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 phytase activity in feed: interlaboratory study

An interlaboratory study was conducted to determine the performance characteristics of a new method for the determination of phytase activity in feed samples. The method is based on the principle that inorganic phosphate is released from the substrate phytate under defined assay conditions and has been validated for its suitability to measure the enzyme activity of various phytase products. Two different experimental designs of the study were applied, allowing for the estimation of the precision of the method under repeatability, intermediate precision and reproducibility conditions, respectively. The relative standard deviation for repeatability (RSDr) ranged from 2.2 to 10.6% and the RSD for reproducibility (RSDR) ranged from 5.4 to 15%. The suitability of the validated method for the intended purpose was demonstrated. The obtained performance profile of the method validated in this study was comparable to that of similar methods that were exclusively validated for one phytase product.     

Determination of aflatoxin B1 in alcoholic beverages: comparison of one- and two-photon-induced fluorescence

The qualitative and quantitative analysis of aflatoxin B₁ in a model system (water/ethanol), in different wines and in beer using one- and two-photon-induced fluorescence is discussed. The absorption and fluorescence properties of aflatoxin B₁ depend on the solvent and pH. The two-photon-absorption cross-section was calculated for aflatoxin B₁ in beer and wine (σ ₂ ∼ 25 GM) for excitation at 720 nm. A comparison of the one- and two-photon- induced fluorescence results showed that the disturbance due to background emission originating from matrix constituents is significantly reduced under two-photon-excitation conditions. The limit of detection for the one- and two-photon-induced fluorescence was determined.     

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 the acid value of instant noodles: interlaboratory study

An interlaboratory study was performed to evaluate the method for determining the acid value of instant noodles, based on the Japanese Agricultural Standard (JAS), with extraction of lipid using petroleum ether at a volume of 100 mL to the test portion of 25 g. Thirteen laboratories participated and analyzed 5 test samples as blind duplicates. Statistical treatment revealed that the repeatability (RSDr) of acid value was <6.5%, and the reproducibility (RSDR) of acid value was <9.6%. The HorRat values (RSDR/predicted RSDR) were 1.2-1.8, where the RSDR and the predicted RSDR were obtained in terms of free fatty acids in the noodles per unit weight, using the equation [acid value = percent free fatty acids (as oleic) x 1.99] and the extracted lipid contents. This method was shown to have acceptable precision by the present study.     

Determination of the moisture content of instant noodles: interlaboratory study

Determination of the moisture content of instant noodles, currently under discussion by the Codex Alimentarius Commission (CAC) requires 2 methods: one for fried noodles and the other for nonfried noodles. The method to determine the moisture content of fried noodles by drying at 105 degrees C for 2 h used in the Japanese Agricultural Standard (JAS) system of Japan can be applied to this purpose. In the present study, the JAS method for fried noodles was modified to be suitable for nonfried noodles by extending the drying time to 4 h. An interlaboratory study was conducted to evaluate interlaboratory performance statistics for these 2 methods. Ten participating laboratories each analyzed 5 test materials of fried and nonfried noodles as blind duplicates. After removal of outliers statistically, the repeatability (RSDr) and the reproducibility (RSD(R)) of these methods were 1.6-2.6 and 3.9-4.8% for fried noodles, and 0.3-1.5 and 1.3-2.9% for nonfried noodles, respectively.     

高效液相色谱-串联质谱法测定山茶油中黄曲霉毒素B1

建立了山茶油中黄曲霉毒素B1含量的高效液相色谱-串联质谱分析方法。通过对前处理方法的优化,选择了甲醇和水作为山茶油中黄曲霉毒素B1的提取溶剂,经免疫亲和柱富集浓缩后,采用高效液相色谱-串联质谱进行分析,经C18色谱柱分离,在电喷雾离子化正离子模式(ESI+)及多反应监测模式(MRM)下进行测定,基质匹配标准溶液外标法定量。在优化条件下,该方法线性范围为0.4～6.4μg/L,相关系数r2>0.998,最低检出限为0.026μg/kg,在添加水平为0.008,0.016和0.032μg时,方法回收率在85.9%～93.8%之间;相对标准偏差为1.8%～5.0%。方法可满足山茶油中黄曲霉毒素B1的检测要求。     

