A critical review of the specifications and performance of antibody and DNA-based methods for detection and quantification of allergens in foods.

Despite the availability of a large number of antibody and DNA based methods for detection and quantification of allergens in food there remain significant difficulties in selecting the optimum technique to employ. Published methods from research groups mostly contain sufficient detail concerning target antigen, calibration procedures and method performance to allow replication by others. However, routine allergen testing by the food industry relies upon commercialised test kits and frequently the suppliers provide disappointingly little specification detail on the grounds that this is proprietary information. In this review we have made a critical assessment of the published literature describing the performance of both commercial and non-commercial test kits for food allergens over the period 2008-2018. Mass spectrometric methods, which have the potential to become reference methods for allergens, are not covered in this review. Available information on the specifications of commercial ELISA and LFD test kits are tabulated for milk, egg and peanut allergens, where possible linking to publications concerning collaborative studies and proficiency testing. For a number of commercial PCR test kits, specifications provided by manufacturers for detection of a small selection of allergen are tabulated. In conclusion we support the views of others of the critical need for allergen reference materials as the way forward to improve the comparability of different testing strategies in foods.

Future perspectives in Orbitrap™-high-resolution mass spectrometry in food analysis: a review.

A literature search from 2007 to 2014 was conducted to identify publications where principally LC-Orbitrap™-high-resolution mass spectrometry (HRMS) has been employed in food analysis. Of a total of 212 relevant references, only 22 papers were from 2007-10, but in subsequent years there has been a steady growth in publications with 38-55 relevant papers being published each year from 2011 to 2014. In the food safety area, over 50% of the published papers were equally divided between pesticides, veterinary drug residues and natural toxins (including mycotoxins) focused primarily on multi-analyte target analysis. LC-Orbitrap-HRMS was also found to be increasingly important for the analysis of bioactive substances, principally phenolic compounds in foods. A number of studies reported for the first time the identification of new fungal metabolites, predominantly various conjugated forms of known mycotoxins. Novel process contaminants were also identified by LC-Orbitrap-HRMS, as were various substances used for food adulteration and bioactive substances in herbal products and dietary supplements. Untargeted analysis is seen as a major future trend where HRMS plays a significant role. Retrospective analysis of scanned high-resolution mass spectra in conjunction with relevant databases can provide new insights. Metabolomics is also being increasingly used where foods are being profiled through fingerprinting using HRMS. All evidence points towards future growth in the number of applications of HRMS in food safety and quality, as the power of this technique gains wider recognition.

A survey of the use of soy in processed Turkish meat products and detection of genetic modification.

To screen for possible illegal use of soybeans in meat products, the performance characteristics of a commercial polymer chain reaction (PCR) kit for detection of soybean DNA in raw and cooked meat products were established. Minced chicken and beef products containing soybean at levels from 0.1% to 10.0% were analysed by real-time PCR to amplify the soybean lectin gene. The PCR method could reliably detect the addition of soybean at a level of 0.1%. A survey of 38 Turkish processed meat products found only six samples to be negative for the presence of soybean. In 32 (84%) positive samples, 13 (34%) contained levels of soy above 0.1%. Of soybean positive samples, further DNA analysis was conducted by real-time PCR to detect whether genetically modified (GM) soybean had been used. Of 32 meat samples containing soybean, two samples were positive for GM modification.

Meat species identification and Halal authentication using PCR analysis of raw and cooked traditional Turkish foods.

The method performance characteristics of commercially available PCR kits for animal species identification were established. Comminuted meat products containing different levels of pork were prepared from authentic beef, chicken, and turkey. These meat products were analysed in the raw state and after cooking for 20 min at 200 °C. For both raw and cooked meats, the PCR kit could correctly identify the animal species and could reliably detect the addition of pork at a level below 0.1%. A survey of 42 Turkish processed meat products such as soudjouk, salami, sausage, meatball, cured spiced beef and doner kebap was conducted. Thirty-six samples were negative for the presence of pork (<0.1%) and four were found to be correctly labelled as containing pork. However, one sausage sample was labelled as containing 5% beef, but beef DNA was not detected and a meatball sample labelled as 100% beef was found to contain chicken. Another turkey meatball sample was predominantly chicken.

