Monique de Nijs

A straightforward method to determine flavouring substances in food by GC-MS

A straightforward GC-MS method was developed to determine the occurrence of fourteen flavouring compounds in food. It was successfully validated for four generic types of food (liquids, semi-solids, dry solids and fatty solids) in terms of limit of quantification, linearity, selectivity, matrix effects, recovery (53-120%) and repeatability (3-22%). The method was applied to a survey of 61 Dutch food products. The survey was designed to cover all the food commodities for which the EU Regulation 1334/2008 set maximum permitted levels. All samples were compliant with EU legislation. However, the levels of coumarin (0.6-63 mg/kg) may result in an exposure that, in case of children, would exceed the tolerable daily intake (TDI) of 0.1mg/kg bw/day. In addition to coumarin, estragole, methyl-eugenol, (R)-(+)-pulegone and thujone were EU-regulated substances detected in thirty-one of the products. The non-EU regulated alkenylbenzenes, trans-anethole and myristicin, were commonly present in beverages and in herbs-containing products.

Inter-laboratory comparison study for pyrrolizidine alkaloids in animal feed using spiked and incurred material

Pyrrolizidine alkaloids (PAs) are hepatotoxic metabolites produced by plants. PAs in animal feed can cause acute or chronic intoxications in animals and can be transferred to milk. An inter-laboratory comparison study among 12 laboratories, using their own methods of analysis, was conducted for the detection and quantification of PAs in animal feed. The participants were asked to quantify PAs in a blank test sample, a blank test sample to be spiked with a provided spiking mixture of seven PA standards, and a test sample contaminated with common groundsel (Senecio vulgaris). Ten of the participating laboratories used an LC-MS/MS method, one used an LC-ToF-MS method, and one used a GC-MS method. None of the laboratories reported false-negative samples, while two laboratories reported false-positive results in the blank sample. z-scores were calculated for each laboratory for seven PAs in test samples B and C. z-scores varied considerably between laboratories for the concentrations of the free bases and less for the N-oxides, probably due to the lower levels of the free bases as compared with the N-oxides in the contaminated feed. Questionable or unsatisfactory results for the z-scores were obtained for 8% of the cases for the spiked sample and for 12% of the incurred sample. Three laboratories scored consequently positive or negative results. No preferred method for quantification of PAs in feed could be identified within the methods used for this study due to the relatively small number of participants. It was concluded that this inter-laboratory study shows that the methods used for PA detection need further development for accurate estimation of PAs in contaminated feed.

Fate of enniatins and deoxynivalenol during pasta cooking

The fate of deoxynivalenol and enniatins was studied during cooking of commercially available dry pasta in the Netherlands in 2014. Five samples containing relatively high levels of deoxynivalenol and/or enniatins were selected for the cooking experiment. Cooking was performed in duplicate on different days, under standardised conditions, simulating house-hold preparation. Samples were extracted with a mixture of acetonitrile/water followed by salt-induced partitioning. The extracts were analysed by LC-MS/MS. The method limits of detection were 8μg/kg for deoxynivalenol, 10μg/kg for enniatin A1 and 5μg/kg for enniatins A, B and B1. During the cooking of the five dry pasta samples, 60% of the deoxynivalenol and 83-100% of the enniatins were retained in the cooked pasta. It is recommended to study food processing fate of mycotoxins through naturally contaminated materials (incurred materials).

Are effects of common ragwort in the Ames test caused by pyrrolizidine alkaloids

It has previously been demonstrated by others that acetone extracts of Senecio jacobaea (syn. Jacobaea vulgaris, common or tansy ragwort) test positive in the Salmonella/microsome mutagenicity test (Ames test). Pyrrolizidine alkaloids (PAs) are thought to be responsible for these mutagenic effects. However, it was also observed that the major PA present in common ragwort, jacobine, produced a negative response (with and without the addition of rat liver S9) in Salmonella test strains TA98, TA100, TA1535 and TA1537. To investigate which compounds in the plant extracts were responsible for the positive outcome, the present study investigated the contents and mutagenic effects of methanol and acetone extracts prepared from dried ground S. jacobaea and Senecio inaequidens (narrow-leafed ragwort). Subsequently, a fractionation approach was set up in combination with LC-MS/MS analysis of the fractions. It was shown that the positive Ames test outcomes of S. jacobaea extracts are unlikely to be caused by PAs, but rather by the flavonoid quercetin. This study also demonstrates the importance of identifying compounds responsible for positive test results in bioassays.

Straightforward analytical method to determine opium alkaloids in poppy seeds and bakery products

A straightforward method to determine the content of six opium alkaloids (morphine, codeine, thebaine, noscapine, papaverine and narceine) in poppy seeds and bakery products was developed and validated down to a limit of quantification (LOQ) of 0.1mg/kg. The method was based on extraction with acetonitrile/water/formic acid, ten-fold dilution and analysis by LC-MS/MS using a pH 10 carbonate buffer. The method was applied for the analysis of 41 samples collected in 2015 in the Netherlands and Germany. All samples contained morphine ranging from 0.2 to 240mg/kg. The levels of codeine and thebaine ranged from below LOQ to 348mg/kg and from below LOQ to 106mg/kg, respectively. Sixty percent of the samples exceeded the guidance reference value of 4mg/kg of morphine set by BfR in Germany, whereas 25% of the samples did not comply with the limits set for morphine, codeine, thebaine and noscapine by Hungarian legislation.

Fate of pyrrolizidine alkaloids during processing of milk of cows treated with ragwort

ABSTRACT To investigate the fate of pyrrolizidine alkaloids (PAs) during milk processing, milk of cows treated via rumen fistula with a mixture of 84% (w/w) ragwort (Jacobaea vulgaris, syn. Senecio jacobaea) and 16% narrow-leaved ragwort (Senecio inaequidens) was processed using laboratory scale heating systems with industrial settings. Pasteurised and sterilised (UHT) milk were produced, as well as set-type yoghurt and cheese. Samples were analysed for 29 PAs using LC-MS/MS, of which 11 PAs were detected above LOQ in the samples (0.1 µg l−1). Alterations in the PA concentration and composition between the standardised milk and the corresponding end-product(s) were evaluated. The heat treatments applied for pasteurisation and UHT sterilisation to prepare semi-skimmed consumption milk did not affect the PA levels in the end-products. In yoghurt, after fermentation of standardised milk (6 h, pH 4.4), 73% of total PAs were recovered. The PA concentration, specifically dehydrojacoline, was decreased, although not quantifiable, during cheese production. A further decrease of 38% during 6 weeks of ripening was observed. The results show that the PA concentration of natural contaminated cow’s milk is not affected by heat treatment applied for pasteurised and sterilised milk, but that microbial fermentation of the milk leads to a lowered PA concentration in yoghurt and cheese. This is probably due to microbiological degradation, since PAs are fairly stable under acidic conditions.