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.     

Determination of deoxynivalenol in cereals and cereal products by immunoaffinity column cleanup with liquid chromatography: interlaboratory study

An interlaboratory study was performed on behalf of the UK Food Standards Agency to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatographic (LC) method for the determination of deoxynivalenol in a variety of cereals and cereal products at proposed European regulatory limits. The test portion was extracted with water. The sample extract was filtered a applied to an immunoaffinity column. After being washed with water, the deoxynivalenol was eluted with acetonitrile or methanol. Deoxynivalenol was quantitated by reversed-phase LC with UV determination. Samples of artificially contaminated wheat-flour, rice flour, oat flour, polenta, and wheat based breakfast cereal, naturally contaminated wheat flour, and blank (very low level) samples of each matrix were sent to 13 collaborators in 7 European countries. Participants were asked to spike test portions of all samples at a range of deoxynivalenol concentrations equivalent to 200-2000 ng/g deoxynivalenol. Average recoveries ranged from 78 to 87%. Based on results for 6 artificially contaminated samples (blind duplicates), the relative standard deviation for repeatability (RSDr) ranged from 3.1 to 14.1%, and the relative standard deviation for reproducibility (RSDR) ranged from 11.5 to 26.3%. The method showed acceptable within-laboratory and between-laboratory precision for all 5 matrixes, as evidenced by HorRat values < 1.3.     

Determination of aflatoxins and ochratoxin A in ginseng and other botanical roots by immunoaffinity column cleanup and liquid chromatography with fluorescence detection

Mycotoxins are toxic secondary metabolites produced by certain molds and are common contaminants of many important food crops, such as grains, nuts, and spices. Some mycotoxins are found in fruits, vegetables, and botanical roots. These contaminants have a broad range of toxic effects, including carcinogenicity, immunotoxicity, neurotoxicity, and reproductive and developmental toxicity. The public health concerns related to both acute and chronic effects of mycotoxins in animals have prompted more than 100 countries to establish regulatory limits for some of the well-known mycotoxins, such as the aflatoxins (AFL). Our research focused on method development for 2 of these toxins, AFL and ochratoxin A (OTA), in ginseng and other selected botanical roots. Methods using an immunoaffinity column (IAC) cleanup, liquid chromatographic separation, and fluorescence detection were modified and evaluated. Two types of IAC cleanup were evaluated: IAC for AFL, and IAC for both AFL and OTA. Three derivatization techniques to enhance the fluorescence of the AFL were compared: precolumn trifluoroacetic acid, postcolumn bromination, and postcolumn ultraviolet irradiation. No derivatization was needed for OTA. Results for AFL using the single analyte IAC cleanup and the 3 derivatization techniques were all comparable for ginseng and for other roots such as ginger, licorice, and kava-kava. Recoveries of added AFL for ginseng at levels from 2 to 16 ng/g were about 80%. Using IAC cleanup for both AFL and OTA recoveries of added AFL for ginseng at 4-16 ng/g were about 70%, and for ginger, licorice, and kava-kava were about 60%. Recoveries of added OTA for ginseng, ginger, and echinacea at 4 ng/g were about 55%.     

Determination of zearalenone in barley, maize and wheat flour, polenta, and maize-based baby food by immunoaffinity column cleanup with liquid chromatography: interlaboratory study

An interlaboratory study was performed on behalf of the UK Food Standards Agency to evaluate the effectiveness of an affinity column cleanup liquid chromatography (LC) method for the determination of zearalenone (ZON) in a variety of cereals and cereal products at proposed European regulatory limits. The test portion is extracted with acetonitrile:water. The sample extract is filtered, diluted, and applied to an affinity column. The column is washed, and ZON is eluted with acetonitrile. ZON is quantified by reversed-phase LC with fluorescence detection. Barley, wheat and maize flours, polenta, and a maize-based baby food naturally contaminated, spiked, and blank (very low level) were sent to 28 collaborators in 9 European countries and 1 collaborator in New Zealand. Participants were asked to spike test portions of all samples at a ZON concentration equivalent to 100 microg/kg. Average recoveries ranged from 91-111%. Based on results for 4 artificially contaminated samples (blind duplicates) and 1 naturally contaminated sample (blind duplicate), the relative standard deviation for repeatability (RSDr) ranged from 6.9-35.8%, and the relative standard deviation for reproducibility (RSDR) ranged from 16.4-38.2%. The method showed acceptable within- and between-laboratory precision for all 5 matrixes, as evidenced by HorRat values <1.7.     

