Analysis Of Ochratoxin A Research Articles

Taking glucose as intermediate bridge-signal-molecule for on-site and convenient detection of ochratoxin A in rice with portable glucose meter

On-site screening of biotoxins is of great importance for food safety. A new electrochemical-biosensing strategy was constructed for ochratoxin A (OTA) detection by direct using ready-made commercial portable-glucose-meter (PGM). Aptamer against OTA was adopted as the recognition probe and pre-immobilized onto the sensing interface. The complementary biotin-modified probe was further decorated by hybridization. Biotinylated invertase was further introduced onto the sensing system with streptavidin, which also acted as the signal amplification unit. The invertase, which was depended on the amount of OTA, produced the glucose from sucrose in the sensing solution. The glucose could be directly and conveniently measured with PGM. Quantitative analysis of OTA was achieved with a linear range from 0.5 ng/mL to 10 ng/mL and detection limit of 0.45 ng/mL. Of significance, it has been successfully applied for OTA analysis in rice with satisfied recoveries. This unique PGM-based electrochemical platform reveals prospective potential in food safety monitoring.

Analysis of aflatoxins and ochratoxin a in chilli powder using ultrahigh performance liquid chromatography with fluorescence detection and tandem mass spectrometry.

Chilli powder, a popular spice, is predominantly contaminated with aflatoxins (AFs) and ochratoxin A (OTA), posing a menace to public health. As no validated method exists for the simultaneous and direct analysis of AFs and OTA in chilli powder, it was imperative to develop one to ensure their effective monitoring and promote trade. In this research, we developed and validated a multi-mycotoxin analysis method that allows the simultaneous determination of AFs (AFB1, AFB2, AFG1 and AFG2) and OTA in chilli powder with high sensitivity, accuracy and precision. The optimised sample preparation workflow started with the extraction of chilli powder (25g) with methanol-water (100mL, 80:20). An aliquot (3mL) was cleaned on a multi-mycotoxin, immunoaffinity column (AFLAOCHRA PREP®) and analysed using ultrahigh performance liquid chromatography with fluorescence (UHPLC-FLD) and tandem mass spectrometric (LC-MS/MS) detection in a single chromatographic run. The method performance was evaluated through intra- and inter-laboratory validation (ILV) studies, and also by analysing a certified reference material. A direct analysis using UHPLC-FLD (without derivatisation) provided the limits of quantification (LOQ) of 0.25 and 1ng/g for AFs and OTA, respectively, while the LOQ for all these mycotoxins in LC-MS/MS was 0.5ng/g. These LOQs are much lower than the maximum levels (MLs) specified by the European Commission. The recoveries of these analytes at LOQ and higher levels were above 75% (RSDr < 12%). The ILV study demonstrated satisfactory method-reproducibility (RSDR < 25%). The analysis of the certified reference material provided accuracies of AFs and OTA in the range of 83-101%. The analysis by UHPLC-FLD and LC-MS/MS provided very similar results. The incurred levels of B1 in market samples were estimated with a precision-RSD of < 6%. Considering its efficiency and alignment with the regulatory requirements, this method can be implemented for the routine analysis of AFs and OTA in chilli powder.

Region separation type bio-photoelectrode based all-solid-state self-powered aptasensor for ochratoxin A and aflatoxin B1 detection

Ochratoxin A (OTA) and Aflatoxin B1 (AFB1) are two highly toxic and naturally coexistence mycotoxins, which have posed a serious threat to food safety. The coexistence of these two mycotoxins can produce significant synergistic effects, so it is necessary to establish an effective analytical method. In this work, a dual bio-photoelectrode based all-solid-state multiplexed self-powered aptasensor was realized for high-throughput analysis of OTA and AFB1. There was a large Fermi level difference between photoanode and photocathode, which ensured the light assisted self-driving of the system. Due to the regional immobilization of aptamers, it could save the dosage of recognition elements, reduce the preparation cost and simplify the operation procedure. Meanwhile, construction strategy of spatial separation could effectively eliminate the overlapping signals and cross interference between targets, achieving the results more accurate and the calculation more convenient. The constructed aptasensor realized OTA and AFB1 detection in corn samples with practicality, good stability, anti-interference ability and repeatability. Therefore, this work not only achieved the high-throughput analysis of mycotoxins, but also provided a new perspective for the construction of all-solid-state multiplexed self-powered sensor.

