Validation of a new enzyme-linked immunosorbent assay to detect the triggering proteins and peptides for celiac disease: interlaboratory study

The performance of Gluten-Tec (EuroProxima, Arnhem, The Netherlands) was tested through an interlaboratory study in accordance with AOAC guidelines. Gluten-Tec is a competitive ELISA that detects an immunostimulatory epitope of a-gliadin in dietary food for celiacs. Fifteen laboratories, representing 14 different countries, announced their interest in taking part in this study. Of the 12 laboratories that sent the results within the established timeframe, two submitted inappropriate standard curves and were excluded from the statistical analysis. Four different food matrixes (rice-based baby food, maize bread, chocolate cake mix, and beer) were selected for preparing the test samples. Two gliadin extraction procedures were used: the conventional 60% ethanol, and a new method based on the reducing reagent dithiothreitol. The 38 samples (19 blind duplicates) tested in this study were prepared by diluting the different extracts in order to cover a wide range of gliadin levels. Both sample extraction and dilution were performed by EuroProxima; the present interlaboratory study was focused only on testing the ELISA part of the Gluten-Tec kit protocol. Repeatability values (within-laboratory variance), expressed as RSD(r) ranged from 6.2 to 25.7%, while reproducibility values (interlaboratory variance), expressed as RSD(R), ranged from 10.6 to 45.9%. Both statistical parameters were in the acceptable range of ELISAs under these conditions, and the method will be presented to the Codex Alimentarius as a preferred method for gluten analysis.     

Determination of domoic acid toxins in shellfish by biosense ASP ELISA--a direct competitive enzyme-linked immunosorbent assay: collaborative study

A collaborative study was conducted on the Biosense amnesic shellfish poisoning (ASP) enzyme-linked immunosorbent assay (ELISA) for the determination of domoic acid (DA) toxins in shellfish in order to obtain interlaboratory validation data for the method. In addition, a method comparison study was performed to evaluate the ASP ELISA as an alternative to the current liquid chromatography (LC) reference method for DA determination. The study material comprised 16 shellfish samples, including blue mussels, Pacific oysters, and king scallops, spiked with contaminated mussel homogenates to contain 0.1-20 mg DA/kg shellfish flesh. The shellfish samples were extracted with 50% aqueous methanol, and the supernatants were directly analyzed. Sixteen participating laboratories in 10 countries reported data from the ASP ELISA, and 4 of these laboratories also reported data from instrumental LC analysis. The participating laboratories achieved interlaboratory precision estimates for the 8 Youden paired shellfish samples in the range of 10-20% for RSD(r) (mean 14.8 +/- 4%), and 13-29% for RSDR (mean 22.7 +/- 6%). The precision estimates for the ELISA data did not show a strong dependence on the DA concentration in the study samples, and the overall precision achieved was within the acceptable range of the Horwitz guideline with HorRat values ranging from 1.1 to 2.4 (mean HorRat 1.7 +/- 0.5). The analysis of shellfish samples spiked with certified reference material (CRM)-ASP-MUS-b gave recoveries in the range of 88-122%, with an average recovery of 104 +/- 10%. The estimate on method accuracy was supported by a correlation slope of 1.015 (R2 = 0.992) for the determined versus the expected DA values. Furthermore, the correlation of the ASP ELISA results with those for the instrumental LC analyses of the same sample extracts gave a correlation slope of 1.29 (R2 = 0.984). This indicates some overestimation of DA levels in shellfish by the ELISA, but it is also a result of apparent low recoveries for the LC methods. This interlaboratory study demonstrates that the ASP ELISA is suitable for the routine determination and monitoring of DA toxins in shellfish, and that it offers a rapid and cost-effective methodology with high sample throughput.     

Selective pressurized liquid extraction of polychlorinated dibenzo-p-dioxins, dibenzofurans and dioxin-like polychlorinated biphenyls from food and feed samples

