About Alternaria toxins in cocoa and chocolate products-method development and monitoring of alternariol, alternariol monomethyl ether and tenuazonic acid.

A quick and selective analytical method was developed via LC-MS/MS for the simultaneous quantitation of alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TeA) which belong to the large group of secondary metabolites produced by fungi of the genus Alternaria. Cocoa is susceptible to a number of toxin-producing microorganisms, including Aspergillus and Penicillium species. The method relies on a single-step extraction, followed by an easy clean up, dilution of the raw extract and direct analysis. To assess whether cocoa and chocolate products can be a source of Alternaria toxins, a monitoring of cocoa and chocolate products (N = 99) as well as cocoa raw and semi-finished materials (cocoa shells, cocoa masses; N = 10) was performed. As the results, cocoa and products made from cocoa (without other ingredients) are no source of the Alternaria toxins considered here.

Analysis and occurrence of matrine in liquorice raw materials - Exclusion of its application as pesticide.

A quick and selective analytical method was developed employing LC-MS/MS for the quantitation of matrine. This is known to be a natural ingredient of Sophora ssp. and is suggested to be a potential contaminant, e.g. in herbal raw materials from liquorice or confectionery products based on liquorice. To prove that the finding of matrine in liquorice roots does not originate from an active use of pesticides, wild collection areas, as well as geographical, legal and economic aspects have been studied with the help of experienced traders and suppliers in cooperation with local liquorice producers. An LC-MS/MS method was successfully developed and applied for monitoring of raw material and semi-finished products (N = 104) and afterwards a model test was performed to show that findings of matrine in liquorice products originates from a co-harvesting of nearby growing Sophora roots during the manual collection of liquorice roots.

Determination of 2-Methylimidazole, 4-Methylimidazole, and 2-Acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole in Licorice Using High-Performance Liquid Chromatography-Tandem Mass Spectrometry Stable-Isotope Dilution Analysis.

A quick and selective analytical method was developed for the simultaneous quantitation of 2-methylimidazole, 4-methylimidazole, and 2-acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole, which are known to be formed by Maillard reactions. The methodology reported here employs stable-isotope dilution analysis (SIDA) using 4-methylimidazole-d6 and [(13)C6]-2-acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole as internal standards. It was successfully applied in a model assay to show that the addition of ammonium chloride during the manufacture of licorice promotes imidazole formation depending on the added amount of ammonium chloride without the well-known impact of present caramel food colorings. Furthermore, a monitoring assay of 29 caramel coloring-free licorice products showed that both 4-methylimidazole and 2-acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole are endogenously generated in detectable quantities. None of the samples showed 2-methylimidazole levels above the limit of detection, 50 µg/kg.

Quantitation of polycyclic aromatic hydrocarbons (PAH4) in cocoa and chocolate samples by an HPLC-FD method.

As a consequence of the PAH4 (sum of four different polycyclic aromatic hydrocarbons, named benzo[a]anthracene, chrysene, benzo[b]fluoranthene, and benzo[a]pyrene) maximum levels permitted in cocoa beans and derived products as of 2013, an high-performance liquid chromatography with fluorescence detection method (HPLC-FD) was developed and adapted to the complex cocoa butter matrix to enable a simultaneous determination of PAH4. The resulting analysis method was subsequently successfully validated. This method meets the requirements of Regulation (EU) No. 836/2011 regarding analysis methods criteria for determining PAH4 and is hence most suitable for monitoring the observance of the maximum levels applicable under Regulation (EU) No. 835/2011. Within the scope of this work, a total of 218 samples of raw cocoa, cocoa masses, and cocoa butter from several sample years (1999-2012), of various origins and treatments, as well as cocoa and chocolate products were analyzed for the occurrence of PAH4. In summary, it is noted that the current PAH contamination level of cocoa products can be deemed very slight overall.

Synthesis and application of (13)C-labeled 2-acetyl-4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)imidazole (THI), an immunosuppressant observed in caramel food colorings.

2-Acetyl-4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)imidazole (THI) is a minor toxic contaminant observed in caramel food colorings and was shown to exert immunosuppressant activity when fed to rodents. Because of this toxicity, maximum levels of THI in caramel food colorings have been defined by international and European authorities. Several reports of THI analysis using external standardization have been published for liquid foods such as beers and soft drinks. However, no suitable internal standard has yet been described allowing THI analysis in more complex samples. In this paper we describe the preparation of a labeled [(13)C6]THI analogue and its application for the successful validation of the first stable isotope dilution assay (SIDA) of THI in caramel food colorings. A brief survey of THI levels in commercially available caramel class III (E 150c) and IV (E 150d) food colorings is also included, corroborating that THI occurs only in caramel class III food colorings.

