Development of a simplified densitometer for the determination of aflatoxins by thin-layer chromatography.

A simple, miniaturised and low power consuming (battery, fully semiconductor based) detector cell (SeBaDeC) was developed for the densitometric measurement of aflatoxins on TLC plates. A UV-light emitting diode (UV-LED) with a peak emission wavelength of 370 nm was used for fluorescence excitation, while a photo diode with a peak sensitivity of 440 nm in combination with a 418 nm cut-off filter was applied for detecting the fluorescence intensity. The resulting signal was further amplified by means of a commonly used operational amplifier integrated circuit (OA) and directly converted into a digital signal with a simple analogue-digital-converter (ADC). This signal was recorded at the serial (RS232) port of a portable PC and processed with a spreadsheet program. The software used for data recording is freeware and available in its source code, and the long lifetime of the UV-LED (up to 10 000 h) permits a maintenance free application of this device. This simplified device has shown to be able to detect concentrations of aflatoxins of 1 ng, thus offering a cheap and sensitive alternative to currently available TCL scanners.

Immunoaffinity column clean-up prior to thin-layer chromatography for the determination of aflatoxins in various food matrices.

A one-dimensional TLC method to determine aflatoxins (B1, B2, G1, G2) in various food matrices was elaborated which abstains fully on the use of chlorinated solvents. It implements an immunoaffinity clean-up step after extraction with methanol. The aflatoxins were quantified by densitometry. The method has shown to be rapid and efficient. In-house performance characteristics were established. The limit of quantification was found to be significantly lower than current regulatory limits for aflatoxin control outside and within the European Community. The obtained recovery and precision data gave a strong indication, that the method is likely to give satisfactory performance if tested in a future collaborative trial.

Analysis of procyanidins in chocolate by reversed-phase high-performance liquid chromatography with electrospray ionisation mass spectrometric and tandem mass spectrometric detection.

The analysis of procyanidins in crude chocolate extracts by reversed-phase high-performance liquid chromatography (HPLC) with electrospray ionisation mass spectrometry (MS) is described in this report. Catechin monomers and procyanidin oligomers (dimers to hexamers) were identified according to the mass of the single charged pseudomolecular ion ([M-H]-). Identification was further confirmed by collision-induced dissociation MS-MS analysis, which in addition, permitted the identification of double charged pentameric, hexameric, and heptameric ions. This study demonstrates the capability of the combination of HPLC and modern ion trap mass analysers to significantly reduce sample preparation and analysis time in combination with high specificity and structural information for compound identification.

Supercritical fluid extraction for liquid chromatographic determination of carotenoids in Spirulina Pacifica algae: a chemometric approach.

An experimental design procedure was used to investigate the effects of some operating parameters on the supercritical fluid extraction of carotenoids beta-carotene, beta-cryptoxanthin and zeaxanthin from Spirulina Pacifica algae, a carotenoid-rich dietary product. Variables tested were temperature and pressure of the supercritical fluid, dynamic extraction time and percentage of ethanol added as the modifier. Each variable was tested at three levels; 31 experiments were performed in random order. Analyses of the extracts were performed by high-performance liquid chromatography with UV-Vis photodiode array detection. Analytical responses (chromatographic peak areas) were processed by using a stepwise multiple regression analysis, in order to find polynomial functions describing the relationships between variables and responses. For all the analytes the experimental conditions providing the highest extraction yield inside the experimental domain considered were found. Supercritical fluid extraction results obtained in these conditions were compared with those obtained by performing solvent extraction in order to evaluate the effectiveness of the supercritical fluid extraction procedure.

Trichothecenes: reference materials and method validation.

The frequent contamination of food and feed with trichothecene mycotoxins, the high consumption of these products, and the potential risk associated herewith, has led to an increasing public awareness and therefore to the establishment of measures to control trichothecene contamination. The analytical difficulty and the economic importance of controlling trichothecenes in food and feed support the need for certified reference materials (CRMs) and validated methods. They form invaluable tools to ensure comparability and traceability in analytical measurements and are very useful for the implementation of written standards, legislation/regulations and laboratory accreditation. The present paper provides an overview of previous work, current strategies and prospectives for the production of CRMs and validation of analytical methods in the field of trichothecene analysis. Additional information is given on methodological demands, normative frameworks and commonly accepted procedures.

