Limit Of Detection For Ochratoxin A Research Articles

Magnetic beads-assisted fluorescence aptasensing approach based on dual DNA tweezers for detection of ochratoxin A and fumonisin B1 in wine and corn.

A magnetic beads (MBs)-assisted fluorescence aptasensing approach based on dual DNA tweezers and magnetic separation was established for the detection of ochratoxin A (OTA) and fumonisin B1 (FB1). A dual DNA tweezers structure with four ends linked with fluorophores (FAM, ROX) and quenchers (BHQ1, BHQ2) was designed, and produced the high initial fluorescence signals because of the long spatial distance between FAM and BHQ1, ROX, and BHQ2. Bio-aptamer/anti-aptamer of OTA and bio-aptamer/anti-aptamer of FB1 were respectively annealed to form dsDNA, and immobilized to MBs coated with streptavidin (SA). With the existence of OTA and FB1, OTA and FB1 preferentially bound with their respective bio-aptamers, which made anti-aptamers dissociate from dsDNA coupled on MBs. After magnetic separation, the dissociated anti-aptamers reacted with dual DNA tweezers, respectively, which made DNA tweezers close and the fluorescence was quenched. The linear ranges of approach for OTA and FB1 detection were 0.05-20ng/mL and 0.1-40ng/mL, respectively. The limit of detection for OTA and FB1 was 0.029ng/mL and 0.061ng/mL. The prepared MBs-assisted fluorescence aptasensing approach was applied to detect OTA and FB1 in spiked red wine and corn samples, which showed good recoveries between 92 and 106%.

A label-free aptasensor for turn-on fluorescent detection of ochratoxin a based on SYBR gold and single walled carbon nanohorns

Ochratoxin A (OTA), is one of the most commonly found mycotoxins in food. It is primarily produced by Aspergillus ochraceus and Penicillium verrucosum. OTA can causes serious damages to liver and kidney, increases the teratogenicity, and has been considered as a potential carcinogen (group 2 B). In this study, we demonstrated a new detection system for the detection of OTA using fluorescence biosensing according to an anti-OTA aptamer and fluorescence quencher using single-walled carbon nanohorns (SWCNHs). In the presence of OTA, the OTA-specific aptamer underwent the conformation change from a random coil to an antiparallel G-quadruplex. The latter one was resistant to being adsorbed onto SWCNHs, leading to the pertection of the fluorescence of SYBR Gold. As a result, the remained fluorescent intensities correlated with the concentrations of OTA. The detection limit of OTA was 2.3 ng/ml, meanwhile the linear detection range was 5–500 ng/ml, with a satisfactory linear correlation (R2 = 0.992) of the fluorescence intensity and the logarithm of OTA concentrations. The aptasensor was subjected to quantify the concentration of OTA in red wine samples. As a result, this label-free aptasensor was suitable for the test of OTA in food samples.

Tailoring the surface coordination of nitrogen doped nano titania to design -A specific fluorescence signal amplification probe for ochratoxin A detection

Metal based nanoprobes for fluorescence signal amplification have become a vital component of biosensing research due to their profound electrical, optical and sensing properties. Because of high specific surface area and photoactive reactions on the surface, nano-Titania (nTiO2) has attracted much attention in the scientific community. Titania can be modulated to be applicable in sensing designs through doping with nonmetallic elements. Thus, it is of great significant to investigate the doped counterparts of nTiO2 for biosensing applications. In this context, we herein integrated surface complexation feature of nitrogen doped nTiO2 (N-nTiO2) to contribute towards enhanced fluorescence signal amplification and subsequently employed it for ultrasensitive detection of Ochratoxin A (OTA). Results indicate that N-doping greatly influenced the surface chemistry and functionalities of nTiO2. The higher amplification efficacy of nitrogen doped nTiO2 can be attributed to its capabilities of good adsorbability and high electron mobility that substantially catalyzes the surface complexation of OTA onto these particales, consequently intensifying the fluorescence of OTA efficiently. Characterization tools including SEM, XRD, FTIR, UV–Vis, RAMAN spectroscopy, photoluminescence (PL) and flouremetric analysis were used to inspect the surface chemistry, morphology and signal amplification activity of prepared nanoprobe. Different parameters such as concentration of nano particles and incubation time were optimized for ultrasensitive detection of OTA. Under optimal experemental conditions, the limit of detection for OTA was as low as to 0.1 ng mL−1 with linear range from 0.5 to 100 ng mL−1. In conclusion, we proposed a highly sensitive and selective fluorescent nanoprobe for analytical applications based on surface complexation affinity and fluorescence signal amplification ability of N doped nTiO2.

