The comparison of two clean-up procedures, multifunctional column and immunoaffinity column, for HPLC determination of ochratoxin A in cereals, raisins and green coffee beans

To evaluate a clean-up method of detecting ochratoxin A (OTA) by HPLC, the performances of two different clean-up columns, an immunoaffinity column and a multifuntional column were compared in an inter-laboratory study. As samples, un-contaminated wheat, corn grits, green coffee beans and naturally contaminated raisins were used. The recovery test was performed at two different concentrations of OTA (0.5 and 5.0 μg/kg) except for naturally contaminated raisins. Using the immunoaffinity column, the recovery rates, and relative standard deviations for repeatability (R.S.D._r) and reproducibility (R.S.D._R) for wheat, corn grits and green coffee beans ranged 59.0-85.8, 4.2-7.8 and 22.9-29.2%, respectively. For naturally contaminated raisins, recovery, R.S.D._r and R.S.D._R were 84.1, 1.8 and 5.1%, respectively. Using the multifunctional column, the recovery rates, R.S.D._r and R.S.D._R for wheat, corn grits and green coffee beans ranged 80.8-185.0, 0.7-6.9 and 15.2-33.9%, respectively. For naturally contaminated raisins, the recovery, R.S.D._r and R.S.D._R were 128.7, 1.1 and 3.7%, respectively. The results suggest that a multifunctional column could be used to detect OTA in wheat and corn grits at a concentration as low as 0.5 μg/kg; however, it was difficult to detect OTA in green coffee beans and raisins at such a low level. Although an immunoaffinity column could be used for all the test samples in this study from a low level to a high level, the recovery rates were lower than with a multifunctional column.     

Combined phenyl silane and immunoaffinity column cleanup with liquid chromatography for determination of ochratoxin A in roasted coffee: collaborative study

A collaborative study was conducted to evaluate a liquid chromatography (LC) method for ochratoxin A using sequential phenyl silane and immunoaffinity column cleanup. The method was tested at 3 different levels of ochratoxin A in roasted coffee, which spanned the range of possible future European regulatory limits. The test portion was extracted with methanol and sodium bicarbonate by shaking for 30 min. The extract was filtered, centrifuged, and then cleaned up on a phenyl silane column before being eluted from the washed column with methanol-water. The eluate was diluted with phosphate-buffered saline (PBS) and applied to an ochratoxin A immunoaffinity column, which was washed with water. The ochratoxin A was eluted with methanol, the solvent was evaporated, and the residue was redissolved in injection solvent. After injection of this solution onto a reversed-phase LC apparatus, ochratoxin A was measured by fluorescence detection. Eight laboratory samples of low-level naturally contaminated roasted coffee and 2 laboratory samples of blank coffee (< 0.2 ng/g ochratoxin A at the signal-to-noise ratio of 3:1), along with ampules of ochratoxin A calibrant and spiking solutions, were sent to 15 laboratories in 13 different European countries. Test portions of the laboratory samples were spiked at levels of 4 ng/g ochratoxin A, and recoveries ranged from 65 to 97%. Based on results for spiked blank material (blind duplicates) and naturally contaminated material (blind duplicates at 3 levels), the relative standard deviation for repeatability (RSDr) ranged from 2 to 22% and the relative standard deviation for reproducibility (RSDR) ranged from 14 to 26%. The method showed acceptable within- and between-laboratory precision, as evidenced by HORRAT values, at the low level of determination for ochratoxin A in roasted coffee.     

Determination and survey of ochratoxin A in wheat, barley, and coffee--1997

Ochratoxin A (OA) is a nephrotoxic and nephrocarcinogenic mycotoxin produced by Aspergillus and Penicillium species. It has been found mainly in cereal grains and coffee beans. The purpose of this study was to investigate the occurrence of OA in cereal grains and in coffee imported to the United States. A modified liquid chromatographic (LC) method for determining OA in green coffee was applied to wheat, barley, green coffee, and roasted coffee. The test sample was extracted with methanol-1% NaHCO3 (7 + 3), and the extract was filtered. The filtrate was diluted with phosphate-buffered saline (PBS), filtered, and passed through an immunoaffinity column. After the column was washed with PBS and then with water, OA was eluted with methanol. The eluate was evaporated to dryness, and the residue was dissolved in acetonitrile-water (1 + 1). OA was separated on a reversed-phase C18 LC column with acetonitrile-water-acetic acid (55 + 45 + 1) as eluant and quantitated with a fluorescence detector. Recoveries of OA from the 4 commodities spiked over the range 1-4 ng/g were 71-96%. The limit of detection was about 0.03 ng/g. OA contamination at > 0.03 ng/g was found in 56 of 383 wheat samples, 11 of 103 barley samples, 9 of 19 green coffee samples, and 9 of 13 roasted coffee samples. None of the coffee samples contained OA at > 5 ng/g; only 4 samples of wheat and 1 sample of barley were contaminated above this level.     