Novel sampling methods for the analysis of mycotoxins and the combination with spectroscopic methods for the rapid evaluation of deoxynivalenol contamination.

A novel non-destructive sampling approach is described for the identification of food matrices contaminated with deoxynivalenol and other mycotoxins. This technique is different from currently applied sampling procedures for this purpose and is based on the principle that surface material from the tested goods is collected on a filter and brought to chemical or spectroscopic analysis. This approach has been applied to several matrices and mycotoxins, with a focus on those mycotoxin-matrix combinations that are of main relevance due to current or future legislation. Tests were carried out with a facility that has been shown to be suitable to process large quantities of materials at points of transaction, such as harbour. Further experiments with a small sampling lance prototype showed that analytical results from the chemical analysis of the tested goods can be correlated with the results obtained with this novel sampling procedure.

Biphenyl and fluorinated derivatives: liver enzyme-mediated mutagenicity detected in Salmonella typhimurium and Chinese hamster V79 cells.

Hepatocarcinogenic polychlorinated and polybrominated biphenyls usually show negative results in in vitro mutagenicity assays. Problems in their testing result from their low water solubility and their slow rate of metabolism. We therefore investigated better soluble model compounds, namely biphenyl and its 3 possible monofluorinated derivatives. In the direct test, these compounds proved to be nonmutagenic in Salmonella typhimurium TA98 and TA100 (reversion to histidine prototrophy) and in Chinese hamster V79 cells (acquisition of resistance to 6-thioguanine). However, when the exposure was carried out in the presence of NADPH-fortified postmitochondrial fraction of liver homogenate from Aroclor 1254-treated rats, all 4 compounds showed mutagenic activity in V79 cells. 3-Fluorobiphenyl produced strong mutagenic effects in S. typhimurium TA100 as well, whereas the other biphenyls were inactive. In strain TA98, 3- and 4-fluorobiphenyl showed mutagenic activity. This mutagenicity was enhanced in the presence of 1,1,1-trichloropropene 2,3-oxide, an inhibitor of microsomal epoxide hydrolase, thus suggesting that epoxides may be active metabolites.

Comparison of HPLC and GLC techniques for the determination of the triglyceride profile of cocoa butter.

Current methods for the authentication of cocoa butter (CB) are mainly based on a knowledge of its triglyceride (TG) composition. The performances of capillary GLC and nonaqueous HPLC with an evaporative light-scattering detector (ELSD) for the quantification of TG of CB of different geographical origins were compared. Use of capillary columns coated with a polarizable stationary phase or two reversed-phase HPLC columns coupled in series efficiently separated the major TG species contained in CB. The velocity of the GLC carrier gas influenced the FID response factors of TG standard compounds, which were linearly related to the retention times of the analytes studied. Within a certain mass range the ELSD response of standard TG solutions did not deviate from unity to a greater extent, independent of the molecular structure of the TG species. The quantities of individual TG as obtained by both methods were in close agreement, and the precisions of the methods were also of comparable magnitude, so that either method can be applied to assess the purity of CB. Capillary GLC has the advantage of higher sample throughput due to a shorter run time and because the consumption of chemicals is negligible.

Development of a rapid method for the detection of cocoa butter equivalents in mixtures with cocoa butter.