Selective pressurized liquid extraction (PLE) of polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (dl-PCBs) from various food and feed samples was performed with a selective PLE method previously developed for bulk PCBs. The method utilizes sulfuric acid impregnated silica inside the extraction cell to oxidize coextracted fat. Extractions were performed at 100 degrees C with n-heptane for 5 min in two cycles. Data obtained by selective PLE combined with gas chromatography/high-resolution mass spectrometry (GC-HRMS) were compared to concentrations derived from reference laboratories applying conventional sample preparation and GC-HRMS. Experiments performed on spiked vegetable oil, naturally contaminated crude fish oil and oil containing compound feed samples showed good results for these relatively simple matrices. The accuracy was generally +/-20% as compared to spiked levels or to values obtained by the reference laboratories. The precision, measured as the relative standard deviation (RSD) for 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalency values (TEQs), was below 10% in all cases. The method was also tested on naturally contaminated herring tissue, chicken tissue, pork tissue and sepiolitic clay, which all caused some trouble. It was observed that sufficient amounts of sodium sulfate should be used for dehydration of tissue samples and additionally, the cells should not be packed too dense in order to avoid suppressed extraction efficiency. Once this was attended to, satisfactory data could be obtained, except for sepiolithic clay. This study demonstrates that selective PLE can be applied with success to a number of food and feed matrices in analysis of PCDD/Fs and dl-PCBs. Since the fat removal step is on-line, the selective PLE method will reduce time and solvent consumption for sample preparation as compared to traditional clean-up.     

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.     

Liquid chromatographic analysis of vitamin B6 in reconstituted infant formula: collaborative study

A liquid chromatographic (LC) method was validated for the determination of total vitamin B6 in infant formula. Total vitamin B6 was quantified by converting the phosphorylated and free vitamers into pyridoxine. Pyridoxine was determined by ion pair reversed-phase LC with fluorescence detection. The method was subjected to an AOAC collaborative study involving a factory-manufactured, milk- and soy-based infant formula. Each was spiked at 3 concentrations in the range of 0-1 microg/g and sent as blind duplicate to participant laboratories. Nine laboratories returned valid data which were statistically analyzed for outliers and precision parameters. The repeatability relative standard deviation (RSD(r)) ranges were 2.0-4.0 and 3.5-5.9% for fortified milk- and soy-based formulas, respectively. The reproducibility relative standard deviation (RSD(R)) ranges were 8.2-8.4 and 6.7-11.2% for fortified milk- and soy-based formulas, respectively. HORRAT values ranged from 0.42 to 0.53, indicating that the precision of the method is acceptable. The mean RSD(r):RSD(R) values were 0.60 and 0.55 for milk- and soy-based formulas, respectively. As expected, RSDs for the unfortified samples were higher, but their HORRAT values (0.81 and 2.06) helped define a realistic limit of quantitation as 0.05 microg/g. Recovery data were quantitative and varied between 81.4 and 98.0% (mean = 89.8%) for each of 6 spiked materials.     

Gluten determination by gliadin enzyme-linked immunosorbent assay kit: interlaboratory study

An interlaboratory study with 10 participants was performed to obtain validation and performance data for an enzyme-linked immunosorbent assay (ELISA) kit developed for quantitative gluten determination in foods. The ELISA kit used for this study is based on 2 monoclonal and 1 polyclonal antibody developed by Immunotech, a Beckman Coulter Co. This kit did not show any false positive results or cross-reactivity with oat, rice, maize, and buckwheat. The gliadin standard from the Working Group on Prolamin Analysis and Toxicity was included in the kit as reference material for calibration. All participants obtained a gliadin ELISA kit with Standard Operational Procedure and a form for recording test results. The study included 13 samples labeled as "gluten-free" and 2 samples spiked by wheat flour. Seven samples had gliadin content below the limit of quantitation (LOQ) of the method, and 1 sample exceeded the highest calibration level. Gliadin content in the range from 10 to 157 mg/kg (1st day) and from 11 to 183 mg/kg (2nd day) was found in 7 samples (including 2 spiked samples). Results of these samples were used for further statistical analysis and evaluation. The Cochran, Dixon, and Mandel statistical tests were applied for detection of outliers. The LOQ of the kit was estimated.     

Determination of glucosamine in raw materials and dietary supplements containing glucosamine sulfate and/or glucosamine hydrochloride by high-performance liquid chromatography with FMOC-Su derivatization: collaborative study

A collaborative study was conducted for determination of glucosamine in raw materials and dietary supplements containing glucosamine sulfate and/or glucosamine hydrochloride by high-performance liquid chromatography (HPLC) with N-(9-fluorenyl-methoxycarbonyloxy) succinimide (FMOC-Su) derivatization. Thirteen blind materials, one pair of which were duplicates, were tested by 12 collaborating laboratories. The test samples consisted of various commercial products, including tablets, capsules, drink mix, and liquids as well as raw materials, blanks, and those for spike recovery analyses. The tests with blank products and products spiked with glucosamine showed good specificity of the method. The average recoveries at spike levels of 100 and 150% of the declared amount were 99.0% with a relative standard deviation (RSD) of 2.1%, and 101% with an RSD of 2.3%, respectively. The test results between laboratories on each commercial product were reproducible with RSD values of no more than 4.0%, and the results were repeatable in the same laboratory with an average RSD of 0.7%. HorRat values ranged from 0.5 to 1.7 on both tests of spike recovery and reproducibility between laboratories on commercial products. The average determination coefficient of the calibration curves from the laboratories was 0.9995 with an RSD of 0.03%. All of the 12 collaborating laboratories succeeded in the study and none of their reported test results were outliers, partly indicating the robustness of the method. It is recommended that the method be accepted by AOAC INTERNATIONAL as Official First Action.     