Determination of ochratoxin A in baby food by immunoaffinity column cleanup with liquid chromatography: interlaboratory study

An interlaboratory study funded by the European Commission, Standards, Measurement and Testing Programme (4th Framework Programme) was performed to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatographic (LC) method for the determination of ochratoxin A in baby food at a possible future European regulatory limit (0.1 ng/g). The test portion is extracted in a blender with tert-butyl methyl ether (chosen to avoid use of chloroform but shown to give equivalent extraction efficiency) after addition of 0.5 mol/L phosphoric acid-2 mol/L sodium chloride solution. The extract is centrifuged and redissolved in a mixture of phosphate buffered saline solution and methanol. After removal of lypophilic substances with hexane, the extract is applied to an immunoaffinity column containing antibodies specific to ochratoxin A. The column is washed with water to remove the interfering compounds and the purified ochratoxin A is eluted with methanol. The separation and determination of ochratoxin A is performed by reversed-phase LC and detected by fluorescence after postcolumn derivatization (PCD) with ammonia. Test materials (baby food infant formulae), both spiked and naturally contaminated with ochratoxin A, were sent to 13 laboratories in 8 different European countries. Test portions were spiked at a level of 0.085 ng/g ochratoxin A. The average recovery for the spiked blank baby food was 108%. Based on results for spiked samples (blind pairs at 0.085 ng/g) as well as naturally contaminated samples (blind pairs at levels between 0.05 and 0.22 ng/g) the relative standard deviation for repeatability (RSDr) ranged from 18-36%. The relative standard deviation for reproducibility (RSDR) ranged from 29-63% and HORRAT values of between 0.4 and 0.9 were obtained.     

Immunoaffinity column cleanup with liquid chromatography for determination of aflatoxin B1 in corn samples: interlaboratory study

An interlaboratory study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatography (LC) method for the determination of aflatoxin B1 levels in corn samples, enforced by European Union legislation. A test portion was extracted with methanol-water (80 + 20); the extract was filtered, diluted with phosphate-buffered saline solution, filtered on a microfiber glass filter, and applied to an immunoaffinity column. The column was washed with deionized water to remove interfering compounds, and the purified aflatoxin B1 was eluted with methanol. Aflatoxin B1 was separated and determined by reversed-phase LC with fluorescence detection after either pre- or postcolumn derivatization. Precolumn derivatization was achieved by generating the trifluoroacetic acid derivative, used by 8 laboratories. The postcolumn derivatization was achieved either with pyridinium hydrobromide perbromide, used by 16 laboratories, or with an electrochemical cell by the addition of bromide to the mobile phase, used by 5 laboratories. The derivatization techniques used were not significantly different when compared by the Student's t-test; the method was statistically evaluated for all the laboratories. Five corn sample materials, both spiked and naturally contaminated, were sent to 29 laboratories (22 Italian and 7 European). Test portions were spiked with aflatoxin B1 at levels of 2.00 and 5.00 ng/g. The mean values for recovery were 82% for the low level and 84% for the high contamination level. Based on results for spiked samples (blind pairs at 2 levels) as well as naturally contaminated samples (blind pairs at 3 levels), the values for relative standard deviation for repeatability (RSDr) ranged from 9.9 to 28.7%. The values for relative standard deviation for reproducibility (RSDR) ranged from 18.6 to 36.8%. The method demonstrated acceptable within- and between-laboratory precision for this matrix, as evidenced by the HorRat values.     

