Aptamer-targeted magnetic nanospheres as a solid-phase extraction sorbent for determination of ochratoxin A in food samples

A sorbent based on the aptamer for ochratoxin A was immobilized onto magnetic nanospheres (MNS) and used to develop a magnetic solid-phase extraction procedure to clean up food samples in conjunction with high-performance liquid chromatography separation and fluorescence detection. Specific retention of ochratoxin A by the sorbent was demonstrated, and the capacity of the MNS-aptamer sorbent was determined. The efficacy of this new approach was successfully evaluated through comparison with solid-phase extraction on commercial C18 cartridge. Several different food samples fortified in the range of with 2.5-50 μg/kg yielded mean recoveries from 67% to 90%, respectively. Finally, this oligosorbent was applied to the selective extraction of ochratoxin A from unfortified food samples.     

Comparison of methods for the determination of ochratoxin A in wine

Different analytical methods for the determination of ochratoxin A (OTA) in wine have been compared. Sample clean-up was based on solid-phase extraction (SPE) with (i) immunoaffinity or (ii) RP-18 sorbent materials applying different experimental protocols. The detection of OTA was accomplished with high-performance liquid chromatography (HPLC) combined either with electrospray ionisation (ESI) tandem mass spectrometry (MS-MS) or fluorescence detection (FL). Comparative method evaluation was based on the investigation of 18 naturally contaminated red wine samples originating from different European countries. The analytical results are discussed in view of the respective method validation data and the corresponding experimental protocols. In general, analytical data obtained with RP-18 SPE combined with LC-MS-MS detection and immunoaffinity extraction combined with FL offered comparable good results in the sub-ppb concentration level indicating that high selectivity of either the sample clean-up or, alternatively the detection system are equally well-suited to guarantee an accurate OTA analysis in wine.     

Non-instrumental immunochemical tests for rapid ochratoxin A detection in red wine

Gel-based and membrane-based flow-through immunoassay formats were investigated for rapid ochratoxin A (OTA) detection in red wine. The flow-through set-up consisted of an antibody containing gel or membrane placed at the bottom of a standard solid-phase extraction column (i.e. the flow-through column), combined with a clean-up column. Different clean-up methods were studied for red wine clarification and purification. The optimal method consisted of passing wine, diluted with an aqueous solution containing 1% polyethylene glycol (PEG 6000) and 5% sodium hydrogencarbonate, through strong anion exchange (SAX) silica. An immunoassay for OTA detection in red wine was optimized and a cut-off level at 2 μg L⁻¹ according to EU legislation was achieved with both formats. A more significant colour difference between blank and spiked samples was observed for the gel-based assay making this superior to the membrane-based assay. The proposed rapid gel-based test was compared with a standard immunoaffinity column - high-performance liquid chromatography - fluorescent detection (IAC-HPLC-FLD) method and a good correlation of the results was obtained for naturally contaminated wine samples.     

Reversed-phase liquid chromatographic method for the determination of ochratoxin A in wine

In this paper, we propose a new, rapid, highly sensitive and reproducible RP-HPLC-FLD method for the detection of ochratoxin A (OTA) in wine, by directly injecting the liquid in the chromatographic system without any extraction or clean-up. An alkaline mobile phase (NH4Cl:CH-CN 85:15 (v/v), 20 mM, pH 9.8) was used to obtain a distinct fluorescence enhancement. This improvement allows to reach, without an immunoaffinity clean-up or concentration, a detection limit of 0.05 ng/ml, which is similar to those commonly obtained after immunoaffinity purification and acidic elution. The method was statistically validated and directly applied to a series of wine samples.     

