Development of certified reference materials for the determination of cadmium and acrylamide in cocoa.

Since 1 January 2019 a maximum content of 0.6 mg kg-1 cadmium (Cd) in cocoa powder sold to the final consumer or as an ingredient in sweetened cocoa powder sold to the final consumer (drinking chocolate) is set by the Commission Regulation (EU) No. 488/2014. Monitoring compliance with the specified limit value requires analytical measuring methods and reference materials for quality control. However, suitable certified reference materials intended for quality assurance and quality control purposes are still lacking. Therefore, three cocoa reference materials (ERM®-BD513, ERM®-514 and ERM®-515) were developed according to the requirements of ISO 17034 and the recommendations of ISO Guide 35. The whole process of reference material development, including material preparation, assessment of homogeneity and stability, characterisation and value assignment is presented. The assignment of the certified mass fractions was based upon an interlaboratory comparison study involving 19 expert laboratories for Cd and 12 laboratories for acrylamide. The certified mass fractions and expanded uncertainties (k = 2) of the reference materials were (0.181 ± 0.009) mg kg-1 Cd (ERM®-BD513), (0.541 ± 0.024) mg kg-1 Cd (ERM®-BD514) and (0.690 ± 0.029) mg kg-1 Cd (ERM®-BD515). Acrylamide contents are given for information.

T-2 and HT-2 toxins in oat flakes: development of a certified reference material.

Trichothecene mycotoxins, with T-2 and HT-2 toxins being the main representatives of the type A subgroup, are naturally and worldwide occurring contaminants frequently found in grain-based food and feed. Due to the high consumption of these products and the potential health risk associated herewith, concerns about the safety and quality of food and feed have increased over the last decades at both governmental and consumer levels. Since it is not possible to avoid their occurrence, tremendous efforts have been performed to identify and monitor mycotoxins in food and feed to make their consumption safe. However, suitable certified reference materials (CRMs) intended for quality assurance and quality control purposes are still lacking for many mycotoxin-matrix combinations. Therefore, in the framework of a European Reference Material (ERM®) project, the first CRM for T-2 and HT-2 toxin in ground oat flakes (ERM®-BC720) was developed according to the requirements of ISO Guide 35. The whole process of ERM®-BC720 development, including sample preparation, homogeneity and stability studies and value assignment, is presented. The assignment of the certified mass fractions was based upon an in-house study using high-performance liquid chromatography isotope-dilution tandem mass spectrometry. Simultaneously, an interlaboratory comparison study involving 24 expert laboratories was conducted in order to support the in-house certification study. The certified values and their corresponding expanded uncertainties (k = 2) for both T-2 and HT-2 toxin in ERM®-BC720, traceable to the international system of units, are (82 ± 4) µg kg(-1) and (81 ± 4) µg kg(-1), respectively.

Development and certification of a reference material for Fusarium mycotoxins in wheat flour.

Deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEN) are toxic secondary metabolites produced by several species of Fusarium fungi. These mycotoxins are often found together in a large variety of cereal-based foods, which are regulated by maximum content levels of DON and ZEN. To date, suitable certified reference materials (CRM) intended for quality control purposes are lacking for these Fusarium mycotoxins. In order to overcome this lack, the first CRM for the determination of DON, NIV and ZEN in naturally contaminated wheat flour (ERM®-BC600) was developed in the framework of a European Reference Materials (ERM®) project. This article describes and discusses the whole process of ERM®-BC600 development, including material preparation, homogeneity and stability studies, and an interlaboratory comparison study for certification. A total of 21 selected expert laboratories from different European countries with documented expertise in the field of mycotoxin analysis took part in the certification study using various gas and liquid chromatographic methods. The certified values and their corresponding expanded uncertainties (k = 2) were assigned in full compliance with the requirements of ISO Guide 35 and are as follows: 102 ± 11 µg kg(-1) for DON, 1000 ± 130 µg kg(-1) for NIV and 90 ± 8 µg kg(-1) for ZEN.

rac-2,3-Dibromo-propionamide.