Determination of aflatoxin B1 in baby food (infant formula) by immunoaffinity column cleanup liquid chromatography with postcolumn bromination: collaborative study

A collaborative study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatography (LC) method for determination of aflatoxin B, in a milk powder based infant formula at a possible future European regulatory limit (0.1 ng/g). The test portion was extracted with methanol-water (8 + 2 [v + v]), filtered, diluted with water, and applied to an immunoaffinity column. The column was washed with water to remove interfering compounds, and the purified aflatoxin B1 was eluted with methanol. The separation and determination of the aflatoxin B1 was performed by reversed-phase LC and detected by fluorescence after postcolumn derivatization (PCD) involving bromination. PCD was achieved with either pyridinum hydrobromide perbromide (PBPB) or an electrochemical (Kobra) cell by addition of bromide to the mobile phase. The baby food (infant formula) test samples, both spiked and naturally contaminated with aflatoxin B1, were sent to 14 laboratories in 13 different European countries. Test portions were spiked at levels of 0.1 and 0.2 ng/g for aflatoxin B1. Recoveries ranged from 101 to 92%. Based on results for spiked test samples (blind pairs at 2 levels) and naturally contaminated test samples (blind pairs at 3 levels), the relative standard deviation for repeatability (RSDr) ranged from 3.5 to 14%. The relative standard deviation for reproducibility (RSDR) ranged from 9 to 23%. Nine participants used PBPB derivatization, and     

Aptamer‐affinity column clean‐up coupled with ultra high performance liquid chromatography and fluorescence detection for the rapid determination of ochratoxin A in ginger powder

Aptamers are single‐stranded oligonucleotides with high affinity and specificity and are widely used in targets separation and enrichment. Here, an aptamer‐affinity column (AAC) was firstly prepared in‐house through a covalent immobilization strategy. Then, ochratoxin A (OTA) in ginger powder was absorbed and enriched using the new aptamer‐based clean‐up technology for the first time, and was further analyzed by ultra high performance liquid chromatography with fluorescence detection. After optimization, the average recoveries for blank samples spiked with OTA at 5, 15, and 45 μg/kg ranged from 85.36 to 96.83%. Furthermore, the AAC exhibited a similar accuracy as an immunoaffinity column to clean up OTA in ginger powder. Above all, it exhibited better reusability, twice that of the immunoaffinity column, had lower toxicity and cost, and took less time. Of 25 contaminated ginger powder samples, OTA contamination levels ranged from 1.51 to 4.31 μg/kg, which were lower than the European Union (EU) regulatory limits. All the positive samples were further confirmed by ultra‐fast LC with MS/MS. In conclusion, the method of clean‐up based on the AAC coupled to ultra‐HPLC with fluorescence detection was rapid, specific, and sensitive for the quantitative analysis of OTA in a complex matrix.     

Liquid chromatographic method with immunoaffinity column cleanup for determination of ochratoxin A in barley: collaborative study

A collaborative study was conducted to evaluate a liquid chromatographic (LC) method with immunoaffinity column cleanup for determination of ochratoxin A. The method was tested at 3 concentration levels of ochratoxin A in barley, which represent possible future European regulatory limits. The test portion was extracted with acetonitrile-water by blending at high speed. The extract was filtered, diluted with phosphate-buffered saline (PBS), and applied to an ochratoxin A immunoaffinity column. The column was washed with water and the ochratoxin A eluted with methanol. The solvent was then evaporated and the residue redissolved in injection solvent. After injection of this solution onto reversed-phase LC column, ochratoxin A was measured by fluorescence detection. Eight samples of low level naturally contaminated barley and 2 samples of blank barley (ochratoxin A not found at the limit of detection of 0.2 microg/kg at the signal-to-noise ratio of 3 to 1) were sent, along with ampules of ochratoxin A, calibrant, and spiking solutions, to 15 laboratories in 13 different European countries. Test portions were spiked with ochratoxin A at levels of 4 ng/g, and recoveries ranged from 65 to 113%. Based on results for spiked samples (blind duplicates) and naturally contaminated samples (blind duplicates at 3 levels), the relative standard deviation for repeatability (RSDr) ranged from 4 to 24%, and the relative standard deviation for reproducibility (RSDR) ranged from 12 to 33%. The method showed acceptable within- and between-laboratory precision, as evidenced by HORRAT values, at the low level of determination for ochratoxin A in barley.     