Fluorescence polarization immunoassay for rapid screening of ochratoxin A in red wine

A fluorescence polarization (FP) immunoassay, based on a monoclonal antibody and an ochratoxin A (OTA)-fluorescein tracer, has been developed for rapid screening of OTA in red wine. Wine samples were diluted with methanol and passed through aminopropyl solid-phase extraction columns prior to the FP assay. Average recoveries from samples spiked with OTA at levels of 2.0 and 5.0 ng/mL were 79% with RDS of 11% (n = 6). The limit of detection of the FP immunoassay was 0.7 ng/mL OTA, and the whole analysis was performed in less than 10 min. The assay was tested on 154 red wine samples (naturally contaminated or spiked at level ranging from 0.1 to 5.0 ng/mL) and compared with an high-performance liquid chromatography/immunoaffinity column clean-up method, showing a good correlation (r = 0.9222). Their compliance with the European regulation (2.0 ng/mL OTA maximum permitted level) was correctly assessed for 70% of the analyzed samples of red wine, whereas confirmatory analyses were required for the remaining ones with OTA levels close to the regulatory limit. No false-negative or positive results were observed using the FP immunoassay. The proposed FP assay is a useful screening method for OTA in red wines, when high throughput is required, that could also be used for white and rosé wines, which are known to contain less interfering compounds such as polyphenols.     

Solid-phase extraction of ochratoxin A from wine based on a binding hexapeptide prepared by combinatorial synthesis

In this paper we describe the preparation of a hexapeptide library by combinatorial synthesis and the identification of a peptide with sequence Ser-Asn-Leu-His-Pro-Lys, which showed good affinity (K(eq)=3.4 x 10(4) M(-1)) towards the mycotoxin ochratoxin A (OTA). An immunoaffinity-like stationary phase supporting such a hexapeptide was used to develop a solid-phase extraction method for the quantification of OTA in wine samples at concentration levels down to 0.10 microg l(-1). Several different wine samples fortified with OTA at 2 and 4 microg l(-1) levels showed recovery of 94.7% and 98.4% at 2.0 and 4.0 microg l(-1), respectively, without any effect on the extraction efficiency of the matrix. The efficacy of this approach was successfully tested by comparison with an immunoaffinity extraction performed on commercial cartridges.     

Worldwide interlaboratory study on the determination of ochratoxin A in different wine type samples

Interlaboratory studies are decisive tools to help the validation of a specific analytical methodology or to assess the reproducibility of the use of different methods to analyze a given compound or compounds in certain sample matrices. In this work, homogeneous samples of two white wines (“White Wine” and “White Liqueur Wine”) and one red wine (“Red Fortified Wine”) from Portugal with different production techniques and characteristics, namely in alcohol strength (10.5%, 16.0% and 19.0% ethanolic content, respectively), were analyzed for their contents in ochratoxin A (OTA), a mycotoxin generated from fungal contamination. White Liqueur Wine was naturally contaminated, whereas the other two wine type were spiked with ethanolic OTA solutions. The participation of 24 laboratories from 17 countries of five continents was ensured for this study. Although with no restrictions in terms of analytical methodology to employ, 75% of the laboratories resorted to immunoaffinity columns clean-up followed by high performance liquid chromatography with fluorescence detection (HPLC-FD), most of them in accordance with the European Standard EN 14133. For White Wine samples, the general mean OTA concentration was 1.96 μg/l (two outliers) with interlaboratorial standard deviation (s_L) of 0.53 μg/l; for White Liqueur Wine, mean of 1.59 μg/l (one outlier), with s_L = 0.59 μg/l; and for Red Fortified Wine, mean of 2.73 μg/l (no outliers), with s_L = 0.96 μg/l. Outliers were determined by Cochran and Grubbs tests. The Horrat index, recommended by the Association of Official Analytical Chemists (AOAC) for the quality assurance of the collaborative study was, on average, 1.7. This study proved that OTA determination in wines is reproducible, regardless of the methodology employed.     