The racemic title compound, C(3)H(5)Br(2)NO, was crystallized from methanol. In the crystal, adjacent mol-ecules are linked through N-H⋯O hydrogen bonds, forming chains along the c-axis direction. These chains are linked through N-H⋯O hydrogen bonds, forming an undulating two-dimensional network lying parallel to the bc plane. There are also short Br⋯Br contacts present [3.514 (3) Å].

Degradation and epimerization of ergot alkaloids after baking and in vitro digestion.

The degradation and epimerization of ergot alkaloids (EAs) in rye flour were investigated after baking cookies and subsequently subjecting them to an in vitro digestion model. Different steps of digestion were analyzed using salivary, gastric, and duodenal juices. The degradation and bidirectional conversion of the toxicologically relevant (R)-epimers and the biologically inactive (S)-epimers for seven pairs of EAs were determined by a HPLC method coupled with fluorescence detection. Baking cookies resulted in degradation of EAs (2-30 %) and a shift in the epimeric ratio toward the (S)-epimer for all EAs. The applied digestion model led to a selective toxification of ergotamine and ergosine, two ergotamine-type EAs. The initial percentage of the toxic (R)-epimer in relation to the total toxin content was considerably increased after digestion of cookies. Ergotamine and ergosine increased from 32 to 51 % and 35 to 55 %, respectively. In contrast, EAs of the ergotoxine type (ergocornine, α- and ß-ergocryptine, and ergocristine) showed an epimeric shift toward their biologically inactive (S)-epimers. Further experiments indicated that the selective epimerization of ergotamine EAs occurs in the duodenal juice only. These results demonstrate that toxification of EAs in the intestinal tract should be taken into consideration.

Stable isotope dilution assay for the accurate determination of mycotoxins in maize by UHPLC-MS/MS.

A fast, easy-to-handle and cost-effective analytical method for 11 mycotoxins currently regulated in maize and other cereal-based food products in Europe was developed and validated for maize. The method is based on two extraction steps using different acidified acetonitrile­water mixtures. Separation is achieved using ultrahigh-performance liquid chromatography (UHPLC) by a linear water­methanol gradient. After electrospray ionisation, tandem mass spectrometric detection is performed in dynamic multiple reaction monitoring mode. Since accurate mass spectrometric quantification is hampered by matrix effects, uniformly [(13)C]-labelled mycotoxins for each of the 11 compounds were added to the sample extracts prior to UHPLC-MS/MS analysis. Method performance parameters were obtained by spiking blank maize samples with mycotoxins before as well as after extraction on six levels in triplicates. The twofold extraction led to total recoveries of the extraction steps between 97% and 111% for all target analytes, including fumonisins. The [(13)C]-labelled internal standards efficiently compensated all matrix effects in electrospray ionisation, leading to apparent recoveries between 88% and 105% with reasonable additional costs. The relative standard deviations of the whole method were between 4% and 11% for all analytes. The trueness of the method was verified by the measurement of several maize test materials with well-characterized concentrations. In conclusion, the developed method is capable of determining all regulated mycotoxins in maize and presuming similar matrix effects and extraction recovery also in other cereal-based foods.

rac-1-(2-Amino-carbonyl-2-bromo-eth-yl)pyridinium bromide.

In the crystal structure of the title compound, C(8)H(10)BrN(2)O(+)·Br(-), inter-molecular N-H⋯Br hydrogen bonds link the mol-ecules into infinite chains along [001]. The inclined angle between the pyridine ring plane and the plane defined by the acid amide group is 63.97 (4)°.

(3S,11Z)-14,16-Dihy-droxy-3-methyl-3,4,5,6,9,10-hexa-hydro-1H-2-benz-oxacyclo-tetra-decine-1,7(8H)-dione (cis-zearalenone): a redetermination.

