LC-MS/MS Validation and Quantification of Cyanotoxins in Algal Food Supplements from the Belgium Market and Their Molecular Origins.

Food supplements are gaining popularity worldwide. However, harmful natural compounds can contaminate these products. In the case of algae-based products, the presence of toxin-producing cyanobacteria may cause health risks. However, data about the prevalence of algal food supplements on the Belgian market and possible contaminations with cyanotoxins are scarce. Therefore, we optimized and validated a method based on Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry to quantify eight microcystin congeners and nodularin in algal food supplements. Our analytical method was successfully validated and applied on 35 food supplement samples. Nine out of these samples contained microcystin congeners, of which three exceeded 1 µg g-1, a previously proposed guideline value. Additionally, the mcyE gene was amplified and sequenced in ten products to identify the taxon responsible for the toxin production. For seven out of these ten samples, the mcyE gene could be amplified and associated to Microcystis sp. EFSA and posology consumption data for algal-based food supplements were both combined with our toxin prevalence data to establish different toxin exposure scenarios to assess health risks and propose new guideline values.

Citrinin Determination in Food and Food Supplements by LC-MS/MS: Development and Use of Reference Materials in an International Collaborative Study.

The development of incurred reference materials containing citrinin (CIT) and their successful application in a method validation study (MVS) in order to harmonize CIT determination in food and food supplements are demonstrated. CIT-contaminated materials made of red yeast rice (RYR), wheat flour, and Ginkgo biloba leaves (GBL), as well as food supplements made of red yeast rice (FS-RYR) and Ginkgo biloba leaves (FS-GBL), were manufactured in-house via fungal cultivation on collected raw materials. The homogeneity and stability from randomly selected containers were verified according to the ISO 13528. CIT was found to be homogenously distributed and stable in all contaminated materials, with no significant degradation during the timescale of the MVS when storage was performed up to +4 °C. Next, an MVS was organized with eighteen international laboratories using the provided standard operating procedure and 12 test materials, including three RYRs (blank, <50 µg/kg, <2000 µg/kg), two wheat flours (blank, <50 µg/kg), two GBL powders (blank, <50 µg/kg), three FS-RYRs (blank, <50 µg/kg, <2000 µg/kg), and two FS-GBLs (blank, <50 µg/kg). The results of seven CIT-incurred materials showed acceptable within-laboratory precision (RSDr) varying from 6.4% to 14.6% and between-laboratory precision (RSDR) varying from 10.2% to 37.3%. Evidenced by HorRat values < 2.0, the results of the collaborative trial demonstrated that the applied analytical method could be standardized. Furthermore, the appropriateness of producing CIT reference materials is an important step towards food and feed quality control systems and the organization of proficiency tests.

Development and Validation of a UHPLC-ESI-MS/MS Method for Quantification of Oleandrin and Other Cardiac Glycosides and Evaluation of Their Levels in Herbs and Spices from the Belgian Market.

Cardiac glycosides (CGs) are naturally occurring plant secondary metabolites that can be toxic to humans and animals. The aim of this work was to develop a targeted analytical method utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS) for quantification of these plant toxins in a herbal-based food and human urine. The method included oleandrin, digoxin, digitoxin, convallatoxin, and ouabain. Samples of culinary herbs were extracted with acetonitrile and cleaned using Oasis® MAX solid-phase extraction (SPE), while samples of urine were diluted with acidified water and purified on Oasis® HLB SPE cartridges. Limits of quantification were in the range of 1.5-15 ng/g for herbs and 0.025-1 ng/mL for urine. The mean recovery of the method complied with the acceptable range of 70-120% for most CGs, and relative standard deviations were at maximum 14% and 19% for repeatability and reproducibility, respectively. Method linearity was good with calculated R² values above 0.997. The expanded measurement uncertainty was estimated to be in the range of 7-37%. The LC-MS/MS method was used to examine 65 samples of culinary herbs and herb and spice mixtures collected in Belgium, from supermarkets and local stores. The samples were found to be free from the analyzed CGs.

Non-invasive in vivo imaging of early metabolic tumor response to therapies targeting choline metabolism.

The cholinic phenotype, characterized by elevated phosphocholine and a high production of total-choline (tCho)-containing metabolites, is a metabolic hallmark of cancer. It can be exploited for targeted therapy. Non-invasive imaging biomarkers are required to evaluate an individual's response to targeted anticancer agents that usually do not rapidly cause tumor shrinkage. Because metabolic changes can manifest at earlier stages of therapy than changes in tumor size, the aim of the current study was to evaluate (1)H-MRS and diffusion-weighted MRI (DW-MRI) as markers of tumor response to the modulation of the choline pathway in mammary tumor xenografts. Inhibition of choline kinase activity was achieved with the direct pharmacological inhibitor H-89, indirect inhibitor sorafenib and down-regulation of choline-kinase α (ChKA) expression using specific short-hairpin RNA (shRNA). While all three strategies significantly decreased tCho tumor content in vivo, only sorafenib and anti-ChKA shRNA significantly repressed tumor growth. The increase of apparent-diffusion-coefficient of water (ADCw) measured by DW-MRI, was predictive of the induced necrosis and inhibition of the tumor growth in sorafenib treated mice, while the absence of change in ADC values in H89 treated mice predicted the absence of effect in terms of tumor necrosis and tumor growth. In conclusion, (1)H-choline spectroscopy can be useful as a pharmacodynamic biomarker for choline targeted agents, while DW-MRI can be used as an early marker of effective tumor response to choline targeted therapies. DW-MRI combined to choline spectroscopy may provide a useful non-invasive marker for the early clinical assessment of tumor response to therapies targeting choline signaling.