New method for determination of (E)-resveratrol in wine based on microextraction using packed sorbent and ultra-performance liquid chromatography

An ultra-fast and improved analytical methodology based on microextraction by packed sorbent (MEPS) combined with ultra-performance LC (UPLC) was developed and validated for determination of (E)-resveratrol in wines. Important factors affecting the performance of MEPS such as the type of sorbent material (C2, C8, C18, SIL, and M1), number of extraction cycles, and sample volume were studied. The optimal conditions of MEPS extraction were obtained using C8 sorbent and small sample volumes (50-250 μL) in one extraction cycle (extract-discard) and in a short time period (about 3 min for the entire sample preparation step). (E)-Resveratrol was eluted by 1×250 μL of the mixture containing 95% methanol and 5% water, and the separation was carried out on a high-strength silica HSS T3 analytical column (100 mm × 2.1 mm, 1.8 μm particle size) using a binary mobile phase composed of aqueous 0.1% formic acid (eluent A) and methanol (eluent B) in the gradient elution mode (10 min of total analysis). The method was fully validated in terms of linearity, detection (LOD) and quantification (LOQ) limits, extraction yield, accuracy, and inter/intra-day precision, using a Madeira wine sample (ET) spiked with (E)-resveratrol at concentration levels ranging from 5 to 60 μg/mL. Validation experiments revealed very good recovery rate of 95±5.8% RSD, good linearity with r(2) values >0.999 within the established concentration range, excellent repeatability (0.52%), and reproducibility (1.67%) values (expressed as RSD), thus demonstrating the robustness and accuracy of the MEPS(C8) /UPLC-photodiode array (PDA) method. The LOD of the method was 0.21 μg/mL, whereas the LOQ was 0.68 μg/mL. The validated methodology was applied to 30 commercial wines (24 red wines and six white wines) from different grape varieties, vintages, and regions. On the basis of the analytical validation, the MEPS(C8)/UPLC-PDA methodology shows to be an improved, sensitive, and ultra-fast approach for determination of (E)-resveratrol in wines with high resolving power within 6 min.     

Fast and sensitive detection of ochratoxin A in red wine by nanoparticle-enhanced SPR

Herein, we present a fast and sensitive biosensor for detection of Ochratoxin A (OTA) in a red wine that utilizes gold nanoparticle-enhanced surface plasmon resonance (SPR). By combining an indirect competitive inhibition immunoassay and signal enhancement by secondary antibodies conjugated with gold nanoparticles (AuNPs), highly sensitive detection of low molecular weight compounds (such as OTA) was achieved. The reported biosensor allowed for OTA detection at concentrations as low as 0.75 ng mL⁻¹ and its limit of detection was improved by more than one order of magnitude to 0.068 ng mL⁻¹ by applying AuNPs as a signal enhancer. The study investigates the interplay of size of AuNPs and affinity of recognition elements affecting the efficiency of the signal amplification strategy based on AuNP. Furthermore, we observed that the presence of polyphenolic compounds in wine samples strongly interferes with the affinity binding on the surface. To overcome this limitation, a simple pre-treatment of the wine sample with the binding agent poly(vinylpyrrolidone) (PVP) was successfully applied.     

New method for determination of ochratoxin A in beer using zinc acetate and solid-phase extraction silica cartridges

A new method for the determination of ochratoxin A (OTA) in beer has been developed. The new method has been compared with a reference method currently accepted as AOAC official first action. The limits of detection and quantification of the proposed method were 0.0008 and 0.0025 ng/ml, respectively, while they were 0.0025 and 0.0075 ng/ml, respectively, in the AOAC method used as reference. The recovery levels in the 0.025-0.40 ng OTA/ml spiking range for the proposed and the reference methods were 80.6-87.6% and 78.2-83.8%, respectively. The relative standard deviations of recoveries were 2.6-7.5% for the proposed method and 0.7-6.1% for the reference method. Passing and Bablok regression analysis of recovery data obtained by the proposed method versus data obtained by the reference method on an OTA-spiked beer sample showed good correlation (r2 = 0.9993), while the slope and intercept were 1.049 and -0.0013, respectively. The advantage of the proposed method is the low cost of the materials used in sample preparation because expensive immunoaffinity columns are not needed to clean-up samples while it maintains or even increases the good performance of the reference method. The proposed method was applied to 69 beer samples from different geographic origins (national and imported) but purchased in the Spanish market. They were found to be contaminated with OTA in the range from 0.008 to 0.498 ng/ml (average: 0.070 ng/ml). Five samples surpassed the limit recommended by the European Union (0.2 ng OTA/g).     