Determination of acrylamide level in popular Iranian brands of potato and corn products.

Acrylamide is a chemical found in starchy foods that have been cooked at high temperatures. These include crisps, chips, bread and crisp breads. It was first discovered by scientists in Sweden in 2002. The Objective of this study is to determine the level of acrylamide in popular Iranian brands of potato crisps and corn products, produced by domestic food industrial factories. For this reason 7 brands of potato and 8 brands of corn products (10 each) were collected, crashed and after preparing the extracts of each sample, using LC-MS-MS spectrometry for measuring acrylamide amount. Results showed that in different brands of potato and corn products there were different amounts (244-1688 microg/kg) and (<30-410 microg/kg) of acrylamide, respectively. As acrylamide is a dangerous toxin for human health, so it needs to reduce the level of acrylamide in these products that are used extensively by people specially children.

Determination of ochratoxin A in liquorice products using HPLC based analytical methods. Part I: proficiency test of methods commonly used by the confectionary industry.

A European proficiency test series was accomplished on behalf of the CAOBISCO (Association of the Chocolate, Biscuit and Confectionery Industries of the EU) expert group on ochratoxin A to assess the performance of laboratories in measuring ochratoxin A in samples of various liquorice products. In addition, the impact of the extraction type (mainly with or without the use of halogenated solvents) was to be evaluated. Four different test samples (two liquorice powders and two liquorice pastes) were tested for sufficient homogeneity and distributed to 15 laboratories in 8 countries in Europe. The results were analysed using standard proficiency testing statistical procedures and laboratories were awarded z-scores on the basis of their reported results. The overall evaluation of the results shows a distinct variation between the participating laboratories. Based on a target standard deviation (σ-value) taken from the Horwitz equation, of the 14 laboratories that reported results, satisfactory results (z-score: |Z| ≤ 2.0) were obtained by 60% and 27% of the laboratories, respectively, for the two liquorice powders and by 93% and 53% of the laboratories, respectively, for the two liquorice pastes. Approximately equal numbers of laboratories used extraction types with and without the use of halogenated solvents. ANOVA testing of the results indicates there was no evident trend using different extraction solvents.

Determination of ochratoxin A in liquorice products using HPLC-based analytical methods. Part II: harmonised method and method validation study.

On the basis of the outcome of an European proficiency test series conducted on behalf of the CAOBISCO (Association of the Chocolate, Biscuit and Confectionery Industries of the EU) expert group on ochratoxin A, a new harmonised method was developed for the analysis of ochratoxin A in liquorice extracts. This method works without the use of halogenated solvents because, as the proficiency test showed, an aqueous extraction solution can be used instead of, for example, chloroform, whose use is restricted in the EU. The main objective of this method validation study was to check the performance of this harmonised method. To carry out the method validation study, a set of three different test samples (one liquorice powder and two liquorice pastes) and a liquorice powder sample with an indicated range of ochratoxin A (a so-called sunshine sample) was distributed to 21 laboratories in ten countries throughout Europe and to one laboratory in the USA. The study was evaluated according to internationally recognised guidelines. In terms of its repeatability and reproducibility for determining ochratoxin A in liquorice extracts with a relative standard deviation for repeatability (RSDr) of between 6.68 and 19.95 and a relative standard deviation for reproducibility (RSDR) of between 17.39 and 29.08 the performance of the harmonised method was found to be in the accepted range of the EU directive for the analysis of mycotoxins in several foodstuffs.

Analysis of acrylamide in food: dedicated to Professor Dr. Werner Baltes.

Since the first discovery of the presence of acrylamide in a variety of food products in April 2002, numerous methods have been developed to determine the acrylamide monomer in heat-treated carbohydrate-rich food. These detection methods are mainly MS-based, coupled with a chromatographic step using LC or GC. The Food Chemistry Institute (LCI) of the Association of the German Confectionery Industry (BDSI) therefore established a detection method by means of aqueous extraction plus a cleaning step and LC-MS/MS detection, making great efforts to ensure internal and external validation. Citing potato crisps as an example, we will in the following show how the German manufacturing companies have gone to great pains to reduce acrylamide levels in their products.