A simple and rapid gas chromatographic (GC) method was developed for the detection of cocoa butter equivalents (CBEs) in cocoa buffer (CB). It is based on the use of a 5 m nonpolar capillary column for the separation of the main triglycerides of CB according to their acyl/carbon numbers. The GC procedure was optimized to avoid thermal degradation of the triglycerides. By computing the ratio C54/C50 and (C54/C50) x C52 and by 2-dimensional plotting of these values, authentic CB samples were clearly distinguished from samples containing various CBEs. The detection of little as 1% CBE in CB (corresponding to about 0.3% CBE in chocolate) in a model system was shown to be possible. Under real conditions, for a wide range of CBs, about 2.5% CBEs in CB were detected. With this method, quantitation was possible at a concentration of 5% CBEs in CB mixtures, which corresponds to around 1% in chocolate; this value is far below the maximum level of 5% CBEs allowed to be added to chocolate.

Validation of an immunoassay for detection and quantitation of a genetically modified soybean in food and food fractions using reference materials: interlaboratory study.

An immunoassay for detection of a specific genetically modified soybean (Roundup-Ready) was validated on dried soybean powder in an interlaboratory study. Different percentages of genetically modified soybeans in nonmodified soybean matrix were evaluated in a blind study. Thirty-eight laboratories from 13 countries participated. The immunoassay was evaluated for 2 endpoints: (1) To give a semiquantitative result, i.e., determination of a given sample above or below a given threshold, or (2) to compute a quantitative result, i.e., percentage of genetically modified soybeans in the sample. Semiquantitative results showed that a given sample which contained <2% genetically modified soybeans was identified as below 2% with a 99% confidence level. Quantitative use of the assay resulted in a repeatability (r) and reproducibility (R) that were computed to be RSDr = 7% and RSDR = 10%, respectively, for a sample containing 2% genetically modified soybeans. Application of this method depends on availability of appropriate reference materials for a specific food matrix. Only matrix-matched reference materials can be used for analysis of food or food fractions.

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 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

Post process product control of rendering plant sterilization conditions by ELISA.

Experiments were conducted in a laboratory autoclave and in a commercial rendering plant to set a limit for the R-value of an enzyme-linked immunosorbent assay (ELISA) to prove that animal meal had been sufficiently heat-treated. The immunoassay method is currently the only technique used to check for appropriate sterilization of animal meal. The results of these experiments were statistically assessed using operation characteristic curves and confirmed that insufficiently heat-treated animal meal has an R-value > 2. Results of the trials also demonstrated that the main parameters of sterilization, temperature, and duration strongy influence the R-value, thus indicating that deviation from the target sterilization conditions is verifiable by the ELISA method.

IUPAC collaborative trial study of a method to detect genetically modified soy beans and maize in dried powder.

This paper presents results of a collaborative trial study (IUPAC project No. 650/93/97) involving 29 laboratories in 13 countries applying a method for detecting genetically modified organisms (GMOs) in food. The method is based on using the polymerase chain reaction to determine the 35S promotor and the NOS terminator for detection of GMOs. reference materials were produced that were derived from genetically modified soy beans and maize. Correct identification of samples containing 2% GMOs is achievable for both soy beans and maize. For samples containing 0.5% genetically modified soy beans, analysis of the 35S promotor resulted also in a 100% correct classification. However, 3 false-negative results (out of 105 samples analyzed) were reported for analysis of the NOS terminator, which is due to the lower sensitivity of this method. Because of the bigger genomic DNA of maize, the probability of encountering false-negative results for samples containing 0.5% GMOs is greater for maize than for soy beans. For blank samples (0% GMO), only 2 false-positive results for soy beans and one for maize were reported. These results appeared as very weak signals and were most probably due to contamination of laboratory equipment.

Determination of an appropriate heat treatment of animal waste using the ELISA technique: results of a validation study.

Effective sterilisation of animal waste is an important prerequisite for the use of animal meal as an ingredient of compound feed for non-ruminants. Specific conditions for the rendering process aimed at the inactivation of the causative agent of Bovine Spongiform Encephalopathy (BSE), and the prevention of other pathogens in feedingstuff, are defined by European law. A validation study encompassing 21 laboratories from 12 European countries was performed using a commercially available test kit based on an enzymelinked immuno sorbent assay (ELISA) which allows proof of the appropriate heat treatment of the processed animal waste. The evaluation of the results regarding the R-value reveals the robustness of the method, a sufficient sensitivity to detect a deviation of the processing conditions from the regulation and a low standard deviation of the data. Moreover the statistical evaluation of the results allows for the estimation of the coefficient of variation which is between 11 and 22% depending on the magnitude of the response of the test.