Development of natural sorbent based micro-solid-phase extraction for determination of phthalate esters in milk samples

In the present study, a natural sorbent based micro-solid phase extraction (μ-SPE) was developed for determination of phthalate esters in milk samples. For the first time, an efficient and cost effective natural material (seed powder of Moringa oleifera) was employed as sorbent in μ-SPE. The sorbent was found to be naturally enriched with variety of functional groups and having a network of interconnected fibers. This method of extraction integrates different steps such as removal of proteins and fatty stuff, extraction and pre-concentration of target analytes into a single step. Thirteen phthalate esters were selected as target compounds for the development and evaluation of method. Some key parameters affecting the extraction efficiency were optimized, including selection of membrane, selection and amount of sorbent, extraction time, desorption solvent, volume of desorption solvent, desorption time and effect of salt addition. Under the optimum conditions, very good linearity was achieved for all the analytes with coefficient of determinations (R²) ranging between 0.9768 and 0.9977. The limits of detection ranged from 0.01 to 1.2 μg L⁻¹. Proposed method showed satisfactory reproducibility with relative standard deviations ranging from 3.6% to 10.2% (n = 7). Finally, the developed method was applied to tetra pack and bottled milk samples for the determination of phthalate esters. The performance of natural sorbent based μ-SPE was better or comparable to the methods reported in the literature.     

Combined use of supported liquid membrane and solid-phase extraction to enhance selectivity and sensitivity in capillary electrophoresis for the determination of ochratoxin A in wine

This paper proposes a novel strategy to enhance selectivity and sensitivity in CE, using supported liquid membrane (SLM) and off-line SPE simultaneously. The determination of ochratoxin A (OA) in wine has been used to demonstrate the potential of this methodology. In the SLM step, the donor phase (either a 20 mL volume of a standard solution at pH 1 or a wine sample at pH 8) was placed in a vial, where a micromembrane extraction unit accommodating the acceptor phase (1 mL water, pH 11) in its lumen was immersed. The SLM was constructed by impregnating a porous Fluoropore Teflon (PTFE) membrane with a water-immiscible organic solvent (octanol). In the off-line SPE step, the nonpolar sorbent (C-18, 4 mg) selectively retained the target ochratoxin, enabling small volumes of acceptor phase (1 mL) to be introduced. The captured analytes were eluted in a small volume of methanol (0.1 mL). This procedure resulted in sample cleanup and concentration enhancement. The method was evaluated for accuracy and precision, and its RSD found to be 5%. The LODs for OA in the standard solutions and wine samples were 0.5 and 30 microg/L, respectively. The results obtained demonstrate that SLM combined with off-line is a good alternative to the use of immunoaffinity columns prior to CE analysis.     

Novel extraction supports based on immobilised aptamers: evaluation for the selective extraction of cocaine

A new kind of selective sorbent based on the use of aptamers and dedicated to the selective solid phase extraction was developed. Cocaine aptamer was chosen as model aptamer to demonstrate the feasibility of this material and to provide a complete evaluation of the synthesized sorbent. The effect of different parameters such as the nature of the immobilisation support (silica, agarose), the type of immobilisation (covalent or non-covalent) and the length of the spacer arm (C₆ or C₁₂) were studied. Therefore, various oligosorbents based on different immobilisation strategies were synthesized and characterised by estimating the extraction recovery and the capacity of cocaine and the binding efficiency of aptamers. Control supports without immobilised aptamers were simultaneously studied in parallel to assess the selectivity brought by the oligosorbents. The oligosorbent based on CNBr-activated sepharose showed the best performances with an extraction recovery for cocaine of 90% while 6% was obtained on the control sorbent. The high selectivity brought by the oligosorbent was then illustrated by applying the oligoextraction followed by LC/MS analysis to a post-mortem blood (cocaine overdose). Results were compared to those resulting from a conventional protein precipitation procedure. The presence of co-extracted interfering compounds was strongly reduced with the treatment by oligoextraction. A limit of quantification of 0.5 ng/mL was obtained that is largely lower than the concentration found after a single intake of cocaine.     