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 of ochratoxin A at parts-per-trillion levels in cereal products by immunoaffinity column cleanup and high-performance liquid chromatography/mass spectrometry

Ochratoxin A (OTA) is a toxic and potentially carcinogenic fungal toxin found in a variety of food commodities. A new sensitive method has been developed to quantify OTA in cereal products by reversed-phase liquid chromatography (LC) with mass spectrometric (MS) detection. Ochratoxin B was used as the internal standard. OTA was extracted from cereal products with acetonitrile-water, and the extract was diluted with a buffer; the diluted extract was cleaned up on an immunoaffinity column before LC/MS analysis. Two multiple-reaction monitoring transitions were used, one for quantification of OTA and one for confirmation of identity. The method was shown to be highly sensitive, with a low decision limit (CCalpha) of 0.012 microg/kg and a detection capability (CCbeta) of 0.021 microg/kg. Within-laboratory repeatability coefficient of variation values were 7.1, 3.7, and 3.1%, and the corresponding recoveries were 104, 106, and 103% for rice samples fortified with OTA at 0.05, 0.10, and 0.15 microg/kg, respectively. Method validation was performed according to the criteria of European Commission Decision 2002/657/EC. All criteria as presented in the Commission Decision were fulfilled. This method is the first fully validated method using immunoaffinity chromatography for cleanup and MS for detection in the analysis of cereals for OTA. The method was also successfully applied to cereal-derived products. The analytical results for determination of the OTA content of cereal products commercially available in Hong Kong are also reported.     

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.     

Determination of ochratoxin A in wine by means of immunoaffinity column clean-up and high-performance liquid chromatography

A new and accurate method to quantify ochratoxin A (OA) in table wine has been developed. The method uses commercial immunoaffinity columns for clean-up and high-performance liquid chromatography (HPLC) with fluorescence detection for quantification of the toxin. Wine was diluted with a solution containing 1% polyethylene glycol (PEG 8000) and 5% sodium hydrogencarbonate, filtered and applied to an OchraTest immunoaffinity column. The column was washed with a solution containing sodium chloride (2.5%) and sodium hydrogencarbonate (0.5%) followed by water. OA was eluted with methanol and quantified by reversed-phase HPLC with fluorometric detection (excitation wavelength 333 nm, emission wavelength 460 nm) using acetonitrile-water-acetic acid (99:99:2) as mobile phase. Average recoveries of OA from white, rosé and red wine samples spiked at levels from 0.04 to 10 ng/ml ranged from 88% to 103%, with relative standard deviations (RSDs) between 0.2 and 9.7%. Detection limit was 0.01 ng/ml based on a signal-to-noise ratio of 3:1. The method was applied successfully to 56 samples of red (38), rosé (8), white (9) and dessert (1) wine. The levels of OA ranged from <0.01 to 7.6 ng/ml with red wines more contaminated than rosé and white wines. A good correlation (r=0.987) was found by comparative analysis of 20 naturally contaminated samples using this method and the method of Zimmerli and Dick with better recoveries of OA and better performances for the new method. Several advantages of this method with respect to the actually available methods have been pointed out, with particular reference to red wine which appears to be the most difficult to analyze.     

Determination of zearalenone in cereal grains, animal feed, and feed ingredients using immunoaffinity column chromatography and liquid chromatography: interlaboratory study

