC. The results indicated the suitability of the developed ELISA for nuts analysis, with recoveries ranging from 87.5% to 102% for ELISA and 87.7% to 97.6% for HPLC. A similar approach was observed by Li et al. [75], where they focused on an ELISA specific for AFG1 and AFG2 detection in peanuts, showing recoveries comparable to HPLC and demonstrating a good correlation between the two techniques. Furthermore, Oplatowska-Stachowiak [76] compared the levels of four aflatoxins in peanuts detected by a developed monoclonal antibody-based ELISA for AFB1 with those obtained using UHPLC-MS/MS.
The results obtained by lateral flow tests (LFTs) may not be as accurate or reliable as more conventional techniques. Therefore, it is generally recommended to confirm the results obtained from a lateral flow assay with a more accurate and quantitative technique, such as HPLC [77]. In a study conducted by Li et al. [80], the levels of mycotoxins (OTA, AFB1, ZEA) in peanut samples were compared using a multi-component immunoaffinity chromatography (ICA) and ELISA as the reference technique. In another study conducted by Wang et al. [83], the levels of mycotoxins (ZEA, AFB1) in contaminated peanut samples were verified using a developed lateral flow immunoassay (IA) and confirmed through HPLC-MS/MS, showing a strong correlation between the two techniques (R2 > 0.88). Zhang et al. [82] developed a nanogold-probe-based immunoassay for the detection of four aflatoxins in peanuts.

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Heba Hellany
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Jean Claude Assaf
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Dalia El-Badan
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Mahmoud Khalil
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