Multicolor colorimetric detection of ochratoxin A via structure-switching aptamer and enzyme-induced metallization of gold nanorods

Colorimetric aptasensors have been intensively studied for the ochratoxin A (OTA) detection, but they mostly exhibit just one-color change, resulting in poor visual resolution and limited use for semi-quantitative analysis. Thus, we designed a high-resolution colorimetric assay on the basis of aptamer structural switching and enzyme-induced metallization of gold nanorods (AuNRs). DNA-alkaline phosphatase (ALP)-immobilized magnetic beads were prepared. The aptamer bounded to OTA to form G-quadruplexes, releasing ALP-labelled complementary DNA (cDNA-ALP). After magnetic separation, cDNA-ALP catalyzed the decomposition of ascorbic acid 2-phosphate to ascorbic acid that reduced Ag+, forming an Ag shell on the surface of AuNRs. This caused a blue-shift of the longitudinal local surface plasmon resonance peak of the AuNRs and a naked eye visible multicolor change. Under optimal conditions, the assay exhibited a 9.0 nM detection limit for OTA, with high specificity. This method is promising for the on-site visual semi-quantitative detection of mycotoxins in foods.

Detachable nanoladders: A new method for signal identification and their application in the detection of ochratoxin A (OTA)

A highly sensitive fluorescence turn-off biosensor for the detection of ochratoxin A (OTA) was developed based on graphene oxide (GO) and an aptamer-induced detachable nanoladders. In this assay, two types of ssDNA (H1 and H2) were involved in the assembly of the DNA nanoladders, in which H1 was labeled with fluorophore, and H2 was the OTA binding aptamer. Self-assembly of the DNA nanoladders with the addition of GO weakened its adsorption and the fluorescence intensity remained strong. In the presence of OTA, the aptamer was specifically recognized and an aptamer-OTA complex was formed, leading to the detached of DNA nanoladders. With the addition of GO, the released H1 was adsorbed on the GO surface, thus efficiently quenching the fluorescence signal (turning off). The detection limit of OTA in this assay was 4.59 nM. To improve the sensitivity of this strategy, we creatively developed an alternative strategy to replace the sturdy nanoladders with frail nanoladders. More precisely, the sequences of H1 had mismatched bases, which, when hybridized with H2 could be used to create long non-perfect complementary nanoladders. For the mismatched bases-based frail nanoladders, it was easier for OTA to bind its aptamer sequence, thus enabling a more thorough and faster detachment of the nanoladders, along with a greater degree of fluorescence quenching. The detection limit for OTA was estimated to be 0.1 nM. The biosensors we developed were sensitive, specific, enzyme-free, cost-effective and can be applied in red wine samples spiked with known concentration of OTA.

Molecularly imprinted polymer based on upconversion nanoparticles for highly selective and sensitive determination of Ochratoxin A

A novel molecularly imprinted polymer (MIP) based on upconversion nanoparticles (UCNPs) was successfully synthesized for determination of Ochratoxin A (OTA). The MIP was developed on the silica-coated UCNPs using N-(1-hydroxy-2-naphthoyl amido)-(L)-phenylalanine (HNA-Phe) as the alternative template. The final composite combined the advantages of the high selectivity of MIP with the high fluorescence intensity of UCNPs which was selective and sensitive to OTA. Under the optimal condition, the fluorescence intensity of UCNPs@SiO2@MIP decreases linearly when the concentration of OTA increases from 0.05 to 1.0 mg/L. The detection limit of OTA with the method was 0.031 mg/L. At three spiked concentration levels (50, 100 and 200 µg/kg), the recovery ranges of OTA in corn, rice and feed are 88.0%–91.6%, 80.2%–91.6% and 89.2%–90.4%, respectively.

A fluorescent aptasensor for ochratoxin A detection based on enzymatically generated copper nanoparticles with a polythymine scaffold.