Detection of nanomaterials in food and consumer products: bridging the gap from legislation to enforcement.

This paper describes the requirements and resulting challenges for the implementation of current and upcoming European Union legislation referring to the use of nanomaterials in food, cosmetics and other consumer products. The European Commission has recently adopted a recommendation for the definition of nanomaterials. There is now an urgent need for appropriate and fit-for-purpose analytical methods in order to identify nanomaterials properly according to this definition and to assess whether or not a product contains nanomaterials. Considering the lack of such methods to date, this paper elaborates on the challenges of the legislative framework and the type of methods needed, not only to facilitate implementation of labelling requirements, but also to ensure the safety of products coming to the market. Considering the many challenges in the analytical process itself, such as interaction of nanoparticles with matrix constituents, potential agglomeration and aggregation due to matrix environment, broad variety of matrices, etc., there is a need for integrated analytical approaches, not only for sample preparation (e.g. separation from matrix), but also for the actual characterisation. Furthermore, there is an urgent need for quality assurance tools such as validated methods and (certified) reference materials, including materials containing nanoparticles in a realistic matrix (food products, cosmetics, etc.).

European Union database of acrylamide levels in food: update and critical review of data collection.

The European Commission's Directorate General Joint Research Centre has been collecting data produced by European Union Member States on the acrylamide content of food since 2003. More than 9000 individual data points have been received from official food control laboratories directly or via their Competent Authorities, and from the food industry. Before being entered into the database, the data were assessed for their reliability. This paper presents an update of the database as well as results of the evaluation of data for selected food commodities in order to establish a trend concerning the content of acrylamide in food. Experience gained with the data collection and data assessment are described and recommendations for future data collection initiatives given.

Determination of dioxins (PCDDs/PCDFs) and PCBs in food and feed using the DR CALUX bioassay: results of an international validation study.

Maximum levels for dioxins in food and feedstuffs have been recently established by the European Commission through two regulations. Dioxin-monitoring programmes of food and feedstuffs will therefore be undertaken by the European Member States to implement these regulations, which would be facilitated by fast and low-cost screening methods. Commission Directives 2002/70/EC and 2002/69/EC describe specific characteristics for such screening methods. In the present study, the performance characteristics of the DR CALUX method from BioDetection Systems were established in a validation study with 14 participants. The study was based on two materials (fish oil and feed), each containing four different levels of dioxins and dioxin-like PCBs around the current limits. The results demonstrate that the test is very promising but that in particular the clean-up procedure was a source of variation and requires further optimization and standardization. In addition the quantification is improved by the use of control samples to correct for background contamination, recovery and differences between the TEF values and REP (relative potency) factors in the test.

Inter-laboratory validation study of five commercial ELISA test kits for the determination of peanut proteins in biscuits and dark chocolate.

The results of an inter-laboratory study with five commercially available peanut ELISA test kits to detect and quantify peanut residues in two food matrices (biscuit and dark chocolate) at four different concentrations (0-10 mg peanut kg(-1) matrix corresponding to about 0-2.5 mg peanut protein kg(-1) matrix) are reported. In general the five ELISA test kits evaluated could detect peanut protein in the two food matrices. In three cases, the study challenged the test kits beyond their intended use for quantification below the manufacturers' defined cut-off limits. Generally, all five ELISA test kits performed well in the concentration range 5-10 mg kg(-1) rather than in the low concentration range (2.0 or 2.5 mg kg(-1)). The variation in the found recoveries of peanut between the different test kits had a spread of 44-191% across all concentrations. The quantification characteristics between test kits differed significantly at the very low mg kg(-1) level. Two test kits performed well even at concentrations below 5 mg kg(-1) with reproducibilities of 27-36% for biscuits and 45-57% for chocolate.