Angelika Preiss-Weigert

Determination of pyrrolizidine alkaloids in tea, herbal drugs and honey

Honey was previously considered to be one of the main food sources of human pyrrolizidine alkaloid (PA) exposure in Europe. However, comprehensive analyses of honey and tea sampled in the Berlin retail market revealed unexpected high PA amounts in teas. This study comprised the analysis of 87 honey as well as 274 tea samples including black, green, rooibos, melissa, peppermint, chamomile, fennel, nettle, and mixed herbal tea or fruit tea. Total PA concentrations in tea ranged from < LOD to 5647 µg kg−1, while a mean value of about 10 µg kg−1 was found in honey samples. Additionally, herbal drugs were investigated to identify the source of PA in teas. Results suggest that PA in tea samples are most likely a contamination caused by co-harvesting of PA-producing plants. In some cases such as fennel, anise or caraway, it cannot be excluded that these plants are able to produce PA themselves.

Sensitive method for the determination of lipophilic marine biotoxins in extracts of mussels and processed shellfish by high-performance liquid chromatography-tandem mass spectrometry based on enrichment by solid-phase extraction.

A solid-phase extraction (SPE) method for the enrichment and clean-up of lipophilic marine biotoxins from extracts of different species of bivalve molluscs and processed shellfish products was developed. Okadaic acid (OA), pectenotoxin2 (PTX2), azaspiracid1 (AZA1) and yessotoxin (YTX) were determined by LC-MS/MS in hydrolyzed and non-hydrolyzed extracts. Applying a concentration factor of 10 the limit of quantification for the four toxins was determined to be 1 microg/kg. An organized in-house ring trial proved transferability of the method protocol and satisfactory results for all four toxins with a relative standard deviation (RSD) of 5-12%. The precision of the whole method including LC-MS detection was determined by processing seven independent extractions analyzed in independent sequences. RSD ranged between 12% and 24%. This SPE method was tested within a concentration range corresponding to the range of the current European Union regulatory limits (up to 160 microg/kg for the OA group), but it would also be applicable to a lower microg/kg range which is important in view of a possible decrease of regulatory limits as proposed by a working group of the European Food Safety Authority. The potential of SPE as a cleaning tool to cope with matrix effects in LC-MS/MS was studied and compared to liquid-liquid portioning.

A basic tool for risk assessment : A new method for the analysis of ergot alkaloids in rye and selected rye products

As a basis for the collection of occurrence and exposure data of ergot alkaloids in food, an HPLC method coupled with fluorimetric detection (HPLC-FLD) for the determination of 12 pharmacologically active ergot alkaloids in rye and rye products was developed. Samples were extracted with a mixture of ethyl acetate, methanol, and aqueous ammonia, followed by centrifugation and purification by solid phase filtration (SPF) with basic alumina. After solvent adjustment, the samples were analyzed by HPLC-FLD using a phenyl-hexyl-column. Recoveries for five major alkaloids were between 89.3% (ergotamine) and 99.8% (alpha-ergokryptine) with a maximum LOQ of 3.3 microg/kg (ergometrine). Precision expressed as RSD ranged from 2.8% (ergocristine) to 12.4% (alpha-ergokryptine) for repeatability, and from 6.5% (ergocornine) to 14.9% (ergotamine) for within-laboratory reproducibility, respectively. In a survey of 39 rye product samples, ergocristine and ergotamine were found to be the major alkaloids in commercially available rye products with contents of 127 microg/kg (ergocristine), and 134 microg/kg (ergotamine) in rye flour, and 152.5 and 117.8 microg/kg in coarse meal, respectively.

Identification Strategy Using Combined Mass Spectrometric Techniques for Elucidation of Phase I and Phase II in Vitro Metabolites of Lipophilic Marine Biotoxins

Combining mass spectrometric tools, a total of 47 in vitro metabolites of okadaic acid (OA), dinophysistoxins 1 and 2 (DTX1 and DTX2), yessotoxin (YTX), azaspiracid1 (AZA1), and pectenotoxin 2 (PTX2) could be detected and confirmed after an incubation with rat liver S9-mix. In a first step, liquid chromatography (LC) combined with tandem mass spectrometry (MS/MS) was used as a screening tool for the identification of in vitro metabolites of lipophilic marine biotoxins. Metabolic phase I and phase II reactions were screened for metabolites by calculating and subsequently monitoring theoretical MS transitions. In a second step, metabolites were confirmed by determination of accurate masses using high resolution MS provided by Orbitrap technology. Subsequently, product ion spectra, precursor ion spectra, and MS3 spectra were recorded for structure elucidation of metabolites. While all investigated toxins were found to form various oxygenated metabolites during the oxidative phase I metabolism, those metabolites varied in the number of added oxygen atoms and in the number of individual isomers. No hints were obtained concerning the formation of glutathione adducts, and a conjugation with glucuronic acid was detected for AZA1 only.

