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 zearalenone content in cereals and feedstuffs by immunoaffinity column coupled with liquid chromatography

The zearalenone content of maize, wheat, barley, swine feed, and poultry feed samples was determined by immunoaffinity column cleanup followed by liquid chromatography (IAC-LC). Samples were extracted in methanol-water (8 + 2, v/v) solution. The filtered extract was diluted with distilled water and applied to immunoaffinity columns. Zearalenone was eluted with methanol, dried by evaporation, and dissolved in acetonitrile-water (3 + 7, v/v). Zearalenone was separated by isocratic elution of acetonitrile-water (50 + 50, v/v) on reversed-phase C18 column. The quantitative analysis was performed by fluorescence detector and confirmation was based on the UV spectrum obtained by a diode array detector. The mean recovery rate of zearalenone was 82-97% (RSD, 1.4-4.1%) on the original (single-use) immunoaffinity columns. The limit of detection of zearalenone by fluorescence was 10 ng/g at a signal-to-noise ratio of 10:1 and 30 ng/g by spectral confirmation in UV. A good correlation was found (R2 = 0.89) between the results obtained by IAC-LC and by the official AOAC-LC method. The specificity of the method was increased by using fluorescence detection in parallel with UV detection. This method was applicable to the determination of zearalenone content in cereals and other kinds of feedstuffs. Reusability of immunoaffinity columns was examined by washing with water after sample elution and allowing columns to stand for 24 h at room temperature. The zearalenone recovery rate of the regenerated columns varied between 79 and 95% (RSD, 3.2-6.3%). Columns can be regenerated at least 3 times without altering their performance and without affecting the results of repeated determinations.     

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.     

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     

Quantitative determination of aflatoxin B1-oxime by column liquid chromatography with ultraviolet detection.

Liquid chromatography was used for the quantification of aflatoxin B1-oxime (AFB1-oxime). The yield of AFB1-oxime in the reaction mixture was 89%, while after purification on silica gel it was 72%. LC analysis of the reaction mixture after silica gel fractionation revealed a retention time of 0.84 min for AFB1-oxime, 8.42 min for AFB1, 1.21 min for unknown 1 and 1.61 min for unknown 2. UV-visible analysis of the reaction mixture after silica gel fractionation showed a lambda(max) of 269 and 361 nm for AFB1-oxime, 263 and 360 nm for AFB1, 273 nm for unknown 1 and 275 nm for unknown 2. Excitation and emission wavelengths were found to be 269 and 368/438 nm for AFB1-oxime, 359/424 nm for AFB1, 270 and 367/450 nm for unknown 1 and 273 and 416/447 nm for unknown 2. The method may find versatile application in monitoring reactions for the preparation of oximes of various analytes for the synthesis of their immunogens.     

Development and application of immunoaffinity column chromatography for atrazine in complex sample media

A rabbit antibody immunoaffinity (IA) column procedure was evaluated as a cleanup method for the determination of atrazine in soil, sediment, and food. Four IA columns were prepared by immobilizing a polyclonal rabbit anti-atrazine antibody solution to HiTrap Sepharose columns. Atrazine was bound to the IA columns when the loading solvents were either 100% water, 2% acetonitrile in water, or 10% methanol in phosphate buffered saline (PBS). Quantitative removal of atrazine from the IA columns was achieved with elution solvents of either 70% ethanol in water, 70% methanol in water, or 100% methanol. One control column was prepared using nonspecific rabbit IgG antibody. This control column did not retain any applied atrazine indicating atrazine did not bind indiscriminately to protein or the Sepharose support. The four IA columns showed reproducible coupling efficiency for the immobilization of the atrazine antibody and consistent binding and releasing of atrazine. The coupling efficiency (4.25 mg of antibody in 1 mL of resin bed) for the four IA columns ranged from 93 to 97% with an average of 96 ± 2% (2.1%). Recoveries of the 500, 50, and 5 ng mL⁻¹ atrazine standard solutions from the four IA columns were 107 ± 7% (6.5%), 122 ± 14% (12%), and 114 ± 9% (8.0%) respectively, based on enzyme-linked immunosorbent assay (ELISA) data. The maximum loading was approximately 700 ng of atrazine for each IA column (∼0.16 μg of atrazine per mg of antibody). The IA columns could withstand 100% methanol as the elution solvent and could be reused more than 50 times with no change in performance. The IA columns were challenged with soil, sediment, and duplicate-diet food samples and effectively removed interferences from these various matrices for subsequent gas chromatography/mass spectrometry (GC/MS) or ELISA analysis. The log-transformed ELISA and GC/MS data were significantly correlated for soil, sediment and food samples although the ELISA values were slightly higher than those obtained by GC/MS. The IA column cleanup procedure coupled with ELISA analysis could be used as an alternative effective analytical method for the determination of atrazine in complex sample media such as soil, sediment, and food samples.     

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.     

