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 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.     

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.     

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 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 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.     

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.     

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 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 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.     

Liquid chromatographic method for the quantification of zearalenone in baby food and animal feed: interlaboratory study

An interlaboratory trial for determination of zearalenone (ZON) in baby food and animal feed was conducted. The study involved 39 participants in 16 European Union member states, as well as Turkey, Uruguay, and China, representing a cross-section of industry, and official food control and research institutes. The method is based on immunoaffinity column cleanup followed by high-performance liquid chromatography using fluorimetry (HPLC-FI). The test portion of the sample is extracted with methanol-water (75 + 25, v/v). The sample extract is filtered, diluted, and passed over an immunoaffinity column. ZON is eluted with methanol. The separation and determination of ZON is performed by reversed-phase HPLC-FI with an excitation wavelength of 274 nm and an emission wavelength of 446 nm. Test portions of the samples were spiked at levels of 20 and 30 microg/kg ZON in baby food and at levels of 100 and 150 microg/kg ZON in animal feed. Mean recoveries from each participant ranged from 78 to 119% with an average value of 92% for baby food and from 51 to 122% with an average value of 74% for animal feed. Based on results for spiked samples (blind duplicates at 2 levels), as well as naturally contaminated samples (blind duplicates at 3 levels), the relative standard deviation for repeatability (RSDr) in baby food ranged from 2.8 to 9.0%. For animal feed, this value ranged from 5.7 to 9.5%. The relative standard deviation for reproducibility (RSDR) in baby food ranged from 8.2 to 13.3%, and for animal feed this value ranged from 15.5 to 21.4%. The Horwitz ratio (HorRat) in baby food ranged from 0.3 to 0.4, and for animal feed this value ranged from 0.6 to 0.9. The method showed acceptable within- and between-laboratory precision for each matrix, as required by European legislation.     

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.     

Automated liquid chromatographic determination of ochratoxin A in cereals and animal products using immunoaffinity column clean-up.

The analysis of the fungal mycotoxin ochratoxin A in cereals and animal products is described using an immunoaffinity column clean-up and high-performance liquid chromatographic determination. The clean-up can be carried out manually or using a commercially available automated sample preparation system. The method has been applied to cereals such as wheat, rye and barley, unprocessed breakfast cereals and animal products such as pigs' kidneys and blood sausages. Recoveries ranged from 70-80% for spiked samples (10 micrograms/kg) and the method had a relative standard deviation of 1.3% (n = 8) for the analysis of a wheat sample naturally contaminated at 13.7 micrograms/kg ochratoxin A and relative standard deviation of 3.0% (n = 8) for a pig kidney sample spiked at 10 micrograms/kg ochratoxin A. The immunoaffinity approach was significantly faster than methods employing conventional chromatographic clean-up, and extracts were freer of co-extractives giving a limit of detection of 0.2 micrograms/kg.     

Immunoaffinity column cleanup with liquid chromatography using postcolumn bromination for the determination of aflatoxins in black and white sesame seed: single-laboratory validation

A single-laboratory validation was conducted to establish the effectiveness of an immunoaffinity column cleanup procedure followed by LC with fluorescence detection for the determination of aflatoxins B1, B2, G1, and G2 in sesame seeds. The sample is homogenized with 50% water (w/w) to form a slurry, then the test portion is extracted with methanol-water (60 + 40, v/v) using a high-speed blender. The sample extract is filtered, diluted with 15% Tween 20 in phosphate-buffered saline solution, and applied to an immunoaffinity column. Aflatoxins are removed with neat methanol, then directly determined by RP-LC with fluorescence detection using postcolumn bromination (Kobra cell). Test portions of blank white sesame seed slurry were spiked with a mixture of aflatoxins to give total levels of 4 and 10 microg/kg. Recoveries for individual and total aflatoxins ranged from 92.7 to 110.3% for spiked samples. Based on results for spiked sesame paste (triplicates at two levels), the RSD for repeatability (RSD(r)) averaged 1.1% for total aflatoxins and 1.4% for aflatoxin B1. The method was demonstrated to be applicable to naturally contaminated samples of black and white sesame seeds obtained from local markets in China.     

Determination of ochratoxin A in currants, raisins, sultanas, mixed dried fruit, and dried figs by immunoaffinity column cleanup with liquid chromatography: interlaboratory study

An interlaboratory study was performed on behalf of the Food Standards Agency to evaluate the effectiveness of an affinity column cleanup liquid chromatographic (LC) method for the determination of ochratoxin A in a variety of dried fruit at European regulatory limits. To ensure homogeneity before analysis, laboratory samples are normally slurried with water in the ratio of 5 parts fruit to 4 parts water, and test materials in this form were used in the study. The test portion was extracted with acidified methanol. The extract was filtered, diluted with phosphate-buffered saline, and applied to an affinity column. The column was washed and ochratoxin A was eluted with methanol. Ochratoxin A was quantified by reversed-phase LC. The use of post-column pH shift to enhance the fluorescence of ochratoxin A by the addition of 1.1 M ammonia solution to the column eluant is optional. Determination was by fluorescence. Currants, sultanas, raisins, figs, and mixed fruit (comprising dried pineapple, papaya, sultanas, prunes, dates, and banana chips), both naturally contaminated and blank (very low level), were sent to 24 collaborators in 7 European countries. Participants were asked to spike test portions of all test samples at a level equivalent to 5 ng/g ochratoxin A. Average recoveries ranged from 69 to 74%. Based on results for 5 naturally contaminated test samples (blind duplicates) the relative standard deviation for repeatability (RSDr) ranged from 4.9 to 8.7%, and the relative standard deviation for reproducibility (RSDR) ranged from 14 to 28%. The method showed acceptable within- and between-laboratory precision for all 5 matrixes, as evidenced by HORRAT values <1.3.     