A method using immunoaffinity column chromatography (IAC) and liquid chromatography (LC) for determination of zearalenone in cereal grains, animal feed, and feed ingredients was collaboratively studied. The test portion is extracted by shaking with acetonitrile-water (90 + 10, v/v) and sodium chloride. The extract is diluted and applied to an immunoaffinity column, the column is washed with water or phosphate-buffered saline or methanol-water (30 + 70, v/v), and zearalenone is eluted with methanol. The eluate is evaporated, the residue is dissolved in mobile phase and analyzed by reversed-phase LC with fluorescence detection. The presence of zearalenone can be confirmed using an alternate excitation wavelength or diode array detection. Twenty samples were sent to 13 collaborators (8 in Europe, 2 in the United States, one in Japan, one in Uruguay, and one in Canada). Eighteen samples of naturally contaminated corn, barley, wheat, dried distillers grains, swine feed, and dairy feed were analyzed as blind duplicates, along with blank corn and wheat samples. The analyses were done in 2 sample sets with inclusion of a spiked wheat control sample (0.1 mg/kg) in each set. Spiked samples recoveries were 89-116%, and for the 18 naturally contaminated samples, RSDr values (within-laboratory repeatability) ranged from 6.67 to 12.1%, RSDR values (among-laboratory reproducibility) ranged from 12.5 to 19.7%, and HorRat values ranged from 0.61 to 0.90.     

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.     

Determination of fumonisins B1 and B2 in corn and corn flakes by liquid chromatography with immunoaffinity column cleanup: collaborative study.

A liquid chromatographic (LC) method for the determination of fumonisins B1 (FB1) and B2 (FB2) in corn and corn flakes was collaboratively studied by 23 laboratories, which analyzed 5 blind duplicate pairs of each matrix to establish the accuracy, repeatability, and reproducibility characteristics of the method. Fumonisin levels in the corn ranged from <0.05 (blank) to 1.41 microg/g for FB1 and from <0.05 to 0.56 microg/g for FB2, whereas in the corn flakes they ranged from <0.05 to 1.05 microg/g for FB1 and from <0.05 to 0.46 microg/g for FB2. The method involved double extraction with acetonitrile-methanol-water (25 + 25 + 50), cleanup through an immunoaffinity column, and LC determination of the fumonisins after derivatization with o-phthaldialdehyde. Relative standard deviations for the within-laboratory repeatability (RSDr) of the corn analyses ranged from 19 to 24% for FB1 and from 19 to 27% for FB2; for the corn flakes analyses, RSDr ranged from 9 to 21 % for FB1 and from 8 to 22% for FB2. Relative standard deviations for the between-laboratories reproducibility (RSDR) of the corn analyses ranged from 22 to 28% for FB1 and from 22 to 30% for the FB2; for corn flakes analyses, RSDR ranged from 27 to 32% for FB1 and from 26 to 35% for FB2. Mean recoveries of FB1 and FB2 from corn spiked with FB1 at 0.80 microg/g and with FB2 at 0.40 microg/g were 76 and 72%, respectively; for corn flakes spiked at the same levels recoveries were 110 and 97% for FB1 and FB2, respectively. HORRAT ratios for the analyses of corn ranged from 1.44 to 1.53 for FB1 and from 0.96 to 1.48 for FB2, whereas for corn flakes they ranged from 1.60 to 1.82 for FB1 and from 1.39 to 1.68 for FB2.     

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.     

Simultaneous determination of aflatoxins, ochratoxin A, and zearalenone in grains by new immunoaffinity column/liquid chromatography

The simultaneous determination of mycotoxins was performed in 3 steps: extraction, cleanup, and detection. For extraction, a mixture of acetonitrile-water (60 + 40, v/v) was proved appropriate. For cleanup, a new Afla-Ochra-Zea immunoaffinity column was used. After derivatization with trifluoroacetic acid, the mycotoxins aflatoxins, ochratoxin A (OTA), and zearalenone (ZEA) were determined simultaneously by liquid chromatography with fluorescence detection. The detection limits in different matrixes after cleanup with the new immunoaffinity column were very low: aflatoxins, 0.002-0.7 microg/kg; OTA, 0.07-0.25 microg/kg; ZEA, 1-3 microg/kg. The limits of determination were: aflatoxins, 0.25 microg/kg; OTA, 0.5 microg/kg; ZEA, 5 microg/kg. The recovery rates for aflatoxins, OTA, and ZEA for rye and rice were between 86 and 93% when a 0.5 g sample matter per immunoaffinity column was used.     