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.     

Proficiency testing of eight French laboratories in using the AOAC mouse bioassay for paralytic shellfish poisoning: interlaboratory collaborative study

In an interlaboratory study, 8 French laboratories were tested for their proficiency in using the AOAC mouse bioassay for paralytic shellfish poisoning (PSP). Each laboratory received 1 saxitoxin (STX) standard solution, 1 STX acidified water solution for determination of the titer, 1 noncontaminated shellfish sample, 1 naturally contaminated shellfish sample, and 2 shellfish samples spiked, respectively, at low (152.8 microg STX/100 g meat) and moderate (334.7 microg STX/100 g meat) levels. All samples were analyzed in duplicate. Mean recoveries were 35.1% for the low level and 46.6% for the moderate level. Relative standard deviations (RSD) for within-laboratory variations (repeatability) ranged from 5.4 to 9.8%; RSD for between-laboratory variations (reproducibility) varied from 7.8 to 39.6%, depending on STX level. On the basis of overall performance, all 8 participating laboratories were proficient in their use of the AOAC mouse bioassay.     

Inter-laboratory comparison of liquid chromatographic techniques and enzyme-linked immunosorbent assay for the determination of surfactants in wastewaters

Seven laboratories participated in an inter-laboratory comparison exercise within the framework of the PRISTINE, SANDRINE and INEXsPORT European Union Projects. Solid-phase extraction (SPE) methodologies were used for the extraction of target analytes from wastewaters. The analytical strategies were based on liquid chromatography (LC) coupled to mass spectrometric (MS) or to fluorescent (FL) detection in all cases with the exception of one laboratory using a test-tube enzyme-linked immunosorbent assay kit. Samples were spiked with the surfactants nonylphenolpolyglycol ether, coconut diethanolamide, linear alkylbenzene sulfonate, nonylphenolpolyglycol ether sulfate, alkylpolyglycol ether and secondary alkane sulfonate. After enrichment on previously conditioned SPE cartridges, the SPE cartridges were distributed among the participating laboratories without the information about the amount of spiked surfactants. In addition, SPE cartridges loaded with a real-world environmental sample containing a tannery wastewater were also analyzed. The results of the programme showed that SPE followed by LC-MS techniques are reliable for the surfactants determination at submicrogram to microgram per liter levels in wastewaters. Inter-laboratory precision values were calculated as the reproducibility relative standard deviation (RSD(R)) which was determined from the reproducibility standard deviation (sR) and the average concentration at a particular concentration level. When data from all laboratories were pooled, the RSD(R) values ranged from 5.1 to 28.3% for the determination of target analytes. The most accurate result corresponded to that given for linear alkylbenzene sulfonates. Taking into account that different methodologies were used (including non-chromatographic techniques) and the complexity of the samples analyzed, it can be considered that acceptable reproducibility values were obtained in this inter-laboratory study.     

Routine analysis of vinicultural relevant fungicides, insecticides and herbicides in soil samples using enhanced solvent extraction (ESE)

Parameters affecting the extraction efficiency of various pesticides from a native contaminated soil sample (C_org = 4.4%) using an enhanced solvent extraction (ESE) technique were investigated. The defined settings of temperature (50/150?°C), pressure (180/240 MPa), static and dynamic extraction time (5/15 min and 0/5 min, respectively) yielded results which did not differ significantly (RSD = 5.9–11.8%). In comparison to a classical shake-out extraction method the yielded quantities were on average 14% higher using ESE. The established method achieved a high precision (RSD = 2.8–9.1%) for the pesticides extracted from native contaminated soil samples. A significant influence of the sample matrix on accuracy was not observed. The RSDs of thirteen pesticides extracted from spiked sea sand varied in a similar range from 1.7 to 9.8% and the recoveries were between 83 and 112%. The method has been applied to soil samples from vineyards routinely.     