The title compound, also known as cis-zearalenone (cis-ZEN), C(18)H(22)O(5), has already been reported elsewhere [Griffin et al. (1981 ▶). ACA Ser.29, 35], but no atomic coordinates are publicly available. The mol-ecule is of inter-est with respect to its toxicity. In the crystal, intra-molecular O-H⋯O hydrogen bonds stabilize the mol-ecular conformation, while inter-molecular O-H⋯O hydrogen bonds link the mol-ecules to form infinite chains along the [110] and [1-10] directions. The absolute configuration has been assigned by reference to an unchanging chiral centre in the synthetic procedure.

Lysergol monohydrate.

IN THE TITLE COMPOUND [SYSTEMATIC NAME: (7-methyl-4,6,6a,7,8,9-hexa-hydro-indolo[4,3,2-fg]quinoline-9-yl)methanol monohydrate], C(16)H(18)N(2)O·H(2)O, the non-aromatic ring (ring C of the ergoline skeleton) directly fused to the aromatic rings is nearly planar, with a maximum deviation of 0.659 (3) Å, and shows an envelope conformation. In the crystal, hydrogen bonds between the lysergol and water mol-ecules contribute to the formation of layers parallel to (10[Formula: see text]).

Ergotaminine.

The title compound {systematic name: (6aR,9S)-N-[(2R,5S,10aS,10bS)-5-benzyl-10b-hy-droxy-2-methyl-3,6-dioxoocta-hydro-8H-oxazolo[3,2-a]pyrrolo-[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a,7,8,9-hexa-hydro-indolo[4,3-fg]quinoline-9-carboxamide}, C(33)H(35)N(5)O(5), was formed by an epimerization reaction of ergotamine. The non-aromatic ring (ring C of the ergoline skeleton) directly fused to the aromatic rings is nearly planar [maximum deviation = 0.317 (4) Å] and shows an envelope conformation, whereas ring D, involved in an intra-molecular N-H⋯N hydrogen bond exhibits a slightly distorted chair conformation. The structure displays chains running approximately parallel to the diagonal of bc plane that are formed through N-H⋯O hydrogen bonds.

Determination of mycotoxins in foods: current state of analytical methods and limitations.

Mycotoxins are natural contaminants produced by a range of fungal species. Their common occurrence in food and feed poses a threat to the health of humans and animals. This threat is caused either by the direct contamination of agricultural commodities or by a "carry-over" of mycotoxins and their metabolites into animal tissues, milk, and eggs after feeding of contaminated hay or corn. As a consequence of their diverse chemical structures and varying physical properties, mycotoxins exhibit a wide range of biological effects. Individual mycotoxins can be genotoxic, mutagenic, carcinogenic, teratogenic, and oestrogenic. To protect consumer health and to reduce economic losses, surveillance and control of mycotoxins in food and feed has become a major objective for producers, regulatory authorities and researchers worldwide. However, the variety of chemical structures makes it impossible to use one single technique for mycotoxin analysis. Hence, a vast number of analytical methods has been developed and validated. The heterogeneity of food matrices combined with the demand for a fast, simultaneous and accurate determination of multiple mycotoxins creates enormous challenges for routine analysis. The most crucial issues will be discussed in this review. These are (1) the collection of representative samples, (2) the performance of classical and emerging analytical methods based on chromatographic or immunochemical techniques, (3) the validation of official methods for enforcement, and (4) the limitations and future prospects of the current methods.

rac-(1R,2R,4S)-1,2-Dibromo-4-[(1R)-1,2-dibromo-eth-yl]cyclo-hexa-ne.

In the title compound, C(8)H(12)Br(4), the cyclo-hexane ring exhibits a chair conformation. The C-Br distances range from 1.964 (6) to 1.985 (5) Šand the C-C distances range from 1.496 (6) to 1.543 (7) Å. Short inter-molecular Br⋯Br contacts [3.467 (4) Å] occur in the crystal.

Ergometrinine.