Toehold-Mediated Cascade Catalytic Assembly for Mycotoxin Detection and Its Logic Applications.

In this work, an enzyme-free biosensor is reported for mycotoxin detection based on a toehold-mediated catalytic hairpin assembly (CHA) and a DNAzyme-cascaded hydrolysis reaction. In the presence of a mycotoxin, the recognition between an aptamer and the mycotoxin releases the trigger DNA. The trigger DNA initiates the toehold-mediated CHA, generating large amounts of partial duplex B/C with four toeholds, which can be used to assemble the DNAzyme-cascaded hydrolysis reaction. Furthermore, through a collaborative autoassembly reaction among the B/C duplex, DNA1, and DNA2, supramolecular nanostructures corresponding to Mg2+-dependent DNAzymes can be formed. With the incubation of Mg2+, the dual-modified (TAMRA/BHQ2) substrate strand DNA2 will be cleaved into two fragments, yielding a high TAMRA fluorescence signal for mycotoxin testing. Under optimal conditions, the sensing system was ultrasensitive and showed low detection limits of 0.2 pM for ochratoxin A (OTA), 0.13 pM for aflatoxin B1 (AFB1), and 0.17 pM for zearalenone (ZEN). The mycotoxin aptasensor also exhibited high selectivity and was successfully applied for the quantitative analysis of OTA, AFB1, and ZEN in wine samples. Due to the advantages of flexibility and versatility, this mycotoxin platform was used to fabricate several concatenated logic gates including "AND-INHIBIT", "INHIBIT-OR", "OR-AND", and "OR-INHIBIT" logic biocomputings. Such multiple functions of the logic system provided a universal sensing strategy for the intelligent detection of multiplex mycotoxins, demonstrating considerable potential in food safety and environmental monitoring.

A signal on-off fluorescence sensor based on the self-assembly DNA tetrahedron for simultaneous detection of ochratoxin A and aflatoxin B1

Highly rigid and versatile functionality of DNA tetrahedron nanostructures is often used in biosensing systems as a potential detection technique. In this work, a signal on-off fluorescence sensor based on the self-assembly DNA tetrahedron was developed for the rapid simultaneous detection of ochratoxin A (OTA) and aflatoxin B1 (AFB1). The fluorophore labeled DNA tetrahedron as probe was successfully synthesized via a simple denaturing annealing, the distance between fluorophore and quencher was regulated according to the change of hairpin structure on the tetrahedron. The fluorescence intensity of Cy3 decreased significantly in the presence of OTA, while the fluorescence intensity of Cy5 kept almost unchanged. And the fluorescence intensity of Cy5 decreased significantly in the presence of AFB1, while the fluorescence intensity of Cy3 kept almost unchanged. In the present of OTA and AFB1, the fluorescence intensity of Cy3 and Cy5 showed decreased significantly simultaneously, indicating the fluorescence sensor can be applied to simultaneous detect OTA and AFB1. Hence, the quantitative analysis of OTA and AFB1 was performed indirectly by the fluorescence intensity changes. The limits of detection (LOD) are as low as 0.005 ng/mL for OTA and 0.01 ng/mL for AFB1. In addition, the novel DNA tetrahedron-based fluorescence sensor possessed a universal applicability, which could be well applied in corn and wine.

Anchoring black phosphorus quantum dots over carboxylated multiwalled carbon nanotubes: A stable 0D/1D nanohybrid with high sensing performance to Ochratoxin a

Exploration of an efficient ochratoxin A (OTA) screening technique is highly important because of its highly toxic effect on human health. Electrochemical sensor, characterized with high accuracy and precise, is an emerging detection technique for toxic agents in food samples. However, the inherent fouling effect and low electrochemical activity from OTA molecular hinder its practical application in OTA detection. Herein, we prepared a novel 0D/1D nanohybrid by anchoring black phosphorus quantum dots (BPQDs) over carboxylated multiwalled carbon nanotubes (CNTs-COOH) via a facile sonication assisted assembly approach. Characterization techniques together with theoretical approximations reveal that BPQDs can anchor onto the CNTs-COOH via strong covalent reaction, endowing the nanohybrid with high conductivity, high stability and high anti-fouling property. The electrochemical sensor constructed with the 0D/1D nanohybrid shows high electrochemical activity and a good antifouling effect in OTA detection. In optimized conditions, the sensor provide a quantitative determination of OTA in a wide concentration range from 0.4 to 10 μM with a detection limit of 0.03 μM. Moreover, the sensor shows high reproductivity and satisfactory anti-interference ability. Satisfactory recoveries that can comparable to those of high-performance liquid chromatography indicate that the sensor is promising for the trace-level analysis of OTA in agricultural crops.