Rapid immunoaffinity-based method for determination of zearalenone in corn by fluorometry and liquid chromatography

An immunoaffinity-based method was developed to determine zearalenone in corn. Corn samples were extracted in acetonitrile-water (90 + 10, v/v), applied to an immunoaffinity column, and eluted with methanol. The isolated toxin was quantitated either by reaction with aluminum chloride hexahydrate (AlCl3.6H2O) prior to measurement with a fluorometer or injection into a liquid chromatographic (LC) system with a fluorescence detector. Performance was evaluated in terms of antibody specificity, limit of detection, percentage recovery, precision, column capacity, assay linearity, and comparison with AOAC Official Method 985.18. With the immunoaffinity column cleanup procedure, only zearalenone and its metabolites were recognized by the antibody (> or = 75% recovery). Limits of detection were 0.10 microgram/g for the fluorometer and 0.10 or 0.0025 microgram/g (sensitive method) for the LC method. Percentage recovery averaged 105% (fluorometer) and 93% (LC method), with average relative standard deviations (RSDs) of 15.7 and 9.3%. Naturally contaminated samples gave comparable RSDs of 8.3 and 9.9% for the fluorometer and LC methods, respectively. Column capacity was 4.0 micrograms with 89% recovery. Assay linearity was comparable for both methods (r2 = 0.998). Optimum assay ranges were 0.10-5.0 micrograms/g for the fluorometer and 0.10-50 or 0.0025-5.0 micrograms/g (sensitive method) for the LC method. Comparative analysis of 17 naturally contaminated corn samples using Zearala Test LC and the official AOAC LC method for detection of zearalenone showed that Zearala Test is statistically comparable to the AOAC Official Method 985.18 (r2 = 0.747).     

Determination of selected neurotransmitter metabolites using monolithic column chromatography coupled with chemiluminescence detection

The oxidation of selected clinically important neurotransmitter metabolites with acidic potassium permanganate in the presence of polyphosphates evokes chemiluminescence of sufficient intensity to enable the sensitive determination of these species. Limits of detection for 5-hydroxyindole-3-acetic acid (5-HIAA), vanilmandelic acid (VMA; α,4-dihydroxy-3-methoxybenzeneacetic acid), 4-hydroxy-3-methoxyphenylglycol (MHPG), homovanillic acid (HVA, 4-hydroxy-3-methoxyphenylacetic acid) and 3,4-dihydroxyphenylacetic acid (DOPAC) were between 5 × 10⁻⁹ and 4 × 10⁻⁸ M, using flow-injection analysis methodology. In addition, we demonstrate the rapid determination of homovanillic acid and 5-hydroxyindole-3-acetic acid in human urine - without the need for extraction procedures - using monolithic column chromatography with chemiluminescence detection.     

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.     

Quantification of deoxynivalenol in wheat using an immunoaffinity column and liquid chromatography

A simple and accurate method to quantify the mycotoxin deoxynivalenol (DON) in wheat is described. The method uses immunoaffinity chromatography for DON isolation and liquid chromatography (LC) for toxin detection and quantification. Wheat samples are extracted in water, filtered twice and applied to an immunoaffinity column. Following a water wash, DON is eluted from the column with methanol and injected onto an LC system with a UV detector for quantification. Test performance was evaluated in terms of antibody specificity, limit of detection, percentage recovery, precision, column capacity, assay linearity and comparison with the GC-electron-capture detection (ECD) method of Tacke and Casper. Specificity of the immunoaffinity column cleanup procedure was confirmed with only DON (>80%) and its 15-C derivatives (40-50%) being recognized by the antibody while 3-C DON derivatives, nivalenol, T-2 and fusarenon-X did not bind. The limit of detection is at least 0.10 microg/g. Percentage recovery for the entire assay range averages 90% with an average relative standard deviation of 8.3%. Naturally contaminated samples showed comparable precision. Column capacity was determined to be 3.3 microg. The assay showed a high degree of linearity (r2=0.999) and an optimum assay range of 0.10 to 10.0 microg/g. Comparative analysis of 28 naturally or artificially contaminated wheat samples using DONtest-HPLC and the GC-ECD method of Tacke and Casper showed that DONtest-HPLC is a statistically significant predictor of the GC-ECD method (r2=0.982).     

A silica-based monolithic column in capillary HPLC and CEC coupled with ESI-MS or electrospray-atmospheric-pressure laser ionization-MS

We describe the successful coupling of CEC and capillary HPLC with the recently developed atmospheric-pressure laser ionization (APLI) method. APLI is suitable for selectively and sensitively ionizing nonpolar aromatic compounds at ambient pressure for subsequent mass-selective detection. The polycyclic aromatic hydrocarbons used as analytes are first separated either by CEC on a silica-based monolithic column or by capillary HPLC. The eluent, along with a sheath flow, is volatilized by microelectrospray and then selectively ionized by excimer laser (KrF*) radiation via two-photon excitation. A QTOF-MS is used as mass-selective detector. This interface combination makes soft ionization of thermally labile nonpolar aromatic analytes possible.     

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.     

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.     