A fluorescence enhancement method is presented for the determination of ochratoxin A (OTA). The interaction of OTA with its aptamer causes structural changes which, in turn, change fluorescence of enzymatically generated polythymine-coated copper nanoparticles (CuNPs) (with excitation/emission maxima at 340/625nm). The OTA-binding aptamer was immobilized on magnetic beads. When it binds OTA, it is partially released and exposes a region with a partly complimentary DNA strand (cDNA). After magnetic separation, the cDNA was employed as a primer to trigger the terminal deoxynucleotidyl transferase-mediated polymerization. This process generates polythymine which act as a template for synthesis of the CuNPs. The method is sensitive in having a 2.0nM detection limit for OTA. It was successfully applied to the determination of OTA in spiked diluted red wine. Graphical abstract Schematic presentation of a fluorometric enhancement method for ochratoxin A assay based on ochratoxin A inducing structure switching of its aptamer and enzymatically generated polythymine for copper nanoparticles formation.

Quantum Dot-Labeled Immunochromatographic Test Strip for Rapid Detection of Ochratoxin A in Cereals

Objective:A quantum dot-labeled immunochromatographic test strip for the determination of ochratoxin A(OTA)in cereals was developed based on the competitive inhibition principle. Methods:Quantum dot antibody conjugates(QD-Ab)were prepared by coupling an carboxy-functionalized quantum dot(QD)with an ochratoxin A polyclonal antibody(Antibody,Ab)via the activated ester method;Different amounts of QD-Ab and working fluid were used to optimize the working conditions of the quantum dot-labeled immunochromatographic test strip;The effectiveness of the method was verified by a commercial kit. Results:When the molar ratio of QD to Ab was 1:10,the fluorescence characteristics of QD-Ab were the best;when QD-Ab and working fluid were added at 1,10 μL,respectively,the best results of quantum dot-labeled immunochromatographic test strips were obtained;The detection limit of the quantum dot-labeled immunochromatographic test strip in the assay buffer was 0.5 μg/L,and the detection limit of OTA in the cereal sample was 5 μg/kg,the entire detection process could be completed within 10 min;The quantum dot-labeled immunochromatographic test strip had good specificity and effectiveness. Conclusion:This assay is simple,rapid,and sensitivity. It can meet the requirements of on-site rapid detection of ochratoxin A in cereals.

Dual flow immunochromatographic assay for rapid and simultaneous quantitative detection of ochratoxin A and zearalenone in corn, wheat, and feed samples

A one-step dual flow immunochromatographic assay (DICGA), based on a competitive format, was developed for simultaneous quantification of ochratoxin A (OTA) and zearalenone (ZEN) in corn, wheat, and feed samples. The limit of detection for OTA was 0.32 ng/ml with a detection range of 0.53‒12.16 ng/ml, while for ZEN it was 0.58 ng/ml with a detection range of 1.06‒39.72 ng/ml. The recovery rates in corn, wheat, and feed samples ranged from 77.3% to 106.3% with the coefficient of variation lower than 15%. Naturally contaminated corn, wheat, and feed samples were analyzed using both DICGA and liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the correlation between the two methods was evaluated using a regression analysis. The DICGA method shows great potential for simple, rapid, sensitive, and cost-effective quantitative detection of OTA and ZEN in food safety control.

SERS based aptasensor for ochratoxin A by combining Fe3O4@Au magnetic nanoparticles and Au-DTNB@Ag nanoprobes with multiple signal enhancement.

A SERS-based aptasensor for ochratoxin A (OTA) is described. It is making use of Fe3O4@Au magnetic nanoparticles (MGNPs) and of Au@Ag nanoprobes modified with theRaman reporter 5,5-dithiobis-(2-nitrobenzoic acid; DTNB). Au-DTNB@Ag NPs were modified with the OTA aptamer (aptamer-GSNPs) and used as Raman signal probes. The SERS peak of DTNB at 1331cm-1 was used for quantitative analysis. MGNPs modified with cDNA (cDNA-MGNPs) were used as capture probes and reinforced substrates. When the Au-DTNB@Ag-Fe3O4@Au complexes are formed through oligonucleotide hybridization, the Raman signal intensity of theRaman probe is significantly enhanced. If the OTA concentration in samples increases, more Raman signal probes (aptamer-GSNPs) will dissociate from the cDNA-MGNPs because more OTA aptamer is bound by OTA. This leads to a lower Raman signal after magnetic separation. Under the optimal conditions, the detection limit for OTA is 0.48pg·mL-1 based on 3σ criterion. This is attributed to the multiple Raman signal enhancement and the good performance of the OTA aptamer. The good recovery and accuracy of the assay was confirmed by evaluating spiked samples of wine and coffee. Graphical abstract Schematic of an aptamer based SERS assay for OTA by integrating Fe3O4@AuNPs (MGNPs) with Au-DTNB@Ag NPs with multiple signal enhancement. Aptamer modified Au-DTNB@Ag NPs are used as Raman probes, and MGNPs modified with cDNA are used as capture probes and reinforced substrates.