Immunoaffinity column cleanup with liquid chromatography using post-column bromination for determination of aflatoxins in hazelnut paste: interlaboratory study

An interlaboratory study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatography (LC) method for determination of aflatoxin B1 and total aflatoxins in hazelnut paste at European regulatory limits. The test portion was extracted with methanol-water (6 + 4). The extract was filtered, diluted with phosphate-buffered saline (PBS) solution to a specified solvent concentration, and applied to an immunoaffinity column containing antibodies specific to aflatoxins. The aflatoxins were removed from the immunoaffinity column with methanol, and then quantified by reversed-phase LC with post-column derivatization (PCD) involving bromination. The PCD was achieved with electrochemically generated bromine (Kobra Cell) followed by fluorescence detection (except for one participant who used pyridinum hydrobromide perbromide for bromination). Hazelnut paste, both naturally contaminated with aflatoxins and blank (<0.1 ng/g) for spiking by participants with aflatoxins, was sent to 14 collaborators in Belgium, The Netherlands, Spain, Turkey, the United Kingdom, and the United States. Test portions were spiked at levels of 4.0 and 10.0 ng/g for total aflatoxins by participants using supplied total aflatoxins standards. Recoveries for total aflatoxins and aflatoxin B1 averaged from 86 to 89%. Based on results for naturally contaminated samples (blind duplicates at 3 levels ranging from 4.0 to 11.8 ng/g total aflatoxins), the relative standard deviation for repeatability (RSDr) ranged from 2.3 to 3.4% for total aflatoxins and from 2.2 to 3.2% for aflatoxin B1. The relative standard deviation for reproducibility (RSD(R)) ranged from 6.1 to 7.0% for total aflatoxins and from 7.3 to 7.8% for aflatoxin B1. The method showed exceptionally good within-laboratory and between-laboratory precision for hazelnut paste, as evidenced by HORRAT values, which in all cases were significantly below target levels, the low levels of determination for both aflatoxin B1 and total aflatoxins.     

Determination of aflatoxins B1, B2, G1, and G2 and ochratoxin A in ginseng and ginger by multitoxin immunoaffinity column cleanup and liquid chromatographic quantitation: collaborative study

The accuracy, repeatability, and reproducibility characteristics of a method using multitoxin immunoaffinity column cleanup with liquid chromatography (LC) for determination of aflatoxins (AF; sum of aflatoxins B1, B2, G1, and G2) and ochratoxin A (OTA) in powdered ginseng and ginger have been established in a collaborative study involving 13 laboratories from 7 countries. Blind duplicate samples of blank, spiked (AF and OTA added) at levels ranging from 0.25 to 16.0 microg/kg for AF and 0.25 to 8.0 microg/kg for OTA were analyzed. A naturally contaminated powdered ginger sample was also included. Test samples were extracted with methanol and 0.5% aqueous sodium hydrogen carbonate solution (700 + 300, v/v). The extract was centrifuged, diluted with phosphate buffer (PB), filtered, and applied to an immunoaffinity column containing antibodies specific for AF and OTA. After washing the column with water, the toxins were eluted from the column with methanol, and quantified by high-performance LC with fluorescence detection. Average recoveries of AF from ginseng and ginger ranged from 70 to 87% (at spiking levels ranging from 2 to 16 microg/kg), and of OTA, from 86 to 113% (at spiking levels ranging from 1 to 8 microg/kg). Relative standard deviations for within-laboratory repeatability (RSDr) ranged from 2.6 to 8.3% for AF, and from 2.5 to 10.7% for OTA. Relative standard deviations for between-laboratory reproducibility (RSDR) ranged from 5.7 to 28.6% for AF, and from 5.5 to 10.7% for OTA. HorRat values were < or = 2 for the multi-analytes in the 2 matrixes.     