Validation according to European Commission Decision 2002/657/EC of a confirmatory method for aflatoxin M1 in milk based on immunoaffinity columns and high performance liquid chromatography with fluorescence detection

A high performance liquid chromatographic method with fluorimetric detection for the determination of aflatoxin M₁ (AFM₁) in milk has been optimized and validated according to Commission Decision 2002/657/EC by using the conventional validation approach. The procedure for determining selectivity, recovery, precision, decision limit (CC_α), detection capability (CC_β) and ruggedness of the method has been reported. The results of the validation process demonstrate the agreement of the method with the provisions of Commission Regulation 401/2006/EC. The mean recovery calculated at three levels of fortification (0.5, 1.0, and 1.5-fold the MRL) was 91% and the maximum relative standard deviation value for the within-laboratory reproducibility was 15%. Limit of detection (LOD) and limit of quantitation (LOQ) values were 0.006 μg kg⁻¹ and 0.015 μg kg⁻¹ while the CC_α and CC_β values were 0.058 μg kg⁻¹ and 0.065 μg kg⁻¹, respectively. The relative expanded measurement uncertainty of the method was 7%. The method was not affected by slight variations of some critical factors (ruggedness minor changes) as pre-treatment and clean-up of milk samples, thermal treatment and different storage conditions, as well as by major changes valued in terms of milk produced by different species (buffalo, goat and sheep). The method allowed accurate confirmation analyses of milk samples, resulted positive by the screening method. In fact, the Z-score values attained in a proficiency test round were well below the reference value of 1, proving the excellent laboratory performances.     

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 fumonisins B1 and B2 in corn by liquid chromatography/mass spectrometry with immunoaffinity column cleanup: single-laboratory method validation

A single-laboratory method validation was conducted to establish the effectiveness of an immunoaffinity column cleanup procedure followed by liquid chromatography/mass spectrometry (LCIMS) for the determination of fumonisins B1 and B2 (FBI + FB2) in corn. The test portion is extracted with acetonitrile-methanol-water (25 + 25 + 50). The extract is filtered, diluted with phosphate-buffered saline solution, and applied to an immunoaffinity column. FB1 + FB2 are removed with methanol and directly determined by reversed-phase LC with MS detection using selected-ion monitoring of 2 characteristic ions in each case. Test portions of blank corn samples were spiked with a mixture of FB1 + FB2 to give total levels of 200 and 500 ng/g, respectively. Recoveries of both FB1 and FB2 from spiked samples averaged 90.4-101%. Based on results for spiked raw corn (triplicates at 2 levels), the relative standard deviation for repeatability ranged from 2.8 to 7.1%. The accuracy of the method was demonstrated by analysis of Food Analysis Performance Assessment Scheme (FAPAS) test material. The method was also applied to a small survey of processed corn products such as corn chips, cornflakes, and popcorn.     

Alkaline treatment of the cellulose fiber affecting membrane column behaviour for high-performance immunoaffinity chromatography

The original cellulose fibers and those treated by alkaline solution were both used to prepare the acrylic membranes. The two kinds of membranes were packed into the columns for high-performance immunoaffinity chromatography by the immobilization of protein A on them. It was observed that the alkaline treatment of the cellulose fiber decreased the pressure resistance of the membrane to the mobile phases and greatly increased the accessible volume to the proteins, but affected the adsorption capacity of human IgG on the protein A membrane columns less. There is little difference between those two kinds of membranes on the adsorption capacities of HIgG, which means that the alkaline treatment of the cellulose fiber only significantly changes the void volume inter-membrane, and the porosity and surface area of membrane less. Alkaline treatment of the cellulose fiber reduced the membrane-column efficiency significantly. Some typical examples for the immunoaffinity analysis of IgG from human and dog plasma on the protein A membrane columns are illustrated.     

Determination of T-2 toxin in cereal grains by liquid chromatography with fluorescence detection after immunoaffinity column clean-up and derivatization with 1-anthroylnitrile

1-Anthroylnitrile (1-AN) has been shown to be an efficient labelling reagent for the determination of T-2 toxin (T-2) by high-performance liquid chromatography (HPLC)-fluorescence detection. This reaction has been used to develop a sensitive, reproducible and accurate method for the determination of T-2 in wheat, corn, barley, oats, rice and sorghum. The method uses immunoaffinity columns containing antibodies specific for T-2 for extract clean-up, pre-column derivatization with 1-AN and HPLC with fluorescence detection for toxin determination. Ground cereal samples were extracted with methanol-water (80:20, v/v), the extracts were purified by immunoaffinity columns and the toxin was quantified by reversed-phase HPLC with fluorometric detection (excitation wavelength 381 nm, emission wavelength 470 nm) after derivatization with 1-AN. Recoveries from the different cereals spiked with T-2 at levels ranging from 0.05 to 1.5 microg/g were from 80 to 99%, with relative standard deviations of less than 6%. The limit of detection was 0.005 microg/g, based on a signal-to-noise ratio of 3:1.