Chitosan-based nanomatrix for the immobilization of ochratoxin-A conjugate on surface plasmon resonance chips

The paper presents the evaluation of chitosan and carboxymethyl chitosan matrix on the gold film of surface plasmon resonance (SPR) chips for the selective detection of ochratoxin-A (OTA) via immobilization of ochratoxin-A-bovine serum albumin (OTA-BSA) conjugate. Surface modification with flexible chitosan-based nanomatrix provides fast and efficient binding of the conjugates. The binding between OTA-BSA conjugate and chitosan was analyzed by localized Fourier transformation infrared reflectance (FTIR) spectroscopy followed by surface plasmon resonance. A clear SPR shift together with the presence of amino (N-H) functional groups on the surface confirmed the binding of conjugates with chitosan and carboxymethyl chitosan. The covalent binding on the surface resulted in a stable and resistant surface to increase the sensitivity of the SPR-based sensor. The limit of detection for OTA on chitosan-based SPR sensor was found to be around 2.52 ng/mL.

Micro-solid phase extraction of ochratoxin A, and its determination in urine using capillary electrophoresis

We describe a simple, environmentally friendly and selective technique for the determination of ochratoxin A (OTA) in urine. It involves (a) the use of a molecularly imprinted polymer as a sorbent in micro-solid-phase extraction in which the sorbent is contained in a propylene membrane envelope, and (b) separation and detection by capillary electrophoresis (CE). Under optimized conditions, response is linear in the range between 50 and 300 ng mL−1 (with a correlation coefficient of 0.9989), relative standard deviations range from 4 to 8 %, the detection limit for OTA in urine is 11.2 ng mL−1 (with a quantification limits of 32.5 ng mL−1) which is lower than those of previously reported methods for solid-phase extraction combined with CE. The recoveries of OTA from urine spiked at levels of 50, 150 and 300 ng mL−1 ranged from 93 to 97 %.

Multiplexed Fluorescence Resonance Energy Transfer Aptasensor between Upconversion Nanoparticles and Graphene Oxide for the Simultaneous Determination of Mycotoxins

We presented a new aptasensor for mycotoxins, which was based on multiplexed fluorescence resonance energy transfer (FRET) between multicolor upconversion fluorescent nanoparticles (UCNPs) as donors and graphene oxide (GO) as the entire and effective acceptor. BaY(0.78)F(5):Yb(0.2), Er(0.02) and BaY(0.78)F(5):Yb(0.7), Tm(0.02) upconversion nanoparticles were synthesized and functionalized, respectively, with immobilized ochratoxin A (OTA)-aptamers and fumonisin B(1) (FB(1))-aptamers. On the basis of the strong π-π stacking effect between the nucleobases of the aptamers and the sp(2) atoms of GO, the aptamer modified-UCNPs can be brought in close proximity to the GO surface. The strong upconversion fluorescence both of BaY(0.78)F(5):Yb(0.2), Er(0.02) and BaY(0.78)F(5):Yb(0.2), Tm(0.02) can be completely quenched by the GO, because of a good overlap between the fluorescence emission of multicolor UCNPs and the absorption spectrum of GO. In contrast, in the presence of OTA and FB(1), the aptamers preferred to bind to their corresponding mycotoxins, which led to changes in the formation of aptamers, and therefore, aptamer modified-UCNPs were far away from the GO surface. Our study results showed that the fluorescence intensity of BaYF(5):Yb Er and BaYF(5):Yb Tm were related to the concentration of OTA and FB(1). We therefore developed a sensitive and simple platform for the simultaneous detection of OTA and FB(1) with multicolor UCNPs and GO as the FRET pair. The aptasensor provided a linear range from 0.05 to 100 ng·mL(-1) for OTA and 0.1 to 500 ng·mL(-1) for FB(1); the detection limit of OTA was 0.02 ng·mL(-1) and FB(1) was 0.1 ng·mL(-1). As a practical application, the aptasensor was used to monitor OTA and FB(1) level in naturally contaminated maize samples with the results consistent with that of a classic ELISA method. More importantly, the novel multiplexed FRET was established for the first time based on multiplexed energy donors to the entire energy acceptor; this work was expected to open up a new field of FRET system applications for various targets.