Structure–activity relationship in the passage of different pyrrolizidine alkaloids through the gastrointestinal barrier: ABCB1 excretes heliotrine and echimidine

SCOPE 1,2-Unsaturated pyrrolizidine alkaloids (PA) are found in plants such as Asteraceae and Boraginaceae families. Acute PA poisoning via contaminated food or feed causes severe damage to liver depending on species-specific oral bioavailability. For assessing PA bioavailability, their passage across the intestinal barrier was investigated using Caco-2 cells. METHODS Differentiated Caco-2 cells were exposed in transport chambers to the PA heliotrine (Hn), echimidine (Em), senecionine (Sc), and senkirkine (Sk). Cell supernatants were analyzed by LC-MS/MS. RESULTS PA pass Caco-2 monolayer from the apical into basolateral compartment depending on their chemical structure. Compared to the cyclic diesters Sc and Sk with a passage rate of 47% ± 4 and 40% ± 3, respectively, the transferred amount of the monoester Hn (32% ± 3) and open-chained diester Em (13% ± 2) was substantially lower. This suggested an active transport of Hn and Em. Using Madin-Darby canine kidney II/P-glycoprotein (ABCB1)-overexpressing cells, the active excretion of Hn and Em by ABCB1 from the gastrointestinal epithelium into the gut lumen was shown. CONCLUSION PA cross the intestinal barrier structure-dependently. The passage of the noncyclic PA Hn and Em is reduced by an ABCB1-driven efflux into the gastrointestinal lumen resulting in a decreased oral bioavailability.

Active elimination of the marine biotoxin okadaic acid by P-glycoprotein through an in vitro gastrointestinal barrier

The consumption of okadaic acid (OA) contaminated shellfish can induce acute toxic symptoms in humans such as diarrhea, nausea, vomiting and abdominal pain; carcinogenic and embryotoxic effects have also been described. Toxicokinetic studies with mice have shown that high cytotoxic doses of OA can pass the gastrointestinal barrier presumably by paracellular passage. However, in vitro studies using human intestinal Caco-2 cell monolayers to represent the intestinal barrier have shown that at low-dose exposure OA is transported against a concentration gradient suggesting an active efflux mechanism. Since P-glycoprotein (P-gp) transports a wide variety of substrates, we investigated its possible influence on the observed elimination of OA. We used two different cellular transwell models: (i) Caco-2 cell monolayer endogenously expressing human P-gp and simulating the intestinal barrier and (ii) MDCK-II cell monolayer stably over-expressing P-gp. Our study demonstrates clearly that OA at non-cytotoxic concentrations passes the monolayer barrier only to a low degree, and that it is actively eliminated by P-gp over the apical membrane. Therefore, our in vitro data indicate that humans appear to have efficient defense mechanisms to protect themselves against low-dose contaminated shellfish by exhibiting a low bioavailability as a result of active elimination of OA by P-gp.

Carryover of maduramicin from feed containing cross-contamination levels into eggs of laying hens

Maduramicin is a coccidiostat authorized as feed additive in the European Union for chickens and turkeys for fattening but not for laying hens, considering the risk of residues in eggs. The unavoidable cross-contamination of non-target feed with coccidiostats is regulated by Commission Directive 2009/8/EC and resulting carry-over in food by Commission Regulation (EC) No. 124/2009. To verify the compliance of the maximum levels for maduramicin in feed (50 μg/kg) and eggs (2 μg/kg), the carry-over from feed into eggs was investigated. Diets containing 10, 30, and 50 μg of maduramicin/kg of feed were fed to laying hens. Feed, egg white, and yolk were analyzed by LC-MS/MS. Maduramicin residues were only detected in in egg yolk. Feeding the 10 μg/kg maduramicin diet resulted in maduramicin concentrations up to 2.5 μg/kg in whole eggs, already exceeding the maximum level. A carry-over rate of 8% maduramicin from feed into eggs was calculated.