A sensitive WS

In this article, we report a tungsten disulphide (WS2) nanosheet sensing platform based on chemiluminescence resonance energy transfer (CRET) for light on detection of ochratoxin A (OTA). The CRET system involves OTA aptamer-12-mer linker-DNAzyme, phosphate-DNA, lambda exonuclease (λexo), hemin, H2O2, luminol and a WS2 nanosheet. Based on the change in chemiluminescence intensity resulting from the OTA aptamer-12-mer linker-G-Quadruplexes/Hemin DNAzymes affinity for or desorption from the WS2 nanosheet surface in the presence and absence of OTA, an ultra-sensitive CRET-based sensor system is proposed, in which the OTA aptamer-12-mer linker-G-Quadruplexes/Hemin DNAzymes–H2O2–luminol and WS2 nanosheets act as CRET donors and acceptors, respectively. Various factors affecting the detection of OTA, including the incubation time of the OTA aptamer-12 mer linker-G-Quadruplexes/Hemin DNAzyme and WS2 nanosheet, the amount of λexo, concentrations of the WS2 nanosheet, luminol and H2O2, and pH value of the chemiluminescence reaction solution were investigated in detail. Under the optimal experimental conditions, the relative luminesecence intensity of the sensing system exhibited a good linear correlation with the OTA concentration in the range of 1.0–10.0 ng mL–1 with a LOD of 0.13 ng mL–1. The proposed CRET-based sensing system also exhibited excellent recoveries of 85.7–93.0% in rice samples and showed good application prospects for the analysis and detection of OTA, which is also easily extended to assay other biomolecules by simply changing the recognition sequence with the substrate aptamer.

Storage fungi and ochratoxin A associated with arabica coffee bean in postharvest processes in Northern Thailand

Arabica coffee is one of the most popular varieties globally; however, during postharvest and storage, it can be contaminated by fungi which produce mycotoxins such as ochratoxin A (OTA). The goal of this study was to determine fungal contamination and OTA levels of arabica coffee produced from Chiang Rai and Chiang Mai provinces in Northern Thailand. Aspergillus and Penicillium were mainly isolated and evaluated from coffee samples in the postharvest process. Coffee cherries, parchment coffee, and green coffee beans were commonly found contaminated with fungi. Coffee beans were contaminated with Penicillium, Aspergillus section Flavi, Aspergillus section Nigri, and Aspergillus section Circumdati at 1.2–10%. Fungal contamination in parchment coffee was studied using three drying methods. The results showed that coffee dried in a greenhouse had higher fungal contamination than samples dried on concrete ground or on a bamboo table. Whether basic storage packaging of green coffee beans affects fungal contamination was also investigated. Green coffee beans in three packaging types showed similar levels of Penicillium, while of the three packaging types, coffee in a polypropylene woven bag showed significantly less Aspergillus than those in a ramie sack or a plastic mesh bag. Fourteen strains of Aspergillus section Circumdati were isolated from coffee samples and 5 isolates showed green-blue fluorescence under ultraviolet light suggesting a potential of OTA production. The morphology of Aspergillus section Circumdati isolates were studied and five isolates with similar morphological characters were further identified to the species level based on ITS and the β-tubulin gene regions. Sequencing and phylogenetic analysis suggested that four isolates were grouped with A. westerdijkiae and one isolate with A. melleus. Analysis of OTA in parchment coffee, green coffee beans, and roasted coffee was performed using HPLC. The results showed that OTA was detected in only one sample of roasted coffee at 0.39 μg kg−1.