Sorbate and benzoate in Turkish retail foodstuffs.

From 2008 to 2011, surveys were conducted to determine the levels of benzoic and sorbic acids and their respective salts in 983 retail food samples which included sauces, vegetable and fruit preparations, flavoured syrups, food supplements, cereals, bakery products, jelly, synthetic cream, sprays, mustards, jam and preserves, molasses, chewing gum, confectionery, non-alcoholic beverages, tea, wine, vinegar, brine and beers. The analysis involved methanol extraction of the foodstuff and direct determination by HPLC with UV detection. Quality assurance was employed with each batch of samples. Accuracy was ensured through regular participation in proficiency tests. Over this four-year period, a total of 23 samples (2.3%), some syrups, tomato sauces and fruit contained individual or combined levels of sorbic and benzoic acids above regulatory limits. Unauthorised use of benzoic acid was also detected in a syrup sample, bakery products and fruit preserves.

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.

Analysis of deoxynivalenol, zearalenone, T-2, and HT-2 toxins in animal feed by LC/MS/MS--a critical comparison of immunoaffinity column cleanup with no cleanup.

A comparison has been made of an LC/MS/MS method using direct analysis of acetonitrile extracts of feed and cereal samples and a method using acetonitrile extraction and subsequent immunoaffinity column (IAC) cleanup. Naturally contaminated samples containing one or more of deoxynivalenol, zearalenone, T-2, and HT-2 toxins were analyzed together with test materials containing known toxin levels. LC/MS/MS ion ratios and peak profiles, repeatability, and LOQs were used as the basis for comparing the two approaches. The method without cleanup had poorer performance than the method with IAC cleanup in terms of identification based on ion ratios compared to standards. Without cleanup, there was more evidence of background interference, and monitored ions were invariably seen against a noisy background. Nevertheless, quantification of samples analyzed without cleanup gave reasonable agreement with the levels found in the same samples that had received IAC cleanup. Repeatability was poorer with no cleanup, and LOQ values were higher for HT-2 and T-2 toxins, but there was no evidence of any adverse effects on MS performance with repeated injections of crude extracts. Overall, it was concluded that LC/MS/MS analysis of samples with no cleanup is adequate for screening, but for definitive measurements (e.g., for food regulatory control purposes) IAC cleanup remains essential.

Detection of porcine DNA in gelatine and gelatine-containing processed food products-Halal/Kosher authentication.

A commercially available real-time PCR, based on a multi-copy target cytochrome b (cyt b) using porcine specific primers, has been validated for the Halal/Kosher authentication of gelatine. Extraction and purification of DNA from gelatine were successfully achieved using the SureFood® PREP Animal system, and real-time PCR was carried out using SureFood® Animal ID Pork Sens kit. The minimum level of adulteration that could be detected was 1.0% w/w for marshmallows and gum drops. A small survey was undertaken of processed food products such as gum drops, marshmallows and Turkish delight, believed to contain gelatine. Of fourteen food products from Germany, two samples were found to contain porcine gelatine, whereas of twenty-nine samples from Turkey twenty-eight were negative. However, one product from Turkey contained porcine DNA and thus was not Halal, and neither was the use of porcine gelatine indicated on the product label.

Determination of fumonisins B1 and B2 in corn by LC/MS with immunoaffinity column cleanup: interlaboratory study.

An interlaboratory validation study was conducted to establish the method performance characteristics of an immunoaffinity column (IAC) cleanup procedure followed by LC/MS for the determination of fumonisins B1 (FB1) and B2 (FB2) and combined FB1 + 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 IAC. FB1 and FB2 are removed with methanol, followed by water, then directly determined by RPLC with MS detection using selected-ion monitoring of two characteristic ions in each case. Naturally contaminated corn samples were milled to a fine powder and mixed to produce three samples with target levels of combined FB1 + FB2 ranging from 350 to 4000 microg/kg. Of 15 initially participating laboratories, two failed to report results and another did not follow the prescribed method. Thus, valid results were obtained from 12 participants located in 11 countries. Statistical analysis of the results produced RSDr values of 4.6-11.9, 1.9-12.6, and 1.4-11.5% for FB1, FB2, and combined FB1 + FB2, respectively; the corresponding RSDR values were 19.8-23.8, 18.2-25.5, and 18.8-23.2%. The three concentration levels of combined FB1 + FB2 were 534, 1194, and 1954 microg/kg. HorRat values for r and R were all < 2.0, indicating that the method is suitable as a regulatory method for the enforcement of European Union limits for fumonisins in corn.