Occurrence of pyrrolizidine alkaloids in animal- and plant-derived food: results of a survey across Europe

ABSTRACT Pyrrolizidine alkaloids (PAs) are secondary metabolites of plant families such as Asteraceae or Boraginaceae and are suspected to be genotoxic carcinogens. Recent investigations revealed their frequent occurrence in honey and particularly in tea. To obtain a comprehensive overview of the PA content in animal- and plant-derived food from the European market, and to provide a basis for future risk analysis, a total of 1105 samples were collected in 2014 and 2015. These comprised milk and milk products, eggs, meat and meat products, (herbal) teas, and (herbal) food supplements collected in supermarkets, retail shops, and via the internet. PAs were detected in a large proportion of plant-derived foods: 91% of the (herbal) teas and 60% of the food supplements contained at least one individual PA. All types of (herbal) teas investigated were found to contain PAs, with a mean concentration of 460 µg kg−1 dry tea (corresponding to 6.13 µg L−1 in [herbal] tea infusion). The highest mean concentrations were found in rooibos tea (599 µg kg−1 dry tea, 7.99 µg L−1 tea infusion) and the lowest in camomile tea (274 µg kg−1 dry tea, 3.65 µg L−1 tea infusion). Occurrence of PAs in food supplements was found to be highly variable, but in comparable ranges as for (herbal) tea. The highest concentrations were present in supplements containing plant material from known PA-producing plants. In contrast, only 2% of the animal-derived products, in particular 6% of milk samples and 1% of egg samples, contained PAs. Determined levels in milk were relatively low, ranged between 0.05 and 0.17 µg L−1 and only trace amounts of 0.10–0.12 µg kg−1 were found in eggs. No PAs were detected in the other animal-derived products.

In vitro biotransformation of pyrrolizidine alkaloids in different species. Part I: Microsomal degradation

Pyrrolizidine alkaloids (PA) are secondary metabolites of certain flowering plants. The ingestion of PAs may result in acute and chronic effects in man and livestock with hepatotoxicity, mutagenicity, and carcinogenicity being identified as predominant effects. Several hundred PAs sharing the diol pyrrolizidine as a core structure are formed by plants. Although many congeners may cause adverse effects, differences in the toxic potency have been detected in animal tests. It is generally accepted that PAs themselves are biologically and toxicologically inactive and require metabolic activation. Consequently, a strong relationship between activating metabolism and toxicity can be expected. Concerning PA susceptibility, marked differences between species were reported with a comparatively high susceptibility in horses, while goat and sheep seem to be almost resistant. Therefore, we investigated the in vitro degradation rate of four frequently occurring PAs by liver enzymes present in S9 fractions from human, pig, cow, horse, rat, rabbit, goat, and sheep liver. Unexpectedly, almost no metabolic degradation of any PA was observed for susceptible species such as human, pig, horse, or cow. If the formation of toxic metabolites represents a crucial bioactivation step, the found inverse conversion rates of PAs compared to the known susceptibility require further investigation.

A case of human poisoning by grayanotoxins following honey ingestion: elucidation of the toxin profile by mass spectrometry

High-resolution mass spectrometry (HRMS) was applied for the detection of grayanotoxins (GrTx) in a contaminated honey sample. This sample was provided by a hospital due to a suspicion of intoxication after a patient had shown the typical symptoms of GrTx poisoning. Subsequent analysis proved the contamination with high amounts of GrTx and other toxins belonging to grayanane-type diterpenoids. This group of natural toxins is synthesised by the plant family Ericaceae and comprises more than 60 individual toxins, but only one compound is available as a reference standard. We applied a screening approach that easily confirms the presence or absence of GrTx without access to standards. By searching for predictable mass spectrometric fragment ions, including typical in-source fragments arising from collision-induced dissociation during electrospray ionisation, the complete toxin profile was screened and allowed the mass spectrometric identification of 15 individual GrTx. The potential of this approach is especially demonstrated by the fact that at least two of these toxins have not been previously described in the literature. A semi-quantitative estimation indicated a total toxin concentration of 358 mg kg−1. An investigation of 49 honeys from the German retail market did not reveal the presence of GrTx. Graphical Abstract