Some interferences in radioimmunoassay of aflatoxin B1

The specificity of radioimmunoassay of aflatoxin B₁ was tested. Relative cross-reactivity of used antiserum with aflatoxins B₁, B₂, G₁, G₂ and M₁ was found to be 100%, 24%, 44.2%, 10.3%, and 1.4%, respectively. The interference of coumarin, albumins, steroids and ethylvanilin was estimated also in radioimmunoassay of aflatoxin B₁. Thus these compounds may cause a false positive finding of aflatoxin B₁.     

Determination of walnut protein in processed foods by enzyme-linked immunosorbent assay: interlaboratory study

Because food allergens from tree nuts, including walnuts, are a frequent cause of adverse food reactions for allergic patients, the labeling of foods containing ingredients derived from tree nuts is required in numerous countries. According to Japanese regulations, the labeling of food products containing walnuts is recommended. To ensure proper labeling, a novel sandwich ELISA kit for the determination of walnut protein in processed foods (Walnut Protein [2S-Albumin] Kit; Morinaga Institute of Biological Science, Inc.; "walnut kit") has been developed. We prepared seven types of incurred samples (model processed foods: biscuits, bread, sponge cake, orange juice, jelly, chicken meatballs, and rice gruel) containing 10 microg walnut soluble protein/g of food for use in interlaboratory evaluations of the walnut kit. The walnut kit displayed sufficient reproducibility relative standard deviations (interlaboratory precision: 5.8-9.9% RSDR) and a high level of recovery (81-119%) for all the incurred samples. All the repeatability relative standard deviation (RSDr) values for the incurred samples that were examined were less than 6.0%. The results of this interlaboratory evaluation suggested that the walnut kit could be used as a precise and reliable tool for determination of walnut protein in processed foods.     

Determination of vegetal proteins in milk powder by enzyme-linked immunosorbent assay: interlaboratory study

Eight laboratories participated in a collaborative study to evaluate an enzyme-linked immunosorbent assay (ELISA) to determine soy, pea, and wheat proteins in pasteurized or ultra-high temperature (UHT) milk powders. To perform this assay, polyclonal antibodies for soy, pea, and wheat proteins were obtained from rabbit sera. Collaborators received calibration standards composed of milk powder containing 0-8% (w/w) vegetal protein in total protein and blind test samples containing approximately 1, 2, and 5% (w/w) vegetal protein. An indirect competitive ELISA was performed with a kit prepared by a participating laboratory; the kit contained plates coated with soy, pea, or wheat proteins, the corresponding specific antisera, enzyme-labeled second antibody, and substrate solution. Test samples and calibrants were extracted with phosphate-buffered saline, pH 7.4, containing 0.05% Tween and assayed with the ELISA kits. The degree of adulteration was affected by the type of heat treatment applied to the samples. The estimated percentage of vegetal protein addition was close to the theoretical value for pasteurized samples but much lower for UHT samples. For pasteurized samples, intralaboratory relative standard deviations ranged from 5 to 22% and interlaboratory relative standard deviations ranged from 14 to 34%.     

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     

Determination of aflatoxins B1, G1, B2 and G2 in medicinal herbs by liquid chromatography-tandem mass spectrometry

An easy method for the determination of aflatoxins B1, G1, B2 and G2 in Rhammus purshiana by LC coupled to mass spectrometry has been developed. Aflatoxins were extracted with a mixture of methanol and water and then it was purified by solid-phase clean-up using a polymeric sorbent, not described previously, for the determination of these toxins. The eluted extract was injected into the chromatographic system using a reversed-phase C18 short column with an isocratic mobile phase composed of methanol-water (30:70). A single-quadruple mass spectrometry using an electrospray ionization source operating in the positive ion mode was used to detect aflatoxins due to derivatization presenting several disadvantages. Recoveries of the full analytical procedure were 110% for aflatoxin B1, 89% for aflatoxin B2, 81% for aflatoxin G1 and 77% for aflatoxin G2. Detection limit (S/N = 3) was 10 ng and quantification limit (S/N = 10) was 25 ng, calculated as amount in medicinal herb.     