Analytical performances of a DNA-ligand system using time-resolved fluorescence for the determination of ochratoxin A in wheat

The analytical performances of a novel DNA-ligand system using the time-resolved fluorescence (TRF) response of ochratoxin A (OTA)-terbium-DNA aptamer interaction were tested for the quantitative determination of OTA in wheat. Wheat was extracted with acetonitrile/water (60:40, v/v) followed by clean-up through affinity columns containing a DNA-aptamer-based oligosorbent. Then, OTA was detected by TRF spectroscopy after reaction with a terbium fluorescent solution containing the DNA-aptamer probe. The entire procedure was performed in less than 30 min, including sample preparation, and allowed analysis of several samples simultaneously with a 96-well microplate reader. The average recovery from samples spiked with 2.5-25 μg kg(-1) OTA was 77%, with a relative standard deviation lower than 6% and a quantification limit of 0.5 μg kg(-1). Comparative analyses of 29 naturally contaminated (up to 14 μg kg(-1)) wheat samples using the aptamer-affinity column/TRF method or the immunoaffinity column/high-performance liquid chromatography method showed good correlation (r = 0.985) in the range tested. The trueness of the aptamer-based method was additionally assessed by analysis of two quality control wheat materials for OTA. The DNA-ligand system is innovative, simple and rapid, and can be used to screen large quantities of samples for OTA contamination at levels below the EU regulatory limit with analytical performances satisfying EU criteria for method acceptability.     

Immunoaffinity column cleanup with liquid chromatography for determination of aflatoxin M1 in liquid milk: collaborative study

A collaborative study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatographic method for determination of aflatoxin M1 in milk at proposed European regulatory limits. The test portion of liquid milk was centrifuged, filtered, and applied to an immunoaffinity column. The column was washed with water, and aflatoxin was eluted with pure acetonitrile. Aflatoxin M1 was separated by reversed-phase liquid chromatography (LC) with fluorescence detection. Frozen liquid milk samples both naturally contaminated with aflatoxin M1 and blank samples for spiking, were sent to 12 collaborators in 12 different European countries. Test portions of samples were spiked at 0.05 ng aflatoxin M1 per mL. After removal of 2 noncompliant sets of results, the mean recovery of aflatoxin M1 was 74%. Based on results for spiked samples (blind pairs at 1 level) and naturally contaminated samples (blind pairs at 3 levels) the relative standard deviation for repeatability (RSDr) ranged from 8 to 18%. The relative standard deviation for reproducibility (RSDR) ranged from 21 to 31%. The method showed acceptable within- and between-laboratory precision data for liquid milk, as evidenced by HORRAT values at the low level of aflatoxin M1 contamination.     

Immunoaffinity column cleanup with liquid chromatography using post-column bromination for determination of aflatoxin B1 in cattle feed: collaborative study

A collaborative study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatography (LC) method for determination of aflatoxin B1 in cattle feed at a possible future European regulatory limit (1 ng/g). The test portion was extracted with acetone-water (85 + 15), filtered, diluted with water, and applied to an immunoaffinity column. The column was washed with water to remove interfering compounds, and the purified aflatoxin B1 was eluted with methanol. Aflatoxin B1 was separated and determined by reversed-phase liquid chromatography (RP-LC) and detected by fluorescence after post column derivatization (PCD) involving bromination. PCD was achieved with either pyridinium hydrobromide perbromide (PBPB), used by 14 laboratories, or an electrochemical cell and addition of bromide to the mobile phase, used by 7 laboratories. Both derivatization techniques were not significantly different when compared by the t-test; the method was statistically evaluated for all laboratories together (bromination and PBPB). The cattle feed samples, both spiked and naturally contaminated with aflatoxin B1, were sent to 21 laboratories in 14 different countries (United States, Japan, and Europe). Test portions were spiked at levels of 1.2 and 3.6 ng/g for aflatoxin B1. Recoveries ranged from 74 to 157%. Based on results for spiked samples (blind pairs at 2 levels) as well as naturally contaminated samples (blind pairs at 3 levels), the relative standard deviation for repeatability (RSDr) ranged from 5.9 to 8.7%. The relative standard deviation for reproducibility (RSDR) ranged from 17.5 to 19.6%. The method showed acceptable within- and between-laboratory precision for this matrix, as evidenced by HORRAT values, at the target levels of determination for aflatoxin B1. No major differences in RSD were observed, showing that the composition of the feeds was not a factor for the samples tested and that the method was applicable for all materials used.     