A CdSe@CdS quantum dots based electrochemiluminescence aptasensor for sensitive detection of ochratoxin A

In this work, a CdSe@CdS quantum dots (QDs) based label-free electrochemiluminescence (ECL) aptasensor was developed for the specific and sensitive detection of ochratoxin A (OTA). Chitosan (CHI) could immobilize abundant QDs on the surface of an Au electrode as the luminescent nanomaterials. Glutaraldehyde was used as the crosslinking agent for coupling a large number of OTA aptamers. Thanks to the excellent stability, good biocompatibility, and strong ECL intensity of CdSe@CdS QDs, as well as the quick reactions of the generated SO4•− in the electrolyte, strong ECL signals were measured. Because of the specific recognition of aptamer toward OTA, the reduced ECL signals caused by OTA in the samples were recorded for quantify the content of OTA. After optimizing a series of crucial conditions, the ECL aptasensor displayed superior sensitivity for OTA with a detection limit of 0.89 ng/mL and a wide linear concentration range of 1–100 ng/mL. The practicability and viability were verified through the rapid and facile analysis of OTA in real Lily and Rhubarb samples with recovery rates (n = 3) of 98.1–105.6% and 97.3–101.5%, respectively. The newly-developed QDs-based ECL aptasensor provided a new universal analytical tool for more mycotoxins in safety assessment of foods and feeds, environmental monitoring, and clinical diagnostics.

Application of the Dimeric G-Quadruplex and toehold-mediated strand displacement reaction for fluorescence biosensing of ochratoxin A

Ochratoxin A (OTA) is one of the most toxic mycotoxins that exists in various agro-products and foods. Here, a non-label and enzyme-free fluorescence biosensor for highly specific detection of OTA has been developed by the combination of toehold-mediated strand displacement reaction (TMSD) and G-quadruplex dimer/ThT (G-dimer/ThT). The DNA duplex (aptamer-IP) is composed of the anti-OTA aptamer and a single stranded initiation probe (IP). In the presence of OTA, the attachment of target to aptamer leads to the liberation of the IP, which activates the cycle TMSD amplifications of two hairpin probes (H1 and H2) accompanied by the production of numerous H1–H2 assemblies. This double-stranded H1–H2 structure results in the proximity between the 5′-end overhang tail of H1 and the 3′-end stem of H2 to liberate the pre-blocked G-dimer sequence for lighting up ThT. In addition, the method displayed a stable fluorescence emission in the high-salt media. It was successfully applied to analyze OTA in real food samples. Hence, the constructed fluorescence biosensing platform might provide a new way for OTA and other toxin analysis detection.

Frontiers in conventional and nanomaterials based electrochemical sensing and biosensing approaches for Ochratoxin A analysis in foodstuffs: A review

Highly sensitive and rapid methods for the Ochratoxin A (OTA) detection as a main class of Ochratoxins (OT) in various food samples is significant. In the present review, novel OTA detection techniques based on electrochemical and conventional methods have been extensively explained, and also compared with each other in food samples. We classified the various electrochemical based techniques of OTA determination creatively into Impedimetric, Voltammetric, Amperometric and Electrochemiluminescence assays and their efficient comparison with conventional, and clinical methods including Capillary Electrophoresis (CE), High Performance Liquid Chromatography (HPLC), Liquid Chromatography/Mass Spectroscopy (LC/MS), and Enzyme Linked Immunosorbent Assay (ELISA) has been performed comprehensively. Moreover, structure, characteristics, and different types of OT and OTA are explained as extensively as possible. A summary of the current limitations and future challenges in OTA analysis are described, which efficiently provide a pathway for further developments and applications.

Recent Advances in Ochratoxin A Electrochemical Biosensors: Recognition Elements, Sensitization Technologies, and Their Applications.

Ochratoxin A (OTA) is a class of mycotoxin that are mainly produced by Aspergillus and Penicillium and widely found in plant origin food. OTA-contaminated foods can cause serious harm to animals and humans, while high stability of OTA makes it difficult to remove in conventional food processing. Thus, sensitive and rapid detection of OTA undoubtedly plays an important role in OTA prevention and control. In this paper, the conventional and novel methods of OTA at home and abroad are summarized and compared. The latest research progress and related applications of novel OTA electrochemical biosensors are mainly described with a new perspective. We innovatively divided the recognition element into single and combined recognition elements. Specifically, signal amplification technologies applied to the OTA electrochemical aptasensor are proposed. Furthermore, summary of the current limitations and future challenges in OTA analysis is included, which provide reference for the further research and applications.