WEFTA interlaboratory comparison on total lipid determination in fishery products using the Smedes method

Lipid determination by the Smedes method was tested in an interlaboratory trial performed by nine laboratories from seven countries belonging to the West European Fish Technologists Association Analytical Methods Working Group. Five samples of fish and fishery products with different lipid contents, including two blind duplicates, were distributed among the participants. All laboratories applied a slightly modified Smedes method, which included extraction of lipids by cyclohexane and isopropanol, transfer of lipids to the cyclohexane phase by addition of water, phase separation by centrifugation, and gravimetric lipid determination. The results indicate that the RSD for reproducibility (RSD(R)) was between 4.11 and 6.31% for samples with moderate (7%) and high (14%) lipid content, depending on the sample. Larger SDs among the laboratories were obtained for a cod sample with low lipid content of 0.5%. The method is judged to be suitable as a routine method for lipid determination in fish and fishery products.     

Immunoreactivity and detection of wheat proteins by commercial ELISA kits

Wheat proteins are responsible for sensitivities, including baker's asthma, immunoglobulin E (IgE)-mediated allergic reaction, wheat-dependent, exercise-induced anaphylaxis, and celiac disease. The detection of gluten/wheat traces in foods is important to safeguard the health of wheat-sensitive individuals and comply with food labeling. Many immunoanalytical-based commercial kits are available for the quantification of gliadin/gluten/wheat proteins. We compared the immunoreactivity of wheat fractions with wheat-allergic human serum IgE and antibody conjugates used in six commercial immunoassay kits. Moreover, the performance of the kits was tested using corn flour spiked with gluten (5, 10, 25, and 50 ppm) and wheat flour (50, 100, 250, and 500 ppm). The albumin, globulin, gliadin, and glutenin fractions reacted with IgE from nine, eight, two, and eight patients' sera, respectively, out of nine wheat allergic patients tested. Among the antibodies from commercial kits, those from R-Biopharm, Morinaga, and Romer Labs reacted strongly with the gliadin fraction, whereas those from BioKits, ALLER-TEK, and ELISA Systems reacted strongly with the glutenin fraction. All kits showed minimal or no reactivity with albumin and globulin fractions. All kits detected the gluten and wheat flour in a corn flour matrix at the lowest spiked levels of 5 and 50 ppm, respectively. However, there was wide variation among the kits when comparing the recovery of gluten and wheat flour. The recovery was also dependent on the source material (gluten or wheat flour) used for spiking the corn flour matrix.     

Stir bar sorptive extraction for the analysis of short‐chain chlorinated paraffins in water

An optimised method using stir bar sorptive extraction (SBSE) and a thermal desorption‐GC‐electron capture detector (GC‐ECD) for the determination of short‐chain chlorinated paraffins from water samples was developed. Recoveries near to 100% were obtained by using 20 mm×0.5 mm (length×film thickness) PDMS commercial stir bars from 200 mL spiked water samples and 20% methanol addition with an extraction period of 24 h. Method sensitivity, linearity and precision were evaluated for surface water and wastewater spiked samples. A LOD of 0.03 and 0.04 μg/L was calculated for surface and wastewater, respectively. The precision of the method given as an RSD was below 20% for both matrices. The developed method was applied for the analysis of two real samples from a contaminated river and a wastewater treatment plant. Results were in accordance with those obtained using a previously developed method based on solid phase microextraction (SPME).     

Routine analysis of vinicultural relevant fungicides, insecticides and herbicides in soil samples using enhanced solvent extraction (ESE)

Parameters affecting the extraction efficiency of various pesticides from a native contaminated soil sample (C_org = 4.4%) using an enhanced solvent extraction (ESE) technique were investigated. The defined settings of temperature (50/150 °C), pressure (180/240 MPa), static and dynamic extraction time (5/15 min and 0/5 min, respectively) yielded results which did not differ significantly (RSD = 5.9–11.8%). In comparison to a classical shake-out extraction method the yielded quantities were on average 14% higher using ESE. The established method achieved a high precision (RSD = 2.8–9.1%) for the pesticides extracted from native contaminated soil samples. A significant influence of the sample matrix on accuracy was not observed. The RSDs of thirteen pesticides extracted from spiked sea sand varied in a similar range from 1.7 to 9.8% and the recoveries were between 83 and 112%. The method has been applied to soil samples from vineyards routinely. Received: 28 December 1998 / Revised: 8 March 1999 / Accepted: 11 March 1999     

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.     

