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

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

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

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

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

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

Determination of 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 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.     

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.     

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 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.     

Immunoaffinity column cleanup with liquid chromatography using post-column bromination for aflatoxins in medicinal herbs and plant extracts

A new and accurate method to quantitate aflatoxins in medicinal herbs is developed. This method consists of extraction of the sample with MeOH-H2O (70:30) followed by clean-up of the extracts with immunoaffinity columns and, finally, high-performance liquid chromatographic determination with fluorescence detection. Aflatoxins B1 and G1 are determined as their bromine derivatives, produced in an online post-column derivatization system. The overall average recoveries for three different medicinal herbs spiked at levels of 1.3 and 2.6 ng/g of total aflatoxins range from 93% to 97%. The detection limit is 0.15 ng/g for both G2 and B2 and 0.20 ng/g for both G1 and B1, based on a signal-to-noise ratio of 3:1 and a precision (within-laboratory relative standard deviation) ranging from 0.8% to 1.4%. The use of immunoaffinity columns provides excellent clean-up of these particular extracts, which are generally difficult to analyze. The method is applied successfully to 96 samples of natural drugs.     

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.     

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 aflatoxin B1 in medical herbs: interlaboratory study

A method was developed for the determination of aflatoxin B1 in medical herbs (senna pods, botanical name Cassia angustifolia; devil's claw, botanical name Harpagophytum procumbens; and ginger roots, botanical name Zingiber officinale). The method, which was tested in a mini-collaborative study by 4 laboratories, is based on an immunoaffinity cleanup followed by reversed-phase high-performance liquid chromatography separation and fluorescence detection after post-column derivatization. It allows the quantitation of aflatoxin B1 at levels lower than 2 ng/g. A second extractant (acetone-water) was tested and compared to the proposed methanol-water extractant. Several post-column derivatization options (electrochemically generated bromine, photochemical reaction, and chemical bromination) as well as different integration modes (height versus area) were also investigated. No differences were found depending on the choice of derivatization system or the signal integration mode used. The method was tested for 3 different matrixes: senna pods, ginger root, and devil's claw. Performance characteristics were established from the results of the study and resulted in HorRat values ranging from 0.12 to 0.75 with mean recoveries from 78 to 91% for the extraction with methanol-water and HorRat values ranging from 0.10-1.03 with mean recoveries from 98 to 103% for the extraction with acetone-water. As a result, the method, with all tested variations, was found to be fit-for-purpose for the determination of aflatoxin B1 in medical herbs at levels of 1 microg/kg and above.     

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 fumonisins B1 and B2 in corn-based foods for infants and young children by LC with immunoaffinity column cleanup: interlaboratory validation study

A liquid chromatographic method for the determination of fumonisins B1 (FB1) and B2 (FB2) in corn-based foods for infants and young children was subjected to an interlaboratory validation study involving 11 laboratories. Five blind duplicate sample pairs of each matrix were analyzed to establish the accuracy, repeatability, and reproducibility of the method. Mass fractions in the baby food samples ranged from 89.1 to 384.4 microg/kg FB1 and from 22.5 to 73.6 microg/kg FB2. The method involved a warm extraction with citrate phosphate buffer-methanol-acetonitrile (50 + 25 + 25, v/v/v), a cleanup through an immunoaffinity column, and an end-determination of fumonisins by LC after automated precolumn derivatization with o-phthaldialdehyde reagent. RSDs for within-laboratory repeatability (RSDr) ranged from 6.8 to 23.5% for FB1 and 7.6 to 22.9% for FB2. RSDs for between-laboratory reproducibility (RSDR) ranged from 15.4 to 26.2% for FB1 and 21.6 to 36.3% for FB2. Mean FB1 recoveries from baby foods spiked at 100.0 and 250.0 microg/kg were 89 and 96%, respectively; for FB2 spiked foods at 25.0 and 62.5 microg/kg recoveries were 90 and 85%, respectively. HorRat values ranged from 0.8 to 1.2 for FB1, whereas for FB2 they ranged from 0.9 to 1.4 when calculated according to Horwitz, and from 1.0 to 1.7 when calculated according to Thompson, indicating an acceptable among-laboratory precision for all matrixes (HorRat values <2).     

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.     

免疫亲和柱净化-高效液相色谱法同时检测鸡蛋中6种玉米赤霉醇类化合物残留量

建立了鸡蛋中6种玉米赤霉醇类化合物(α-玉米赤霉醇、β-玉米赤霉醇、α-玉米赤霉烯醇、β-玉米赤霉烯醇、玉米赤霉酮和玉米赤霉烯酮)残留量的免疫亲和柱净化-高效液相色谱检测方法。样品酶解后用叔丁基甲醚提取、氢氧化钠反萃取,经免疫亲和柱富集和净化后,采用高效液相色谱-紫外检测器进行测定。色谱柱: Agilent Eclipse XDB-C18(150 mm×4.6 mm, 3.5 μm);流动相: 甲醇-乙腈-水(50:15:35, v/v/v);流速: 1.0 mL/min;检测波长: 270 nm。结果表明,6种目标物在0.01～0.2 mg/L范围内线性关系良好,相关系数(r)≥0.9998,检出限(LOD,S/N≥3)为1.0 μg/kg,平均回收率为73.2%～95.7%,相对标准偏差小于8%。该方法灵敏度高、重现性好,适用于鸡蛋样品中痕量玉米赤霉醇类药物残留的测定。     

Immunoaffinity column as sample cleanup method for determination of the beta-adrenergic agonist ractopamine and its metabolites

A monoclonal antibody-based immunoaffinity column (RAC-IAC) was developed as a cleanup method for the determination of ractopamine and ractopamine glucuronides. [14C]Ractopamine (5 microg) and [14C]ractopamine glucuronides (5 microg) were fortified into 10 mL cattle urine, and loaded onto an RAC-IAC (5 mg IgG/mL) column. The column was washed and the bound analytes were eluted. In the initial loading and washing, 22% of the radioactivity was washed off and the subsequent elution step recovered 78%. A blank column prepared from nonspecific IgG retained <10% of the radioactivity. The RAC-IACs were damaged by high methanol concentrations, preventing reuse. Elution of the analytes with 50mM glycine buffer, pH 2.8, prevented damage, and the columns could be reused at least 20 times with no change in performance. They were stored >3 months in phosphate-buffered saline with 0.02% sodium azide at 4 degrees C. The method was used with fortified cattle muscle, liver, and kidney samples with recoveries of 82.1+/-7.6, 87.8+/-1.9, and 92.5+/-0.4%, respectively (n = 3). Similar studies with sheep muscle, liver, and kidney samples gave recoveries of 91.8+/-0.2, 91.7+/-0.3, and 92.3+/-0.3, respectively (n = 3). Liver and kidney samples were diluted to prevent column plugging, but all of the eluants were suitable for liquid chromatography analysis. This IAC is a selective, efficient, and economical cleanup method in a variety of matrixes for ractopamine determination.     

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.