Aptamer-modified pencil graphite electrodes for the impedimetric determination of ochratoxin A

Ochratoxin A (OTA), a widespread food contaminant, is considered as one of the most hazardous mycotoxins commonly occurring in beer. Herein, we describe a novel impedimetric aptasensor based on Pencil Graphite Electrodes (PGE) for the analysis of OTA. Taking advantage of its affordability and high active surface area, a PGE was modified with a specific aptamer, to achieve sensitive detection of OTA. This strategy relies on the target-induced increase in the electron transfer resistance (Rct) upon OTA recognition using ferro/ferricyanide as a redox probe. With the addition of OTA, the formation of OTA–aptamer complex resulted in an increase of Rct. The change of Rct strongly depends on OTA concentration, which is applied for mycotoxin quantification. An acceptable linear range was obtained from 0.1 to 2.0 ng mL−1 with a very good limit of detection as low as 0,1 ng mL−1. This simple-to-design aptasensor was successfully tested in spiked beer samples, with OTA concentrations in the range of 0.4–1.6 ng mL−1, attesting a recovery percentage of 93.4 ± 6.6%. It also showed a high level of stability and reproducibility thanks to the electro-grafting method via in-situ generation of 4-carboxyphenyl diazonium salts. This PGE-based platform is a versatile approach for the design of low-cost aptasensors for food safety control.

Development of the signal amplification based on Au@Pt/MIL-101(Cr) as mimetic enzyme and RecJf exonuclease-assistant target recycling

A novel electrochemical aptasensor was developed for multiple signal amplification based on Au@Pt/MIL-101(Cr) as mimetic enzyme and RecJf Exo-assisted target recycling. For the detection of ochratoxin A (OTA), the catalytic ability of Au@Pt/MIL-101(Cr) was better than that of Au@Pt toward hydroquinone oxidation in the presence of H2O2, and the peak current increased by 48.3 %. The peak current obtained with RecJf Exo incubation increased by 140 % than that without RecJf Exo incubation. These results demonstrated that the synergistic effect of Au@Pt/MIL-101(Cr) and RecJf Exo realized the signal amplification and enhanced the sensitivity. The prepared aptasensor exhibited a wider linear range of OTA concentration from 0.001 ng/mL to 5 ng/mL with a lower LOD of 0.17 pg/mL. The aptasensor also showed good specificity, reproductibility, and repeatability toward OTA detection. For the application in the spiked white spirit, the obtained recovery was from 97 % to 106 %, indicating that the proposed aptasensor can be applied for OTA analysis in real samples.

Transcriptome Analysis of Ochratoxin A-Induced Apoptosis in Differentiated Caco-2 Cells.

Ochratoxin A (OTA), an important mycotoxin that occurs in food and animal feed, has aroused widespread concern in recent years. Previous studies have indicated that OTA causes nephrotoxicity, hepatotoxicity, genotoxicity, immunotoxicity, cytotoxicity, and neurotoxicity. The intestinal toxicity of OTA has gradually become a focus of research, but the mechanisms underlying this toxicity have not been described. Here, differentiated Caco-2 cells were incubated for 48 h with different concentrations of OTA and transcriptome analysis was used to estimate damage to the intestinal barrier. Gene expression profiling was used to compare the characteristics of differentially expressed genes (DEGs). There were altogether 10,090 DEGs, mainly clustered into two downregulation patterns. The Search Tool for Retrieval of Interacting Genes (STRING), which was used to analyze the protein–protein interaction network, indicated that 24 key enzymes were mostly responsible for regulating cell apoptosis. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis was used to validate eight genes, three of which were key genes (CASP3, CDC25B, and EGR1). The results indicated that OTA dose-dependently induces apoptosis in differentiated Caco-2 cells. Transcriptome analysis showed that the impairment of intestinal function caused by OTA might be partly attributed to apoptosis, which is probably associated with downregulation of murine double minute 2 (MDM2) expression and upregulation of Noxa and caspase 3 (CASP3) expression. This study has highlighted the intestinal toxicity of OTA and provided a genome-wide view of biological responses, which provides a theoretical basis for enterotoxicity and should be useful in establishing a maximum residue limit for OTA.

