Sorting out the plants responsible for a contamination with pyrrolizidine alkaloids in spice seeds by means of LC-MS/MS and DNA barcoding: Proof of principle with cumin and anise spice seeds.

High value commodities such as spices suffer from occasional contaminations of both chemical and biological origin. Consequently, quality control and safety monitoring has become a pressing issue for the spice industry. Two recent independent studies showed that at least one third of the analyzed cumin and green anise spice seeds samples surpassed the by the European Union recently established threshold value for toxic pyrrolizidine alkaloids (PAs) and their corresponding N-oxides (PANOs). These heterocyclic secondary plant metabolites are produced by a large number of different plant families. In those spice seeds, it was found by means of DNA metabarcoding, that predominant contamination was due to the presence of herbal material from the Heliotropium genus (Boraginaceae). Unfortunately, the use of this specific type of DNA-based identification remains controversial for the majority of the official instances and preference is still given to the use of more tangible classical approaches, including microscopy and chemical analysis. However, these methodologies often suffer from inherent drawbacks. Here we demonstrate that at least for spice seeds, a combinatory approach of microscopy, chemical analysis and classical DNA barcoding of the isolated contaminants using the matK and trnH-psbA loci, provides qualitative and quantitative information on the amount of plant material responsible for the contaminations and the extent of the contamination. The generated data also demonstrates that the presence of a very limited number of Heliotropium sp. seeds in a standard commercially available canister is sufficient to surpass the allowed threshold value, illustrating once more the importance of weed control.

Targeted LC-MS/MS combined with multilocus DNA metabarcoding as a combinatory approach to determine the amount and the source of pyrrolizidine alkaloids contamination in popular cooking herbs, seeds, spices and leafy vegetables.

Pyrrolizidine alkaloids (PAs) and their corresponding N-oxides (PANOs) are natural protoxins biosynthesised by many plant species and are responsible for occasional fatal intoxication outbreaks due to the consumption of contaminated food. However, only in rare cases has the origin of the contamination been determined. Although their presence has been studied in many matrices, occurrence data on popular widely used cooking herbs, seeds, spices and leafy vegetables is very scarce. Therefore, a systematic study on the occurrence of PA/PANOs contaminations in these popular herbal items, available on the Belgian market, was performed, by means of a validatedtargeted LC-MS/MS analysis, followed by multilocus DNA metabarcoding to track back the origin of the contamination for seven highly to moderately contaminated samples. Our results clearly indicate that 21% of the seed spices and 25% seed-based aromatic mixes contain an amount higher than 400 µg of the 30 summed targeted PAs and PANOs per kg, the value which is currently under discussion by the European member states to be set as a maximum threshold. For both the herbs and the herb-based mixes only 7% of analysed samples exceeded these levels. As a proof of concept, multilocus DNA metabarcoding was performed on six highly contaminated samples, belonging to each subtype matrix, containing high levels of heliotrine type of PA/PANOs. Each time the analysis demonstrated the presence of DNA from a plant species belonging to the plant genus Heliotropium. Moreover, a contaminated leafy vegetable sample, containing solely senecionine type PA/PANOs, contained DNA from Senecio vulgaris. Taken together, it can be stated that the proposed combinatory chemical and molecular techniques could be used to verify if a PA/PANO(s) contamination occurred in these popular cooking items and to pinpoint the origin the contamination, which is pivotal in the case of a detrimental intoxication or intoxication outbreak.

Monitoring of pyrrolizidine alkaloids in beehive products and derivatives on the Belgian market.

Pyrrolizidine alkaloids (PAs) and related N-oxides (PANOs) are secondary plant metabolites thought to be found in approximately 3% of the flowering plants worldwide and exhibiting hepatotoxic properties to humans. As a consequence, beehive products are prone to be contaminated with those compounds by bees foraging PA-producing plants. Downstream contamination can also occur through food items containing honey. Analytical methods based on UHPLC separation and MS/MS detection were developed with a focus on very low LOQs and validated for the analysis of 16 PAs and 14 PANOs in honey, honey-based candies and snacks, as well as beehive product-based food supplements. A maximum level of 182 ng/g of PAs was detected in a Mediterranean honey, and high levels of heliotrine-type compounds were reported for the first time. An extensive sampling of honeys harvested in Belgium was performed (N = 374), the concentration levels were more limited with a maximum of 60 ng/g, and the contamination pattern was dominated by senecionine-type PAs. The PA levels in honey-based candies and snacks were very low, with respective maxima of 7.61 ng/g and 0.36 ng/g. Seventy-five percent among the pre-dosed food supplements based on beehive products were contaminated, with a maximum of 43 ng/g. The highest level was detected in a bee-collected pollen sample (1672 ng/g). The analytical results were consistent with the previously reported data for beehive products and confirmed that PA/PANO contamination in these food commodities is recurrent.