Determination of aflatoxins in Chinese medicinal herbs by high-performance liquid chromatography using immunoaffinity column cleanup Improvement of recovery

Although analytical methods are available for the determination of aflatoxins in medicinal herbs, none of them can be applied satisfactorily to all sample matrices. The difficulty arises from the complex chemical composition of the herbs. Recovery is generally low by using immunoaffinity column cleanup due to the acidity of the water extractive leading to a weakened binding affinity. As a solvent for dilution and neutralization, phosphate buffer saline is useful for certain herbs but not for others that have high acidity. The problem can be solved by using 0.1 M phosphate buffer, which has a higher buffering capacity and eliminates sodium chloride. The modified method was validated by the analysis of a certified reference material and shown to be useful for the determination of aflatoxins in herbal samples of high acidity.     

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     

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.     

Analysis of ochratoxin A in milk after direct immunoaffinity column clean-up by high-performance liquid chromatography with fluorescence detection

The present work describes a new analytical method for direct immunoaffinity column clean-up of ochratoxin A (OTA) in milk samples followed by determination of the toxin using high-performance liquid chromatography with fluorescence detection (HPLC-FD). Two different immunoaffinity cartridges (IAC) were investigated, and Ochraprep columns were chosen because they showed the best results. An average recovery of 89.8% and a mean RSD of 5.8% for artificially contaminated cow's milk in the range of 5-100 ng/L were attained. The calculated limit of detection (LOD) and limit of quantitation (LOQ) were as low as 0.5 and 5 ng/L, respectively. This new easy and fast method avoids a previous liquid-liquid extraction step and therefore the use of toxic chlorinated solvents. Chromatograms of the final extracts were clean and OTA could be easily detected at a retention time of 8.4 min without interferences. To assess the presence of the toxin in cow's milk eight samples of skimmed and four samples of whole milk were analysed and OTA was not detected over the established detection limit.     

Determination of halofuginone in eggs by liquid chromatography/tandem mass spectrometry after cleanup with immunoaffinity chromatography

A sensitive and very selective high-performance liquid chromatography/tandem mass spectrometric (LC/MS/MS) method for the detection of halofuginone in whole egg has been developed. After deproteinisation with acetonitrile and evaporation of the organic solvent, halofuginone was further isolated by applying immunoaffinity chromatography. The concentrated eluent was injected into the LC/MS/MS system on a C18 column. The precursor ion ([M+H]+) produced by positive electrospray ionisation was selected for fragmentation with argon. Validation parameters such as recovery, linearity and repeatability, decision limit (CCalpha) and detection capability (CCbeta) were determined.     

Determination of zearalenone in botanical dietary supplements, soybeans, grains, and grain products by immunoaffinity column cleanup and liquid chromatography: single-laboratory validation

The accuracy, repeatability, and reproducibility characteristics of a method for measuring levels of zearalenone (ZON) in botanical root products, soybeans, grains, and grain products were determined by an AOAC single-laboratory validation procedure. Replicates of 10 test portions of each powdered root product (black cohosh, ginger, ginseng), brown rice flour, brown rice grain, oat flour, rice bran, soybeans, and wheat flour at each spiking level (ZON at 0, 50, 100, and 200 microg/kg) were analyzed on 3 separate days. Test samples were extracted with methanol-water (75 + 25, v/v). The extracts were centrifuged or filtered, diluted with phosphate-buffered saline (PBS) containing 0.5% Tween 20, and filtered; the filtrates were applied to an immunoaffinity column containing antibodies specific for ZON. After the column was washed with methanol-PBS (15 + 85, v/v) containing 0.5% Tween 20 and then with water, the toxin was eluted from the column with methanol, and the eluate was diluted with water. The eluate containing the toxin was then subjected to RPLC with fluorescence detection. All commodities that were found to contain ZON at < 10 microg/kg were used for the recovery study. The average within-day and between-days recoveries of ZON added at levels of 50-200 microg/kg ranged from 82 to 88% and from 81 to 84%, respectively, for all test commodities. The total average of within- and between-day SD and RSDr values for all test commodities ranged from 2.5 to 7.3 microg/kg and from 4.6 to 6.2%, respectively. HorRat values were <1.3 for all matrixes examined. The tested method was found to be acceptable for the matrixes examined.