Simultaneous Direct Analysis of Aflatoxins and Ochratoxin A in Cereals and Their Processed Products by Ultra-High Performance Liquid Chromatography with Fluorescence Detection

Abstract Background: Mycotoxins such as aflatoxins (AFs) and ochratoxin A (OTA) can pose severe health hazards because of their toxicity. Given a wide range of food matrices susceptible to fungal infections and possible cooccurrence of mycotoxins at different concentrations, validated multimycotoxin and multimatrix methods are strongly warranted. Objective: The aim of this research was to develop a simple and fast ultra-high performance LC (UHPLC) fluorescence detection (FLD)–based method to simultaneously determine AFs (B1, G1, B2, and G2) and OTA and, furthermore, to carry out single-laboratory validation in a range of cereals and processed product matrices. Methods: The sample preparation involved homogenization and extraction with methanol–water (80 + 20). For cleanup, an aliquot (3 mL) was diluted with phosphate-buffered saline, loaded on an immunoaffinity column (AFLAOCHRA PREP®), and eluted with methanol (1 mL). The cleaned extract was diluted with 0.2% acetic acid (at a 1:1 ratio) before injection into an ultra-high performance liquid chromatograph. To perform simultaneous analysis of AFs and OTA, the FLD program was developed by switching the excitation wavelength in a single chromatographic run. Results: The method provided LOQs of 0.25 and 1 ng/g for AFs and OTA, respectively, without involving any derivatization. In rice, the recoveries of AFs ranged from 84 to 106%, whereas OTA had a recovery above 72%, with the repeatability relative SDs &amp;lt;12% for both analytes. The method was successfully applied to a range of naturally contaminated market samples. Conclusions: The method is suitable for regulatory testing because of its significant time and cost effectiveness and sensitivity in compliance with the regulatory maximum levels. Highlights: The study achieves high-throughput analysis of AFs and OTA in raw and processed cereals using simultaneous extraction, cleanup, and UHPLC-FLD. Method sensitivity complies with the regulatory maximum levels. Single-laboratory validation results meet analytical QC requirements.

Label-free fluorescent aptasensing of mycotoxins via aggregation-induced emission dye

Aggregation-induced emission (AIE) dyes now serve as powerful tools for fluorescence analysis and biological imaging due to their light-up property in the aggregated state. Herein, we proposed an AIE dye-based aptasensor, enabling label-free and turn-on detection of mycotoxin. The positively charged AIE dyes would confer high-affinity to negatively charged aptamers and be lighted up by the aggregation process of dye molecules. Attributed to the resistance of ochratoxin A (OTA)-binding aptamer complex to enzyme digestion, label-free analysis of OTA could be realized via the synergetic utilization of AIE dyes and enzymatic digestion process. The digestion process could contribute to a signal to background ratio (S/B) of 1.71 for OTA detection. Remarkably, the adoption of AIE dyes would lead to a dramatic enhancement of over 104.5% of S/B compared to conventional nucleic acid dyes, thus favoring the sensitivity of the aptasensor. Only one aptamer sequence was involved in the assay, thus avoiding the complex structure design of aptamer probes. The AIE dye-based aptasensor could confer a limit of detection of 0.4 ng/mL for OTA detection. Besides, it exerted remarkable specificity for identifying OTA, and has been successfully applied for OTA analysis in wine and coffee. AIE dyes would be a compelling candidate in biohazard screening filed, and the proposed aptasensor would potentially provide a universal platform for on-site detection of food and environment contaminations.

Simultaneous Direct Analysis of Aflatoxins and Ochratoxin A in Cereals and Their Processed Products by Ultra-High Performance Liquid Chromatography with Fluorescence Detection.