Determination of ochratoxin A in wine grapes: comparison of extraction procedures and method validation

A method for determination of ochratoxin A (OTA) in wine grapes is described, using extraction with a hydrogen carbonate and polyethylene glycol (PEG) solution (5% NaHCO₃ and 1% PEG 8000), followed by immunoaffinity clean-up and liquid chromatography with fluorescence detection. Validation was made with spiked samples, in levels of 0.05 and 1 μg kg⁻¹, with average recovery rates of 76% and relative standard deviations in repeatability and intermediate precision conditions of 8 and 12%, respectively. The limit of detection and limit of quantification in grapes were established at 0.004 and 0.007 μg kg⁻¹, respectively. To evaluate further the accuracy and efficiency of this method, naturally contaminated grapes were also analysed by another method that involves extraction with acidified methanol, at levels ranging from 0.05 to 37 μg kg⁻¹, and the results compared. A good correlation (r=0.9996) was found, with better performances in terms of precision for the new method. A survey was conducted on wine grapes from 11 Portuguese vineyards, during the harvest of 2002, using the proposed method. OTA was detected in three out of the 11 samples, at levels ranging from 0.035 to 0.061 μg kg⁻¹.The new method meets all the criteria of the European Commission directive 2002/26/CE, that lays down the sampling and the analysis methods for the official control of OTA levels in foodstuffs. It is reliable for low levels of contamination (ng kg⁻¹), and avoids the use of organic solvents in the extraction step.     

Monoclonal antibody based electrochemical immunosensor for the determination of ochratoxin A in wheat

Competitive electrochemical enzyme-linked immunosorbent assays based on disposable screen-printed electrodes have been developed for quantitative determination of ochratoxin A (OTA). The assays were carried out using monoclonal antibodies in the direct and indirect format. OTA working range, I₅₀ and detection limits were 0.05-2.5 and 0.1-7.5 μg L⁻¹, 0.35 (±0.04) μg L⁻¹ and 0.9 (±0.1) μg L⁻¹, 60 and 100 μg L⁻¹ in the direct and indirect assay format, respectively. The immunosensor in the direct format was selected for the determination of OTA in wheat. Samples were extracted with aqueous acetonitrile and the extract analyzed directly by the assay without clean-up. The I₅₀ in real samples was 0.2 μg L⁻¹ corresponding to 1.6 μg/kg in the wheat sample with a detection limit of 0.4 μg/kg (calculated as blank signal −3σ). Within- and between-assay variability were less than 5 and 10%, respectively. A good correlation (r = 0.9992) was found by comparative analysis of naturally contaminated wheat samples using this assay and an HPLC/immunoaffinity clean-up method based on the AOAC Official Method 2000.03 for the determination of OTA in barley.     

Modified magnetic nanoparticles in an electrochemical method for the ochratoxin A determination in Vitis vinifera red grapes tissues

This work described the development and characterization of an electrochemical method using square wave voltammetry (SWV) combined with the use of modified magnetic nanoparticles (MNPs), which had shown a rapid and sensitive determination of ochratoxin A (OTA) in wine grapes (Cabernet Sauvignon, Malbec and Syrah) post-harvest tissues. The wine grapes were inoculated with Aspergillus ochraceus to obtain OTA in artificially infected samples. The OTA was directly determined using square-wave voltammetry. The current obtained is directly proportional to the concentration of OTA present in the samples. This method has been used for OTA determination in wine grapes and it was validated against a commercial ELISA test kit. The limits of detection calculated for electrochemical detection and the ELISA were 0.02 and 1.9 μg kg⁻¹, respectively and the coefficients of variation for accuracy and precision dates were below 5.5%. This method promises to be suitable for the detection and quantification of OTA in apparently healthy fruits post-harvest for assuring safety and quality of food as well as consumer's health.     