Determination of nitrogen solubility in dilute pepsin hydrochloric acid solution of fishmeal: interlaboratory study

A revised method to determine solubility of nitrogen in dilute pepsin, using 0.0002% pepsin in place of 0.2% in AOAC Official Method 971.09, was tested in 16 laboratories with 12 samples of fishmeal. Results were calculated according to 2 procedures: AOAC Official Method 971.09 and a method described in 1964 by researchers at the Torry Research Station (Aberdeen, Scotland), and generally referred to as the modified Torry method. Variations in the method of shaking and source of pepsin were also investigated. Pepsin solubility values were lower and more variable when calculated by the Torry procedure. The method of shaking apparently affected the result when calculated according to the Torry but not the AOAC method. The source of pepsin had no significant effect on between-laboratory variability, but a comparison of the 2 main sources within one laboratory resulted in highly significant differences. Based on this study, the International Fishmeal and Fish Oil Organization has adopted this new method, using 0.0002% pepsin but keeping the AOAC method of calculation. The type of shaker and source of pepsin are recommended but are not mandatory. The repeatability and reproducibility limits of this new method are 1.6 and 3.3% units of solubility, respectively.     

Determination of docosahexaenoic acid and n-3 fatty acids in refined fish oils by 1H-NMR spectroscopy: IUPAC interlaboratory study

A high-resolution proton nuclear magnetic resonance (NMR) method for determining the concentration (mg/g) of docosahexaenoic acid (DHA), the molar proportion (mol%) of DHA, and the molar proportion of total n-3 fatty acids in fish oils was validated by an IUPAC interlaboratory study (the Commission VI-6 on Oils, Fats, and Derivatives WG 3/98). Thirteen laboratories from 5 countries tested 6 pairs of blind duplicate fish oils: a refined tuna oil, 2 extracted tuna oils, an extracted bonito oil, an extracted salmon oil, and an extracted sardine oil ranging from 9 to 30 mol% DHA and from 20 to 35 mol% n-3 fatty acids. Before 1 D-proton NMR measurements with 300-500 MHz instruments, oil samples were weighed and diluted with deuterochloroform solution containing ethylene glycol dimethyl ether as internal standard. To achieve precise performance, a detailed procedure for signal area measurement was described in the protocol, and all participants were instructed about the critical importance of following the protocol. Statistical performances with invalid and outlier data removed were as follows: repeatability relative standard deviations (RSDr) ranged from 0.91 to 2.62% and reproducibility relative standard deviation (RSDR) ranged from 1.73 to 4.27% for DHA concentration (mg/g); RSDr ranged from 0.39 to 2.06%, and RSDR ranged from 0.59 to 3.46% for mol% DHA; RSDr ranged from 0.23 to 0.90% and RSDR ranged from 0.85 to 2.01 % for mol% total n-3 fatty acids. The method is expected to be recommended by IUPAC.     

Photostimulated luminescence detection of irradiated herbs, spices, and seasonings: international interlaboratory trial