Background: Mycotoxins such as aflatoxins (AFs) and ochratoxin A (OTA) can pose severe health hazards because of their toxicity. Given a wide range of food matrices susceptible to fungal infections and possible cooccurrence of mycotoxins at different concentrations, validated multimycotoxin and multimatrix methods are strongly warranted. Objective: The aim of this research was to develop a simple and fast ultra-high performance LC (UHPLC) fluorescence detection (FLD)-based method to simultaneously determine AFs (B1, G1, B2, and G2) and OTA and, furthermore, to carry out single-laboratory validation in a range of cereals and processed product matrices. Methods: The sample preparation involved homogenization and extraction with methanol-water (80 + 20). For cleanup, an aliquot (3 mL) was diluted with phosphate-buffered saline, loaded on an immunoaffinity column (AFLAOCHRA PREP®), and eluted with methanol (1 mL). The cleaned extract was diluted with 0.2% acetic acid (at a 1:1 ratio) before injection into an ultra-high performance liquid chromatograph. To perform simultaneous analysis of AFs and OTA, the FLD program was developed by switching the excitation wavelength in a single chromatographic run. Results: The method provided LOQs of 0.25 and 1 ng/g for AFs and OTA, respectively, without involving any derivatization. In rice, the recoveries of AFs ranged from 84 to 106%, whereas OTA had a recovery above 72%, with the repeatability relative SDs <12% for both analytes. The method was successfully applied to a range of naturally contaminated market samples. Conclusions: The method is suitable for regulatory testing because of its significant time and cost effectiveness and sensitivity in compliance with the regulatory maximum levels. Highlights: The study achieves high-throughput analysis of AFs and OTA in raw and processed cereals using simultaneous extraction, cleanup, and UHPLC-FLD. Method sensitivity complies with the regulatory maximum levels. Single-laboratory validation results meet analytical QC requirements.

Determination of Aflatoxins and Ochratoxin A in Traditional Turkish Cereal-Based Fermented Food by Multi-Affinity Column Cleanup and LC Fluorescence Detection: Single-Laboratory Validation.

Background: Tarhana is a traditional fermented, sun-dried Turkish food containing yogurtand cereals. There are several potential sources of mycotoxins in tarhana, such as contamination of ingredients or formation during preparation, when water activity is suitable for fungal growth and may lead to mycotoxin production during fermentation or subsequent sun-drying. Objective: To optimize an immunoaffinity column method and carry out single-laboratory validation for the determination of aflatoxins B1, B₂, G1, and G₂ together with ochratoxin A (OTA) in tarhana. Method: A homogenized sample was extracted with methanol-acetonitrile-water (25 + 25 + 50) using a high-speed blender. The sample extract was filtered, diluted with phosphate buffered saline (PBS) solution, and applied to a multi-immunoaffinity column (AFLAOCHRA PREP®). Aflatoxins and OTA were removed with neat methanol and then directly analyzed by reverse-phase LC with fluorescence detection using post-column bromination (Kobra® Cell). Results: Test portions of blank tarhana were spiked with a mixture of total aflatoxins and OTA to give levels ranging from 2.5 to 10.0 and 1.5 to 6.0 μg/kg, respectively. Recoveries for total aflatoxins and OTA ranged from 82 to 93 and 78 to 94%, respectively, for spiked samples. Based on results for spiked tarhana (30 replicates, each at three levels), the relative standard deviation for repeatability ranged from 1.4 to 7.2 and 3.6 to 7.7% for total aflatoxins and OTA, respectively. Conclusions: The performance characteristics for recovery, repeatability, and sensitivity have demonstrated that the method meets method performance criteria for use for official purposes. The method was demonstrated as being applicable to naturally contaminated samples of tarhana of varied composition obtained from local markets in Turkey. Highlights: This is the first immunoaffinity column method for simultaneous analysis of aflatoxins and OTA in traditional Turkish food (tarhana). Suitability was demonstrated by single-laboratory validation for official purposes in Turkey. The method was demonstrated as suitable for naturally contaminated samples of tarhana of varied composition.

Automated analysis of ochratoxin A in cereals and oil by immunoaffinity magnetic beads coupled to UPLC-FLD

A rapid and accurate method based on automated immunoaffinity magnetic beads (IMB) clean-up and ultra-performance liquid chromatography with fluorescence detection (UPLC-FLD) was developed for the determination of ochratoxin A (OTA) in cereals and oils. The main parameters affecting the method were studied in detail. Under optimized conditions, the recoveries of spiked analytes at four different concentrations were ranged from 86.3 to 95.4% and the inter-day relative standard deviations were below 7.9%. Coupling with UPLC-FLD allowed the limits of detection and quantification to be 0.24 and 0.80 μg/kg, respectively. The results demonstrate that the proposed method is reliable to determine OTA in cereals and oils. This novel protocol utilizes a simple and automatic sample pretreatment procedure for high-throughput and rapid clean-up of OTA in complex samples. Moreover, this method improves the performance of OTA analysis in cereals and oils. By consuming less time, cost and labor, as well as fewer errors from operation, the developed protocol overcomes the drawbacks of traditional column-based clean-up methods and thus offers a great prospect for OTA analysis mainly in the applications of food safety and public health protection.