Immunoaffinity column clean-up techniques in food analysis: A review.

This review provides a critical assessment of the applications of immunoaffinity columns for sample clean-up in the field of food safety. The performance of immunoaffinity columns are compared in terms of specificity, binding capacity and recovery, and commercial disposable columns are contrasted with home-made columns. Areas covered include multiple-use of columns, multiple-analyte columns, use with automated systems and validation of IAC methods. Publications illustrating the many varied applications of IAC for sample clean-up in the areas of mycotoxins, veterinary drug residues, pesticide residues, environmental contaminants and vitamins have been compiled, comparing extraction methods, achievable recovery, and illustrating the variety of end-detection methods that have been employed.

Determination of aflatoxins and ochratoxin A in high-sugar-content traditional Turkish foods by affinity column cleanup and LC fluorescence detection.

The effectiveness of an affinity column cleanup procedure followed by LC with fluorescence detection was established for the determination of aflatoxins and ochratoxin A in high-sugar-content traditional Turkish foods. Traditional foods, such as baklava (finely layered pastry filled with nuts and steeped in syrup), halvah (containing sesame paste and pistachios), cevizli sucuk (a confection made of grape juice boiled and dried on strings of nuts), Turkish delight (containing hazelnuts, pistachios, or walnuts), and pismaniye (candy made of sugar, butter, and flour), were tested, and the performance of the method was established with spiked samples. To examine the robustness of the methodology, baklava was prepared from raw materials and spiked at the initial stage of dry ingredients and through subsequent stages of preparation of dough, after cooking, and after addition of syrup and nuts. For all products, the analytical method required grinding the composite foodstuff under liquid nitrogen to form a fine powder, which was then thoroughly mixed before subsampling. After vortex extraction into methanol-water (aflatoxins) and aqueous sodium bicarbonate (ochratoxin A), the sample was filtered, diluted with phosphate-buffered saline, and then passed through either an aflatoxin or ochratoxin A affinity column before HPLC analysis with fluorescence detection (using post-column bromination for the aflatoxins). In all the traditional Turkish products, the recovery of aflatoxin B1 ranged from 77 to 98%, and LODs were <0.1 microg/kg. For ochratoxin A, the recoveries were from 88 to 93% and LODs were similarly <0.1 microLg/kg. Despite the complex nature of these traditional Turkish foods, which frequently contain products from sugar caramelization, there was no evidence of any interfering co-extractives, and the method has proved to be robust enough to be used for food control purposes.

Profiling Turkish honeys to determine authenticity using physical and chemical characteristics.

Seventy authentic honey samples of 9 different floral types (rhododendron, chestnut, honeydew, Anzer (thymus spp.), eucalyptus, gossypium, citrus, sunflower, and multifloral) from 15 different geographical regions of Turkey were analyzed for their chemical composition and for indicators of botanical and geographical origin. The profiles of free amino acids, oligosaccharides, and volatile components together with water activity were determined to characterize chemical composition. The microscopic analysis of honey sediment (mellissopalynology) was carried out to identify and count the pollen to provide qualitative indicators to confirm botanical origin. Statistical analysis was undertaken using a bespoke toolbox for Matlab called Metabolab. Discriminant analysis was undertaken using partial least-squares (PLS) regression followed by linear discriminant analysis (LDA). Four data models were constructed and validated. Model 1 used 51 variables to predict the floral origin of the honey samples. This model was also used to identify the top 5 variable important of projection (VIP) scores, selecting those variables that most significantly affected the PLS-LDA calculation. These data related to the phthalic acid, 2-methylheptanoic acid, raffinose, maltose, and sucrose. Data from these compounds were remodeled using PLS-LDA. Model 2 used only the volatiles data, model 3 the sugars data, and model 4 the amino acids data. The combined data set allowed the floral origin of Turkish honey to be accurately predicted and thus provides a useful tool for authentication purposes. However, using variable selection techniques a smaller subset of analytes have been identified that have the capability of classifying Turkish honey according to floral type with a similar level of accuracy.