The absolute configuration of ergometrinine, C(19)H(23)N(3)O(2) {systematic name: (6aR,9S)-N-[(S)-1-hy-droxy-propan-2-yl]-7-methyl-4,6,6a,7,8,9-hexa-hydro-indolo[4,3-fg]quinoline-9-carb-ox-amide}, was established based on epimerization reaction of ergometrine, which was followed by preparative HPLC. The non-aromatic ring (ring C of the ergoline skeleton) directly fused to the aromatic rings is nearly planar [maximum deviation = 0.271 (3) Å] and shows an envelope conformation, whereas ring D, involved in an intra-molecular N-H⋯N hydrogen bond, exibits a slightly distorted chair conformation. The structure displays undulating layers in the ac plane formed by O-H⋯O and N-H⋯O hydrogen bonds.

Photodegradation of the novel brominated flame retardant 2,4,6-Tris-(2,4,6-tribromophenoxy)-1,3,5-triazine in solvent system: Kinetics, photolysis products and pathway.

In this study the direct and indirect photolysis of the novel brominated flame retardant 2,4,6-Tris-(2,4,6-tribromophenoxy)-1,3,5-triazine (TTBP-TAZ) in an organic solvent mixture (60:30:10, ACN:MeOH:THF) under UV-(C) and simulated sunlight irradiation was investigated, and the formed photo-transformation products were identified for the first time. TTBP-TAZ was almost completely degraded within 10 min under UV-(C) irradiation. Due to the fast degradation no specific kinetic order could be observed. In comparison, the reaction under simulated sunlight irradiation was much slower and thus, the kinetic first-order could be determined. The observed photolysis rate constant k as well as the half-life time t1/2 were estimated to be k = (0.0163 ±â€¯0.0002) h-1 and t1/2 = 42.3 h, respectively. The addition of 2-propanol and hydrogen peroxide to investigate the influence of indirect photolysis under UV-(C) irradiation causes no influence on the degradation of TTBP-TAZ. Nevertheless, the removal of TTBP-TAZ under UV-(C) and simulated sunlight without additional chemicals (except solvent) indicates that the direct photolysis plays a significant role in the degradation mechanism of TTBP-TAZ. In both irradiation experiments, TTBP-TAZ was quantitatively degraded that involve the formation of previously unknown PTPs. Overall, two main PTPs were determined when irradiated with UV-(C) and eight sequential debromination products were observed when irradiated by simulated sunlight. These were determined by HPLC-DAD and - MS/(MS), respectively. Based on the chosen experimental conditions the consecutive debromination as well as photo-Fries rearrangement was confirmed as the main degradation pathway by high resolution mass spectrometry and X-ray diffraction.

1,3,5-Tris-(2,3-dibromopropyl)-1,3,5-triazine-2,4,6-trione: kinetic studies and phototransformation products.

1,3,5-Tris-(2,3-dibromopropyl)-1,3,5-triazine-2,4,6-trione (TDBP-TAZTO) is an emerging brominated flame retardant which is widely used in several plastic materials (electric and electronic equipment, musical instruments, automotive components). However, until today, no photochemical studies as well as the identification of possible phototransformation products (PTPs) were described in literature. Therefore, in this study, UV-(C) and simulated sunlight irradiation experiments were performed to investigate the photolytic degradation of TDBP-TAZTO and to identify relevant PTPs for the first time. The UV-(C) irradiation experiments show that the photolysis reaction follows a first-order kinetic model. Based on this, the photolysis rate constant k as well as the half-life time t1/2 were calculated to be k = (41 ± 5 × 10-3) min-1 and t1/2 = (17 ± 2) min. In comparison, a minor degradation of TDBP-TAZTO and no formed phototransformation products were obtained under simulated sunlight. In order to clarify the photochemical behavior, different chemicals were added to investigate the influence on indirect photolysis: (i) H2O2 for generation of hydroxyl radicals and (ii) two quenchers (2-propanol, sodium azide) for scavenging oxygen species which were formed during the irradiation experiments. Herein, nine previously unknown PTPs of TDBP-TAZTO were detected under UV-(C) irradiation and identified by HPLC-(HR)MS. As a result, debromination, hydroxylation, and dehydrobromination reactions could be presumed as the main degradation pathways by high-resolution mass spectrometry. The direct as well as the OH radical-induced indirect photolysis were observed. Graphical abstract .

