Determination of ochratoxin A in green coffee by immunoaffinity column cleanup and liquid chomatography: collaborative study.

A collaborative study was conducted to evaluate a method using immunoaffinity column cleanup with liquid chromatography (LC) for the determination of ochratoxin A (OTA) in green coffee at levels that could be included in possible future regulations of the European Union. The test portion was extracted with methanol-3% aqueous sodium hydrogen carbonate solution (50 + 50, v/v). The extract was filtered, and the filtrate was diluted with phosphate-buffered saline and applied to an immunoaffinity column containing antibodies specific for OTA. After washing, the toxin was eluted from the column with methanol and quantified by LC with fluorescence detection. Pairs of 4 homogeneous noncontaminated and naturally contaminated materials (mean levels of < 0.12, 2.44, 5.15, and 13.46 ng/g) and blank samples (< 0.12 ng/g) for spiking were sent to 20 participant laboratories from 8 countries. The materials were analyzed according to the method description and all difficulties encountered in the analysis were reported. Statistical analysis was carried out according to the Harmonized Protocol of the International Union of Pure and Applied Chemistry. The relative standard deviation for repeatability (RSDr) ranged from 7.42 to 20.94%, and the relative standard deviation for reproducibility (RSDR) ranged from 16.34 to 29.17%. The method showed acceptable within-laboratory and between-laboratories precision for green coffee materials, as evidenced by HorRat values of < or = 0.85, at the studied range, for spiked and naturally contaminated materials. The mean recovery was 92.8% for green coffee material spiked with OTA at a level of 4.82 ng/g.

Trace level determination of acrylamide in cereal-based foods by gas chromatography-mass spectrometry.

A quantitative method has been developed for the determination of trace levels (<50 microg/kg) of acrylamide in cereal-based foods. The method is based on extraction of acrylamide with water, acidification and purification with Carrez I and II solutions, followed by bromination of the acrylamide double bond. The reaction product (2,3-dibromopropionamide) is extracted with ethyl acetate/hexane (4:1, v/v), dried over sodium sulfate, and cleaned up through a Florisil column. The derivative is then converted to 2-bromopropenamide by dehydrobromination with triethylamine and analyzed by gas chromatography coupled to mass spectrometry (GC-MS), employing (13C3)acrylamide as internal standard. In-house validation data for commercial and experimental cereal products showed good precision of the method, with repeatability and intermediate reproducibility relative standard deviations below 10%. The limit of detection and limit of quantitation are estimated at 2 and 5 microg/kg, respectively, and recoveries of acrylamide from samples spiked at levels of 5-500 microg/kg ranged between 93 and 104% after correction of analyte loss by the internal standard. Finally, a comparative test organized with two independent laboratories provided additional confidence in the good performance of the method, particularly at very low concentration levels.

Rapid, low cost thin-layer chromatographic screening method for the detection of ochratoxin A in green coffee at a control level of 10 microg/kg.

A thin-layer chromatographic (TLC) screening method was developed for the detection of ochratoxin A (OTA) in green coffee at a control level of 10 microg/kg. The method is based on extraction of OTA with a mixture of phosphoric acid and dichloromethane, purification by liquid-liquid partition into sodium hydrogen carbonate, separation by normal-phase TLC, and detection by visual estimation of fluorescence intensity under a UV lamp at 366 nm. The method was validated by performing replicate analyses of uncontaminated green coffee material spiked at 3 different levels of OTA (5, 10, and 20 microg/kg), and also by comparing results obtained on a series of test trial green coffees naturally contaminated with OTA (range 0.2 to 136.7 microg/kg) with those measured by a quantitative immunoaffinity/HPLC method. The agreement between the two methods was excellent, and neither false positive nor false negative results were recorded. This screening method is rapid, simple, robust, and very cheap, which makes it particularly well adapted for implementation in coffee-producing countries.