Determination of ochratoxin A at part-per-trillion level in Italian salami by immunoaffinity clean-up and high-performance liquid chromatography with fluorescence detection

A fast high-performance liquid chromatography method has been devised for the determination of ochratoxin A (OTA) in Italian salami in the low part-per-trillion (pg/g) level. The samples were extracted with ethyl acetate and purified by immunoaffinity column (IAC). The IAC eluate could be directly injected or previously concentrated 10-fold. Recovery at 0.5 and 1 ng/g was 77 +/- 4%. The between-day coefficient of variation measured over 5 days on samples spiked at 1 ng/g was 8%. The developed method required a relatively small volume of non-halogenated organic solvent and the whole procedure was simpler and faster compared to other existing procedures. The limit of detection was 0.06 ng/g that could be even lowered using a preconcentration step. A total of 30 salami samples were analysed using this procedure; the most contaminated sample was found to have OTA concentration at 0.4 ng/g level.     

Novel gel-based rapid test for non-instrumental detection of ochratoxin A in beer

A rapid easy-to-use immunoassay was optimised for the non-instrumental detection of ochratoxin A (OTA) in beer. The analytical method involves preconcentration on the immunoaffinity layer inside a column followed by direct competitive ELISA detection in the same layer. The visual cut-off value, i.e. the lowest OTA concentration resulting in no colour development, was 0.2 μg L^-1. Assay validation was performed using samples spiked with OTA. Thirty-seven naturally contaminated samples were screened with the gel-based method developed and no false-negative results were obtained. The method described offers a simple, rapid and cost-effective screening tool, thus contributing to better health protection of consumers. Figure Gel-based immunoassay of spiked beer samples.     

Immunoaffinity column cleanup with liquid chromatography using post-column bromination for determination of aflatoxins in peanut butter, pistachio paste, fig paste, and paprika powder: collaborative study

A collaborative study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatography (LC) method for the determination of aflatoxin B1 and total aflatoxins at European regulatory limits. The test portion is extracted with methanol-water (8 + 2) for dried figs and paprika, and with methanol-water (8 + 2) plus hexane (or cyclohexane) for peanut butter and pistachios. The sample extract is filtered, diluted with phosphate buffer saline, and applied to an immunoaffinity column. The column is washed with water and the aflatoxins are eluted with methanol. Aflatoxins are quantitated by reversed-phase LC with post-column derivatization (PCD) involving bromination. PCD is achieved with either an electrochemical cell (Kobra cell) and addition of bromide to the mobile phase or pyridinium hydrobromide perbromide. Determination is by fluorescence. Peanut butter, pistachio paste, dried fig paste, and paprika powder samples, both naturally contaminated with aflatoxins and containing added aflatoxins, were sent to 16 collaborators in 16 European countries. Test portions of samples were spiked at levels of 2.4 and 9.6 ng/g for total aflatoxins which included 1.0 and 4.0 ng/g aflatoxin B1, respectively. Recoveries for total aflatoxins ranged from 71 to 92% with corresponding recoveries for aflatoxin B1 of 82 to 109%. Based on results for spiked samples (blind duplicates at 2 levels) as well as naturally contaminated samples (blind duplicates at 4 levels, including blank), the relative standard deviation for repeatability ranged from 4.6 to 23.3% for total aflatoxins and from 3.1 to 20.0% for aflatoxin B1. The relative standard deviation for reproducibility ranged from 14.1 to 34.2% for total aflatoxins, and from 9.1 to 32.2% for aflatoxin B1. The method showed acceptable within-laboratory and between-laboratory precision for all 4 matrixes, as evidenced by HORRAT values <1, at the low levels of determination for both total aflatoxins and aflatoxin B1.     

Determination of ochratoxin A in wine and beer by immunoaffinity column cleanup and liquid chromatographic analysis with fluorometric detection: collaborative study.