Performance Tested Method multiple laboratory validation study of ELISA-based assays for the detection of peanuts in food

Performance Tested Method multiple laboratory validations for the detection of peanut protein in 4 different food matrixes were conducted under the auspices of the AOAC Research Institute. In this blind study, 3 commercially available ELISA test kits were validated: Neogen Veratox for Peanut, R-Biopharm RIDASCREEN FAST Peanut, and Tepnel BioKits for Peanut Assay. The food matrixes used were breakfast cereal, cookies, ice cream, and milk chocolate spiked at 0 and 5 ppm peanut. Analyses of the samples were conducted by laboratories representing industry and international and U.S governmental agencies. All 3 commercial test kits successfully identified spiked and peanut-free samples. The validation study required 60 analyses on test samples at the target level 5 microg peanut/g food and 60 analyses at a peanut-free level, which was designed to ensure that the lower 95% confidence limit for the sensitivity and specificity would not be <90%. The probability that a test sample contains an allergen given a prevalence rate of 5% and a positive test result using a single test kit analysis with 95% sensitivity and 95% specificity, which was demonstrated for these test kits, would be 50%. When 2 test kits are run simultaneously on all samples, the probability becomes 95%. It is therefore recommended that all field samples be analyzed with at least 2 of the validated kits.     

Determination of aromatic hydrocarbons in asphalt release agents using headspace solid-phase microextraction and gas chromatography-mass spectrometry

The possibility of quantitative analysis of aromatic hydrocarbons in oil-based asphalt release agents was investigated using headspace solid-phase microextraction (HS-SPME) followed by gas chromatography-mass spectrometry (GC-MS). The target analytes studied were benzene, toluene, ethylbenzene, p-, m-, and o-xylene (BTEX) and 1,3,5-trimethylbenzene and 1,2,4-trimethylbenzene. Experimental parameters influencing HS-SPME efficiency were studied (equilibration time between sample and headspace and between headspace and SPME fiber, sample amount and sample matrice effects). A HS-SPME method using hexadecane as a surrogate matrice was developed. The detection limit was estimated as 0.03-0.08 ppm (w/w) for the target analytes investigated. Good linearity was observed (R2 > 0.999) for all calibration curves at high, medium and low concentration level. The repeatability of the method (RSD, relative standard deviation) was found to be less than 10% (generally less than 5%) in triplicate samples and approximately 2% at eight consecutive tests on one and the same sample. The accuracy of the method given by recovery of spiked samples was between 85 and 106% (generally between 95 and 105%). The HS-SPME method developed was applied to four commercially available asphalt release agents. External calibration and standard addition approaches were investigated regarding accuracy. The results showed that standard addition generates higher accuracy than external calibration. The contents of target aromatic hydrocarbons in the asphalt release agents studied varied greatly from approximately 0.1-700 ppm. The method described looks promising, and could be a valuable tool for determination of aromatic hydrocarbons in different types of organic matrices.     

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 fat in raw and processed milks by the Gerber method: collaborative study

The Gerber method is used worldwide as a simple and rapid method for determining fat in raw and processed milks. However, the volume of the test portion used in the method has not been internationally agreed upon. A collaborative study was conducted to evaluate performance of the Gerber method using either a weighed test portion (11.13 g) or by a 10.77 mL test portion delivered by pipet. For each method, laboratories received 10 test samples: 5 raw and 5 pasteurized homogenized milks, 2 of which were blind duplicate pairs. Eleven and 10 laboratories participated in the evaluation of aliquot addition by weight and pipet, respectively. Mojonnier ether extraction (Method 989.05) was used as the reference method. Interlaboratory study statistics were similar between methods of test portion addition and between raw and processed materials; therefore, summary interlaboratory study statistics were pooled. The fat content of milk samples ranged from 0.96 to 5.48%. Absolute reproducibility and repeatability were not affected by fat level, and pooled statistical performance (invalid and outlier data removed) was (g fat/100 g milk) s(r) = 0.026, s(R) = 0.047, r = 0.074, and R = 0.132. Relative standard deviations increased with decreasing fat content, and were summarized by fat level: 1-2% fat milk, mean = 1.437, RSD(r) = 1.809%, RSD(R) = 3.271%; 2-6% fat milk, mean = 4.156, RSD(r) = 0.626%, RSD(R) = 1.131%. Compared with ether extraction, test results by the Gerber method were slightly lower (0.02% fat) using a weighed test portion and significantly lower (0.06% fat) using a 10.77 mL volume addition by pipet. A trend toward underestimating fat content at lower fat concentrations (1-2% fat) was observed with the weighed test portion but not when a pipet was used. The Associate Referee recommends that the Gerber method using a weighed test portion be adopted as First Action with applicability limited to whole milk.