Liquid chromatographic method for determination of patulin in clear and cloudy apple juices and apple puree: collaborative study

A collaborative trial was conducted to validate the effectiveness of a liquid chromatographic (LC) procedure for determination of patulin in both clear and cloudy apple juices and apple puree. The test portion of clear apple juice was directly extracted with ethyl acetate; cloudy apple juice and apple puree were treated with pectinase enzyme before extraction. After back-extraction into sodium carbonate to remove interfering acidic compounds, the extract was dried and concentrated, and patulin was determined by LC with UV detection. Clear and cloudy apple juices, apple puree test samples naturally contaminated with patulin, and blank test samples for spiking with patulin were sent to 14 collaborators in 12 different European countries. Test portions of each of the 3 test sample types were spiked with patulin at 75 ng/g. Recoveries of patulin ranged from 80 to 92%. Based on the results for spiked test samples (blind pairs) and naturally contaminated test samples (blind pairs at 3 levels), the relative standard deviations for repeatability (RSDr) and reproducibility (RSDR) ranged from 8 to 35% and 11 to 36%, respectively. Although HORRAT values of <1.4 were obtained for all 3 matrixes at patulin levels ranging from 26 to 121 ng/g, better performance values (RSDr values 6-10% and RSDR values 11-25%) were obtained for clear and cloudy apple juice spiked above 50 ng/g, which is either the statutory limit or the advisory level for patulin contamination in apple juices in many countries.     

Liquid chromatographic method for quantitation of patulin at 10 ng/mL in apple-based products intended for infants: interlaboratory study

An interlaboratory trial for the determination of patulin in apple juice and fruit puree was conducted, involving 17 participants representing a cross section of industry, official food control, and research facilities. Mean recoveries reported ranged from 74 (10 ng/g) to 62% (25 ng/g) for apple juice and from 72 (25 ng/g) to 74% (10 ng/g) for fruit puree. 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) in juice ranged from 8.0 to 14.3% and in puree from 3.5 to 9.3%. The relative standard deviation for reproducibility (RSD(R)) in juice ranged from 19.8 to 39.5% and in puree from 12.5 to 35.2%, reflecting HORRAT values from 0.6 to 1.0 for juice and 0.4 to 0.9 for puree. The method showed acceptable within-laboratory and between-laboratory precision for each matrix, as required by current European legislation.     

Analysis of T-2 and HT-2 toxins in cereal grains by immunoaffinity clean-up and liquid chromatography with fluorescence detection

A sensitive, precise and accurate method has been developed for the simultaneous determination of T-2 and HT-2 toxins in cereal grains at ppb levels using high-performance liquid chromatography (HPLC) with fluorescence detection and 1-antroylnitrile (1-AN) as labeling reagent after immunoaffinity clean-up. Cereal samples were extracted with methanol/water (90:10, v/v), and the extracts were cleaned-up through commercially available immunoaffinity columns containing monoclonal anti-T-2 antibodies (T-2 test HPLC, Vicam). T-2 and HT-2 toxins were quantified by reversed-phase HPLC with fluorometric detection (excitation wavelength 381 nm, emission wavelength 470 nm) after derivatization with 1-AN. The monoclonal antibody showed 100% cross-reactivity with both T-2 and HT-2 toxin, and the immunoaffinity column clean-up was effective up to 1.4 microg of both toxins. The method was successfully applied to the analysis of T-2 and HT-2 toxins in wheat, maize and barley. Recoveries from spiked samples with toxin levels from 25 to 500 microg/kg ranged from 70% to 100%, with relative standard deviation generally lower than 8%. The limit of detection of the method was 5 microg/kg for T-2 toxin and 3 microg/kg for HT-2 toxin, based on a signal-to-noise ratio 3:1. HT-2 toxin was detected in ten naturally contaminated wheat samples out of 14 samples analyzed, with toxin levels ranging from 10 to 71 microg/kg; three of them contained also T-2 toxin up to 12 microg/kg.     

Determination of aflatoxin M1 using a dialysis-based immunoaffinity sample pretreatment system coupled on-line to liquid chromatography. Reusable immunoaffinity columns.

A liquid chromatographic column-switching system containing a dialysis unit and an anti-aflatoxin immunoaffinity precolumn (immuno precolumn) is described for the automated determination of aflatoxin M1 in milk samples. Both a flat membrane dialysis unit working according to the flowing donor-flowing acceptor principle and a laboratory made hollow-fibre dialysis unit working according to the stagnant donor-flowing acceptor principle were evaluated. The hollow-fibre unit is superior with respect to repeatability (3% relative standard deviation) and detection limit (10 ng/l for aflatoxin M1 in milk), in spite of the fact that the overall recovery is only 6%. Interfering compounds, which would destroy the activity of the immuno precolumn, are efficiently removed from the system by the dialysis step; a single immuno precolumn can then be used for over 70 milk analyses. No decrease in the performance of either the immuno precolumn or the hollow-fibre dialysis unit is observed.