High-performance liquid chromatography/electrospray mass spectrometry analysis of the mycotoxin aurofusarin.

High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) can be used for simultaneous quantification of various mycotoxins in contaminated food samples. Therefore, multi-mycotoxin methods have been developed in the last couple of years. To enlarge these methods for further analytes, we have developed a LC-MS/MS method for the quantification of the mycotoxin aurofusarin. Additionally, further LC- MS(n) experiments were performed to demonstrate the fragmentation pattern of aurofusarin. Applicable multiple reaction monitoring (MRM) transitions of aurofusarin were found and optimized by parameter variation of the tandem mass spectrometer. The applicability of the developed method was tested by analysis of naturally contaminated wheat.

On the thermally induced isomerisation of hexabromocyclododecane stereoisomers.

The interconversion of the stereoisomers contained in technical 1,2,5,6,9,10-hexabromocyclododecane, a major brominated flame retardant increasingly found in the environment and in biota, was investigated at elevated temperatures. The application of pure enantiomers of the three constituents alpha-, beta-, and gamma-HBCD enabled the unambiguous elucidation of the individual isomerisation reactions as well as the quantification of all respective rate constants. At 160 degrees C the rate constants range over two orders of magnitude from 1.50x10(-3) to 1.88x10(-5)mol(%)s(-1). A preliminary mechanistic explanation for the differences of the rate constants which govern the composition of HBCD diastereomers at equilibrium is given.

Synthesis of M + 4 Stable Isotopomers of Ergometrine and Ergometrinine.

The priority ergot alkaloids ergometrine and ergometrinine are highly toxic mycotoxins naturally occurring in different types of grains (i.e. rye, wheat, rice), as well as grain-based foods and, therefore, have gained increasing importance for food safety over the last years. The application of HPLC-MS/MS for the analysis of ergot alkaloids in food presupposes the availability of isotopically labelled internal standards. Thus, a multistep synthesis was developed for ergometrine-(N-13CD3) and its epimer ergometrinine-(N-13CD3) with a mass shift of four units compared with the parent compounds. The synthesis is based on the preparation of stable isotope labelled lysergic acid that was coupled with (S)-alaninol. The chemical synthesis of both compounds has been achieved in six steps with an overall yield of 1 % (ergometrine-(N-13CD3)) and 0.6 % (ergometrinine-(N-13CD3)), respectively. Structural identification was performed by MS analysis as well as 1H and 13C NMR.

Novel solid-phase extraction for epimer-specific quantitation of ergot alkaloids in rye flour and wheat germ oil.

Ergot alkaloids and their epimer-specific determination have gained increasing importance for food safety. A solid-phase extraction and cleanup method based on sodium-neutralized strong cation exchange (Na(+)-SCX) was developed to quantitate 12 priority ergot alkaloids in rye flour and wheat germ oil by HPLC fluorescence analysis. Sample preparation is achieved by omitting acidic and alkaline conditions enabling minimized epimerization, which is necessary to determine ergot alkaloids according to their natural distribution in foods. Ergot alkaloids are eluted from SCX-column by forming ion pairs using a sodium hexanesulfonate containing solution which prevents epimerization for at least 96 h. Method validation yielded recoveries of 80-120% (rye flour) and 71-96% (wheat germ oil) with a maximum limit of quantitation (LOQ) of 2.0 µg kg(-1) per ergot alkaloid for both matrices. The applicability of the developed method was demonstrated by analyzing 16 samples from German retail markets: 9 rye flours (max 178 ± 5 µg kg(-1)) and, reported for the first time, 7 wheat germ oils (max 56.8 ± 2.7 µg kg(-1)) expressed as the sum of 12 ergot alkaloids.