Changes in free amino acid and sugar levels of dried figs during aflatoxin B1 production by Aspergillus flavus and Aspergillus parasiticus.

An aqueous slurry of gamma-irradiated sterilized dried figs was inoculated with toxigenic strains of Aspergillus flavus and Aspergillus parasiticus. During incubation at 28 degrees C, pH, fructose, glucose, and free amino acids were determined by high-performance liquid chromatography and liquid chromatography (LC)/mass spectrometry, respectively, over 13 time points (1-20 days). At the same 13 time points using a LC/time-of-flight mass spectrometry screening method, aflatoxin B 1 and other secondary metabolites were simultaneously monitored. During the course of incubation, the pH significantly decreased and aflatoxin B 1 formation correlated with a reduction in proline content for both fungi. Of the 22 free amino acids that were monitored, only proline and cystine were found to be critical in supporting aflatoxin production. Levels of fructose and glucose steadily declined during incubation, until glucose was almost exhausted after 21 days. These time-course experiments confirmed the need for carbon and nitrogen sources for aflatoxin production in dried figs, and the favorable composition of figs as a fungal growth medium.

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.

Rapid analysis of fungal cultures and dried figs for secondary metabolites by LC/TOF-MS.

A liquid chromatography-time-of-flight mass spectrometry (LC/TOF-MS) method has been developed for profiling fungal metabolites. The performance of the procedure in terms of mass accuracy, selectivity (specificity) and repeatability was established by spiking aflatoxins, ochratoxins, trichothecenes and other metabolites into blank growth media. After extracting, and carrying out LC/TOF-MS analysis, the standards were correctly identified by searching a specially constructed database of 465 secondary metabolites. To demonstrate the viability of this approach 11 toxigenic and four non-toxigenic fungi from reference collections were grown on various media, for 7-14 days. The method was also applied to two toxigenic fungi, A. flavus (200-138) and A. parasiticus (2999-465) grown on gamma radiation sterilised dried figs, for 7-14 days. The fungal hyphae plus a portion of growth media or portions of dried figs were solvent extracted and analysed by LC/TOF-MS using a rapid resolution microbore LC column. Data processing based on cluster analysis, showed that electrospray ionization (ESI)-TOF-MS could be used to unequivocally identify metabolites in crude extracts. Using the elemental metabolite database, it was demonstrated that from culture collection isolates, anticipated metabolites. The speed and simplicity of the method has meant that levels of these metabolites could be monitored daily in sterilised figs. Over a 14-day period, levels of aflatoxins and kojic acid maximised at 5-6 days, whilst levels of 5-methoxysterigmatocystin remained relatively constant. In addition to the known metabolites expected to be produced by these fungi, roquefortine A, fumagillin, fumigaclavine B, malformins (peptides), aspergillic acid, nigragillin, terrein, terrestric acid and penicillic acid were also identified.

Improved and simplified liquid chromatography/atmospheric pressure chemical ionization mass spectrometry method for the analysis of underivatized free amino acids in various foods.

An improved analytical method which offers rapid, accurate determination and identification of 22 amino acids in a variety of matrices, e.g. baby foods, juices, honey is reported. The amino acids were extracted from the matrixes using acidified water. Simultaneous determination of 22 underivatized amino acids was carried out by a liquid chromatography-mass spectrometry (LC/MS). A narrow-bore column allowed rapid screening and quantitative analysis by positive LC/atmospheric pressure chemical ionization (APCI) MS with only acidified mobile phase. Retention times of the 22 amino acids were in the range of ca. 0.9-7.5 min. Sample preparation without clean-up followed by fast chromatographic analysis allowed the analysis to be completed in <25 min.