Analytical strategies for the determination of pyrrolizidine alkaloids in plant based food and examination of the transfer rate during the infusion process.

Two sample preparation methods were developed (graphitised carbon and C18 solid phase extraction clean-up) and validated in house using liquid chromatography and tandem mass spectrometry (MS/MS) for the determination of 30 pyrrolizidine alkaloids (PAs) in salads, herbs, tea, herbals teas as well as tea infusion and ice-tea beverages. Total PAs concentration of samples purchased on the Belgian market varied greatly with matrix type ranging from

Fluorescent chemosensors for anions and contact ion pairs with a cavity-based selectivity.

The association of a concave macrocyclic compound to one or multiple fluorophores is an appealing strategy for the design of chemosensors. Indeed, as with biological systems, a cavity-based selectivity can be expected with such fluorescent receptors. Examples of calix[6]arene-based systems using this strategy are rare in the literature, and to our knowledge, no examples of fluorescent receptors that can bind organic contact ion pairs have been reported. This report describes the straightforward synthesis of fluorescent calix[6]arene-based receptors 4a and 4b bearing three pyrenyl subunits and the study of their binding properties toward anions and ammonium salts using different spectroscopies. It was found that receptor 4a exhibits a remarkable selectivity for the sulfate anion in DMSO, enabling its selective sensing by fluorescence spectroscopy. In CDCl3, the receptor is able to bind ammonium ions efficiently only in association with the sulfate anion. Interestingly, this cooperative binding of ammonium sulfate salts was also evidenced in a protic environment. Finally, a cavity-based selectivity in terms of size and shape of the guest was observed with both receptors 4a and 4b, opening interesting perspectives on the elaboration of fluorescent cavity-based systems for the selective sensing of biologically relevant ammonium salts such as neurotransmitters.

Ipso-Nitration of calix[6]azacryptands: intriguing effect of the small rim capping pattern on the large rim substitution selectivity.

The ipso-nitration of calix[6]arene-based molecular receptors is a important synthetic pathway for the elaboration of more sophisticated systems. This reaction has been studied for a variety of capped calixarenes, and a general trend for the regioselective nitration of three aromatic units out of six in moderate to high yield has been observed. This selectivity is, in part, attributed to the electronic connection between the protonated cap at the small rim and the reactive sites at the large rim. In addition, this work highlights the fact that subtle conformational properties can drastically influence the outcome of this reaction.

Allosterically driven self-assemblies of interlocked calix[6]arene receptors.

The construction of self-assembled receptors based on flexible concave subunits is a challenging task and constitutes an interesting approach to mimic binding processes occurring in biological systems. The receptors studied herein are based on flexible calix[6]arene skeletons bearing three (or more) acid-base functionalities at their narrow rim. When complementary, they self-assemble in a tail-to-tail manner to give a diabolo-like complex, provided that each calixarene subunit hosts a guest. The allosterically-driven multi-recognition pattern is highly selective and leads to stable quaternary adducts. In order to evaluate the scope of this system, various polyamino and polyacidic calix[6]arenes have been studied. It is shown that modifications of the nature of the wide rim substituents do not alter the efficiency of the quaternary self-assembling process, even with the more flexible macrocycles that lack tBu substituents. On the contrary, the replacement of the latter by smaller groups led to receptors with broader scope, as larger guests such as tryptamine and dopamine derivatives were stabilized in the cavities. Implementation of extra-functionalities at the narrow rim were revealed also to be of high interest. Indeed, it is shown that secondary interactions take place between the two calix-subunits when they present additional and complementary functions such as carboxylate and ureido moieties. The ureido arms are also capable of binding the counter anion Cl(-) of the ammonium guest, thus leading to a quinternary neutral complex. Such remarkable behavior is due to the versatility of the calix[6]arene platform, which allows the implementation of a high number of functions, leading to multiple non-covalent attractive interactions, whereas the macrocycle remains flexible, thus allowing induced-fit processes to occur.