Competitive direct ELISA based on a monoclonal antibody for detection of Ochratoxin A in dried fig samples

A monoclonal antibody (MAb) generated against Ochratoxin A (OTA) has been used in a competitive direct enzyme linked immunosorbent assay (cdELISA) for the detection of OTA in dried figs obtained from the Spanish retail market. Fifty per cent inhibition of the maximum binding was obtained with an OTA concentration of 2 ng/mL, and the detection limit for OTA in solution was 0.18 ng/mL, corresponding to 3.15 ng OTA per gram of sample. OTA was detected in 19 (54.3%) out of 35 samples of dried figs analysed, with concentrations that ranged from 3.15 to 277 ng/g. Five samples contained OTA concentrations above the tolerable level set by EC regulations for dried vine fruits (10 ng/g). The MAb-based cdELISA assay developed in this work could be effectively applied for OTA screening in dried figs.

Immunomagnetic microbeads for screening with flow cytometry and identification with nano-liquid chromatography mass spectrometry of ochratoxins in wheat and cereal

Multi-analyte binding assays for rapid screening of food contaminants require mass spectrometric identification of compound(s) in suspect samples. An optimal combination is obtained when the same bioreagents are used in both methods; moreover, miniaturisation is important because of the high costs of bioreagents. A concept is demonstrated using superparamagnetic microbeads coated with monoclonal antibodies (Mabs) in a novel direct inhibition flow cytometric immunoassay (FCIA) plus immunoaffinity isolation prior to identification by nano-liquid chromatography–quadrupole time-of-flight-mass spectrometry (nano-LC-Q-ToF-MS). As a model system, the mycotoxin ochratoxin A (OTA) and cross-reacting mycotoxin analogues were analysed in wheat and cereal samples, after a simple extraction, using the FCIA with anti-OTA Mabs. The limit of detection for OTA was 0.15 ng/g, which is far below the lowest maximum level of 3 ng/g established by the European Union. In the immunomagnetic isolation method, a 350-times-higher amount of beads was used to trap ochratoxins from sample extracts. Following a wash step, bound ochratoxins were dissociated from the Mabs using a small volume of acidified acetonitrile/water (2/8 v/v) prior to separation plus identification with nano-LC-Q-ToF-MS. In screened suspect naturally contaminated samples, OTA and its non-chlorinated analogue ochratoxin B were successfully identified by full scan accurate mass spectrometry as a proof of concept for identification of unknown but cross-reacting emerging mycotoxins. Due to the miniaturisation and bioaffinity isolation, this concept might be applicable for the use of other and more expensive bioreagents such as transport proteins and receptors for screening and identification of known and unknown (or masked) emerging food contaminants. FigureMicrobead coated with antibody

Surface plasmon resonance biosensor for the detection of ochratoxin A in cereals and beverages

Ochratoxins are a group of mycotoxins produced as secondary metabolites by fungi which contaminate a large variety of food and feed commodities. Due to their teratogenic and carcinogenic properties, ochratoxins present a serious hazard to human and animal health. There is an increasing need to establish a simple sensitive method to detect these toxins. Here we report a rapid and highly sensitive surface plasmon resonance (SPR) assay of ochratoxin A (OTA) using Au nanoparticles for signal enhancement on a mixed self-assembled monolayer (mSAM) surface. A competitive immunoassay format was used for the development of the OTA immunoassay, which is based on the immobilization of target OTA through its ovalbumin (OVA) conjugate with a polyethylene glycol (PEG) linker. The new OTA conjugate (OTA-PEG-OVA) showed remarkably enhanced performance characteristics compared with those based on the immobilization of a commercial bovine serum albumin BSA-OTA conjugate without a PEG linker. Although OTA concentrations as low as 1.5 ng mL −1 could be directly detected on this surface, the limit of detection (LOD) can be dramatically improved to 0.042 ng mL −1 for OTA by applying large gold nanoparticles (40 nm) for signal enhancement. Various chemical conditions to minimize the influence of the food matrix on assay performance were also investigated. Grain samples were simply extracted with 50% methanol and liquid samples treated with poly(vinylpyrrolidone) (PVP) (3 or 5%), without any sample clean-up or pre-concentration step prior to analysis. The LODs for OTA in oats and corn were 0.3 and 0.5 ng g −1, respectively, while in wine and other beverages, LODs ranged from 0.058 to 0.4 ng mL −1. No cross-reactivity was observed with three other common mycotoxins. In addition, the mSAM/OTA-PEG-OVA surface exhibited high stability with over 600 binding/regeneration cycles. This approach with simple sample preparation provides a powerful tool for the rapid and sensitive quantitative determination of OTA in food matrices.