Certification of reference materials for ochratoxin A analysis in coffee and wine

Mycotoxins are important non-anthropogenic food and feed contaminants, which can be present on almost every agricultural commodity. Effective consumer protection therefore essentially depends on food surveillance by reliable quantitative analysis enabled by appropriate quality control. Certified (matrix) reference materials (CRMs) are versatile tools to support quality assurance. However, in the case of ochratoxin A (OTA), a hepato- and nephrotoxic mycotoxin, which is regulated in various foods, there is a lack of suitable CRMs. This lack has now been overcome by the development of two European Reference Materials (ERM^?) for the determination of OTA in roasted coffee (ERM^?-BD475) and red wine (ERM^?-BD476). This article discusses the material preparation process as well as the results of homogeneity and stability testing. Furthermore, the results of the in-house certification studies carried out at BAM Federal Institute for Materials Research and Testing are presented and discussed. Interlaboratory comparison studies involving selected expert laboratories with documented expertise in the field of mycotoxin analysis were conducted to confirm the certified values determined by BAM. The certified ochratoxin A values and their corresponding expanded uncertainties (k?=?2) were assigned in full compliance with the requirements of ISO Guide 35 and are as follows: (6.0?±?0.6)???g?kg^?1 for roasted coffee, ERM^?-BD475, and (0.52?±?0.11)???g?L^?1 for red wine, ERM^?-BD476.     

Molecularly imprinted polymer‐based solid phase clean‐up for analysis of ochratoxin A in beer,red wine,and grape juice

A simple, reliable, and low‐cost method based on molecularly imprinted polymer as a selective sorbent of SPE was proposed for the determination of ochratoxin A (OTA) in beer, red wine, and grape juice by HPLC coupled with fluorescence detection (HPLC‐FLD). Samples were diluted with water and cleaned up with an AFFINIMIP® SPE OTA column. After washing and eluting, the analyte was analyzed by HPLC‐FLD. Under the optimized conditions, LOD and LOQ for OTA were 0.025 and 0.08 ng/mL, respectively. The recoveries of OTA from beer, red wine, and grape spiked at 0.1, 2, and 5 ng/mL ranged from 91.6 to 101.7%. Furthermore, after a simple regenerated procedure, the molecularly imprinted polymer based SPE column could be reused at least 14 times to achieve more than 80% recoveries of OTA in real samples. The developed method was applied to the detection of 30 beer, red wine, and grape juice samples and only four samples were contaminated by OTA with levels below the legal limits.     

Solid-phase extraction applied to the determination of ochratoxin A in wines by reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method is described for the analysis of Ochratoxin A at low microg l(-1) levels in samples of artificially contaminated wines. The method involves solid-phase extraction of samples using octadecylsilane cartridges and an additional preconcentration step prior to chromatography with isocratic elution and fluorimetric detection. The method was evaluated for accuracy and precision with relative standard deviations lower than 10%. Recoveries of ochratoxin A added to commercial wines over the range 0.1-3.0 microg l(-1) were higher than 80% in the assays. The performance of the octadecylsilane cartridge method tested compared very favourably with results of other published studies of ochratoxin A which use immunoaffinity columns or solvent extraction techniques.     

Lanthanides determination in red wine using ultrasound assisted extraction,flow injection,aerosol desolvation and ICP-MS

This paper deals with the determination of the fourteen naturally occurring elements of the lanthanide series in red wine. Ultrasound (US) was used for sample preparation prior lanthanides determination using ICP-MS. Flow injection (FI) and pneumatic nebulization/aerosol desolvation were used for nebulization of aliquots of 50 μL of sample and its subsequent transportation to plasma. Sample preparation procedures, matrix interference and time of sonication were evaluated. Better results for lanthanides in red wine were obtained by sonication with US probe for 90 s and sample 10-fold diluted. The limits of detection of La, Ce, Nd, Sm, Gd, Pr, Eu, Tb, Dy, Ho, Er, Tm, Lu and Yb were 6.57, 10.8, 9.97, 9.38, 2.71, 1.29, 1.22, 0.52, 2.35, 0.96, 2.30, 0.45, 0.24 and 1.35 ng L⁻¹, respectively. Red wines of different varieties from three countries of South America were discriminated according to the country of origin by means of multivariate analysis of lanthanides concentration.