Computer vision based analysis of potato chips--a tool for rapid detection of acrylamide level.

In this study, analysis of digital color images of fried potato chips were combined with parallel LC-MS based analysis of acrylamide in order to develop a rapid tool for the estimation of acrylamide during processing. Pixels of the fried potato image were classified into three sets based on their Euclidian distances to the representative mean values of typical bright yellow, yellowish brown, and dark brown regions using a semiautomatic segmentation algorithm. The featuring parameter extracted from the segmented image was NA2 value which was defined as the number of pixels in Set-2 divided by the total number of pixels of the entire fried potato image. Using training images of potato chips, it was shown that there was a strong linear correlation (r = 0.989) between acrylamide level and NA2 value. Images of a number of test samples were analyzed to predict their acrylamide level by means of this correlation data. The results confirmed that computer vision system described here provided explicit and meaningful description from the viewpoint of inspection and evaluation purpose for potato chips. Assuming a provisional threshold limit of 1000 ng/g for acrylamide, test samples could be successfully inspected with only one failure out of 60 potato chips.

Improved method for the determination of hydroxymethylfurfural in baby foods using liquid chromatography-mass spectrometry.

An improved analytical method for the rapid, reliable, and sensitive determination of hydroxymethylfurfural (HMF) in baby foods is described. It entailed aqueous extraction from food matrix with simultaneous clarification using Carrez I and II reagents, solid-phase extraction cleanup using Oasis HLB, and analysis by liquid chromatography-mass spectrometry. A narrow-bore column allowed fast chromatographic separation with good resolution of HMF and matrix coextractives. In positive atmospheric pressure chemical ionization conditions, precursor and compound-specific ions were sensitively detected in selected ion monitoring mode. Sample preparation with efficient cleanup followed by fast chromatographic analysis allowed the analysis to be completed in <20 min. Recovery ranged between 91.8 and 94.7% for spiking levels of 0.25, 1.0, and 5.0 mg/kg HMF in cereal-based baby foods. The method was shown to be successful when using liquid chromatography coupled to ultraviolet detection at 285 nm.

A simplified approach for the kinetic characterization of acrylamide formation in fructose-asparagine model system.

The potential of acrylamide formation and degradation was studied in fructose-asparagine reaction system at different temperatures (120-200 degrees C). Kinetic data for concurrent formation and degradation of acrylamide was analysed based on a simplified form of chemical reaction in series in which acrylamide occurred as an intermediate. Experimental results revealed that the reaction proceeds zero order and first order with respect to asparagine and fructose, respectively. The thermal degradation of acrylamide was determined to be first order in fructose-glycine reaction system. The concurrent formation and degradation of acrylamide followed a typical kinetic pattern at the temperatures studied. Thermal degradation was observed within 60 min at T>150 degrees C, while only the accumulation was noted at T<150 degrees C. The mathematical model fitted to experimental data very well within temperature range of 120-200 degrees C. The temperature dependence of both acrylamide formation and degradation were found to obey Arrhenius law, and the activation energies were 52.1 kJ/mol and 72.9 kJ/mol, respectively.

A generic method for the determination of acrylamide in thermally processed foods.

A generic sample preparation method for the determination of acrylamide in foods was developed. The method entails extraction with methanol, purification with Carrez I and II solutions, evaporation and solvent change to water, and cleanup with Oasis HLB solid-phase extraction (SPE) cartridge. The final extract was analyzed by liquid chromatography-mass spectrometry (LC-MS) for quantitation. The chromatographic separation was performed on ODS-3 column using the isocratic mixture of 0.01 mM acetic acid in 0.2% aqueous solution of formic acid at a flow rate of 0.6 ml/min at 25 degrees C. The recoveries of acrylamide from potato chips, biscuits and coffee ranged between 92.8 and 101.5% with relative standard deviations of 4.1% or less. The limit of detection (LOD) and the limit of quantitation (LOQ) were 2 ng/g and 6 ng/g in the basis of signal to noise ratios of 3:1 and 9:1, respectively.