The accuracy, repeatability, and reproducibility characteristics of a liquid chromatographic method for the determination of ochratoxin A (OTA) in white wine, red wine, and beer were established in a collaborative study involving 18 laboratories in 10 countries. Blind duplicates of blank, spiked, and naturally contaminated materials at levels ranging from < or =0.01 to 3.00 ng/mL were analyzed. Wine and beer samples were diluted with a solution containing polyethylene glycol and sodium hydrogen carbonate, and the diluted samples were filtered and cleaned up on an immunoaffinity column. OTA was eluted with methanol and quantified by reversed-phase liquid chromatography with fluorometric detection. Average recoveries from white wine, red wine, and beer ranged from 88.2 to 105.4% (at spiking levels ranging from 0.1 to 2.0 ng/mL), from 84.3 to 93.1% (at spiking levels ranging from 0.2 to 3.0 ng/mL), and from 87.0 to 95.0% (at spiking levels ranging from 0.2 to 1.5 ng/mL), respectively. Relative standard deviations for within-laboratory repeatability (RSDr) ranged from 6.6 to 10.8% for white wine, from 6.5 to 10.8% for red wine, and from 4.7 to 16.5% for beer. Relative standard deviations for between-laboratories reproducibility (RSDR) ranged from 13.1 to 15.9% for white wine, from 11.9 to 13.6% for red wine, and from 15.2 to 26.1% for beer. HORRAT values were < or =0.4 for the 3 matrixes.     

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.     

Use of multitoxin immunoaffinity columns for determination of aflatoxins and ochratoxin A in ginseng and ginger

Conditions were optimized for the simultaneous, alkaline, aqueous methanol extraction of aflatoxins (AFL), i.e., B1 (AFB1), B2 (AFB2), G1 (AFG1), and G2 (AFG2), and ochratoxin A (OTA) with subsequent purification, isolation, and determination of the toxins in ginseng and ginger. Powdered roots were extracted with methanol-0.5% NaHCO3 solution (7 + 3). After shaking and centrifugation, the supernatant was diluted with 100 mM phosphate buffer containing 1% Tween 20 and filtered through glass microfiber filter paper. The filtrate was then passed through an immunoaffinity column, and the toxins were eluted with methanol. The AFL were separated and determined by reversed-phase liquid chromatography (RPLC) with fluorescence detection after postcolumn UV photochemical derivatization. OTA was separated and determined by RPLC with fluorescence detection. Recoveries of AFL added at 2-16 ng/g and OTA added at 1-8 ng/g to ginseng were 72-80 and 86-95%, respectively. Recoveries of AFL and OTA added to ginger were similar to those for ginseng. A total of 39 commercially available ginger products from 6 manufacturers were analyzed. Twenty-six samples were found to be contaminated with AFL at 1-31 ng/g and 29 samples, with OTA at 1-10 ng/g. Ten samples contained no AFL or OTA. Ten ginseng finished products were also analyzed; 3 contained AFL at 0.1 ng/g and 4 contained OTA at levels ranging from 0.4 to 1.8 ng/g. LC/tandem mass spectrometry with multiple-reaction monitoring of 3 collisionally induced product ions from the protonated molecular ions of OTA, AFB1, and AFG1 was used to confirm the identities of the toxins in extracts of the finished products.     

A survey of ochratoxin A and aflatoxins in domestic and imported beers in Japan by immunoaffinity and liquid chromatography.

Analyses of ochratoxin A (OTA) and aflatoxins (AFs) in 94 imported beer samples from 31 producing countries and in 22 Japanese beer samples were performed by immunoaffinity column and reversed-phase liquid chromatography (LC) with fluorescence detection. Recoveries of OTA from beer samples spiked at 25 and 250 pg/mL were 86.1 and 88.2%, respectively. Recoveries of AFs were 98.4 and 98.9%, 95.4 and 95.5%, 101.2 and 97.8%, and 98.9 and 96.0%, respectively, from beer samples spiked at 4.1 and 41 pg AF B1, 4.45 and 44.5 pg AF B2, 4.7 and 47 pg AF G1, and 4.65 and 46.5 pg AF G2/mL. Detection limits were 1.0 pg/mL for OTA, 0.5 pg/mL for AFs B1 and B2, and 1.0 pg/mL for AFs G1 and G2. OTA was detected in 86 (91.5%) of 94 imported beer samples at a mean level of 10.1 pg/mL and in 21 (95.5%) of 22 Japanese beer samples at a mean level of 12.5 pg/mL. AF B1 was detected in 11 of 94 imported beer samples at a level of 0.5-83.1 pg/mL and in 2 of 22 Japanese beer samples at 0.5 and 0.8 pg/mL. Except for one beer sample from Peru, the samples contaminated with AFs were also contaminated with OTA. Although OTA was detected in most samples from various countries, AFs were detected in the beer samples from only a limited number of countries where AF contamination might be expected to occur because of their warm climate.