Natural occurrence of ochratoxin A in dried figs

Ochratoxin A (OTA) contamination in dried figs was investigated using high performance liquid chromatography (HPLC) with fluorescence detection after extraction with methanol and orthophosphoric acid and clean up by an immunoaffinity column. The limit of detection for OTA was 0.12 μg kg −1. One hundred and fifteen samples were taken during the drying stage from 7 different districts in the Aegean Region in 2003 and 2004. Fifty-five (47.2%) of the 115 samples were found to contain detectable levels of ochratoxin A, ranging from 0.12 to 15.31 μg kg −1. However, the OTA level for a majority of the samples was low, with only 4 samples containing OTA exceeding 1 μg kg −1. The calculated overall median for the OTA level was below the limit of detection and the overall mean was estimated as 0.52 μg kg −1. Frequency of ochratoxin A contamination in dried figs harvested in 2003 and 2004 are 47 and 50%, respectively. Highest contamination ratio was determined in dried figs from Erbeyli (60%), followed by Selcuk (56%), and Ortaklar (50%).

Five-year survey of ochratoxin A in processed sultanas from Turkey

The results of surveillance for ochratoxin A (OTA) in 1885 samples of sultanas taken during five crop years between 1999 and 2003 are reported. The analytical method was based on extraction with methanol + sodium bicarbonate and clean-up by immunoaffinity column chromatography followed by high-performance liquid chromatography with fluorescence detection. The limit of detection for OTA was 0.3 µg kg−1. The results show that 9.3% of the samples contained no detectable levels of OTA, whereas 0.6% had concentrations exceeding 10 µg kg−1; the remaining 90.3% had levels within the range 0.3–10 µg kg−1. The overall mean OTA concentration in the total number of 1885 samples taken was 1.36 ± 2.91 µg kg−1; the overall median was calculated as 0.90 µg kg−1.

Array Biosensor for Detection of Ochratoxin A in Cereals and Beverages

Contamination of food by mycotoxins occurs in minute quantities, and therefore, there is a need for a highly sensitive and selective device that can detect and quantify these organic toxins. We report the development of a rapid and highly sensitive array biosensor for the detection and quantitation of ochratoxin A (OTA). The array biosensor utilizes a competitive immunoassay format. Immobilized OTA derivatives compete with toxin in solution for binding to fluorescent anti-OTA antibody spiked into the sample. This competition is quantified by measuring the formation of the fluorescent immunocomplex on the waveguide surface. The fluorescent signal is inversely proportional to the concentration of OTA in the sample. Analyses for OTA in buffer and a variety of food and beverage samples were performed. Samples were extracted with methanol, without any sample cleanup or preconcentration step prior to analysis. The limit of detection for OTA in several cereals ranged from 3.8 to 100 ng/g, while in coffee and wine, detection limits were 7 and 38 ng/g, respectively.

Determination of Ochratoxin a in coffee beans and coffee products by monoclonal antibody affinity chromatography

A clean‐up method using a monoclonal antibody affinity column was developed for the analysis of ochratoxin A (OTA) in coffee products. Monoclonal antibody specific for OTA was covalently bound to Sepharose‐4B and the resultant affinity column was used for the clean‐up of sample extracts. OTA was quantitatively recovered from the affinity column. The subsequent high performance liquid chromatography (HPLC) of the eluted sample revealed no marked interference peaks when compared with those extracts obtained by conventional approaches. The HPLC peak coinciding with OTA was confirmed by derivatization into ethyl and n‐propyl esters. Furthermore, the present affinity column could be regenerated at least 30 times without significant loss of the binding activity. The detection limit of OTA in the present affinity column‐HPLC procedure was 0.5 μg/kg for coffee beans and instant coffee powder, and 0.025 μg/kg for a canned coffee drink. The recoveries of OTA from coffee products were more than 98%, with coefficient variations in the range of 1.11%‐4.56%.