An interlaboratory trial was conducted to validate photostimulated luminescence (PSL) methods for herbs, spices, and seasonings. Forty products (11 herbs, 17 spices, and 12 seasonings) were purchased from a local commercial source, and randomly selected samples were irradiated with 10 kGy. Four blended products were prepared at Scottish Universities Research and Reactor Centre, mixing varying proportions of irradiated material with the untreated product. Precharacterization against a predefined threshold identified low sensitivity products (black and white peppers) and products with high natural signals (thyme, sage, parsley, and mixed herbs), both of which might be susceptible to misclassification. Precharacterization also revealed whether calibration was likely to resolve overlap between classification categories. Eight sets of screening data and 5 sets of calibrated data were returned by participants. Of the 840 samples sent, 1593 screening measurements and 788 calibrated measurements were received from 662 samples. In screening mode, participants reached definitive conclusions in 87% of cases, 99% of which were correct. Of the remaining 13%, calibration to identify low-sensitivity resolved 60% of cases. Overall, 94% of samples were correctly identified by either screening alone, or screening plus calibration; 6% remained unclassified and therefore required further investigation by thermoluminescence. The results confirm the validity of the PSL method for herbs, spices, seasonings, and blends, and emphasize the need for calibration to identify low-sensitivity samples. This method has now been adopted by the European Committee for Standardization (CEN) and the Codex Alimentarius Commission.     

Determination of vegetal proteins in milk powder by sodium dodecyl sulfate-capillary gel electrophoresis: interlaboratory study

An interlaboratory study, with the participation of 8 laboratories, was conducted to evaluate a sodium dodecyl sulfate-capillary gel electrophoresis method for determination of adulteration of milk powder with soy and pea proteins. Calibration standards (0-8%, w/w, soy and pea protein in total protein) and adulterated skim milk powders (0-5%, w/w, soy and pea proteins in total protein) were produced. Vegetal proteins were determined after removal of milk proteins by pretreatment of the samples with tetraborate-EDTA buffer, pH 8.3. Repeatability standard deviations ranged from 9 to 15% and reproducibility standard deviations ranged from 25 to 30% in the samples containing 5% vegetal protein in total protein.     

Determination of aflatoxin B1 by biomass derived porous carbon modified electrode with molecularly imprinted polymer

Aflatoxin determination is imperative among agricultural and food products as the pollution of the toxic aflatoxin could cause severe hazards towards animals and human beings. Here, we prepared Elaeagnus gum derived porous carbon material as electrode modified material. In situ electropolymerization of molecularly imprinted polymer utilizing phenol as monomer and aflatoxin B₁ as target template was further processed onto the porous carbon surface to achieve aflatoxin B₁ sensing ability. This sensitive MIP sensor has a detection range from 5 pM to 100 pM of aflatoxin B₁, an electrochemical sensitivity of 82.4 μA log(pM)⁻¹ cm⁻² with a detection limit of 1.7 pM, and a recovery rate of real sample measurement is 98.21 %. Good selectivity, fair repeatability and stability was confirmed. This work demonstrates the promising application of Elaeagnus gum derived porous carbon modified electrode sensor for food and drug monitoring.     

Qualitative method for determination of aflatoxin B1 in nuts

The proper characterization of a commercial qualitative method for determining aflatoxin B1 in some nuts is described. A qualitative method that provides binary responses of the yes/no type means that the performance parameters have been properly adapted and defined. Performance characteristics such as the cut-off limit, the detection limit, sensitivity, specificity, false-positive and negative rates, and the unreliability or uncertainty region are defined and then estimated by means of the performance characteristic curves. The commercial test kit showed the cut-off limit at 1.6 ng/g, with a sensitivity rate of 95% and a false-negative rate of zero. A modification can be performed to shift the cut-off to 2.0 ng/g, keeping the same values for the sensitivity and false-negative rate.     

固相萃取-高效液相色谱/串联质谱检测谷物中黄曲霉毒素B1、B2、G1、G2和M1

建立了高效液相色谱/串联质谱(LC-MS/MS)同时检测谷物中的黄曲霉毒素B1、B2、G1、G2和M1的方法.并优化了液相色谱条件和质谱的相关参数.谷物样品经研磨成粉末后,直接经甲醇-水( V∶V=10∶90)提取,Oasis HLB固相萃取净化,乙腈-水(0.2％甲酸)梯度洗脱,选择电喷雾离子源(ESI),正离子扫描...