Development, Validation and Application of a Targeted LC-MS Method for Quantification of Microcystins and Nodularin: Towards a Better Characterization of Drinking Water

peer reviewedCyanotoxins can be produced in surface waters by cyanobacterial blooms, mostly during summer and early autumn. Intoxications would result from consumption of water contaminated with the potent hepatotoxins, microcystins and nodularin. Therefore, the WHO has set a guideline value for drinking water quality concerning one congener of microcystin. Consequently, the design of a validated, public reference method to detect and quantify the hepatotoxins in drinking water is necessary. During this study, a method was developed to quantify cyanotoxins (eight microcystin congeners and nodularin) in water using liquid chromatography coupled with tandem mass spectrometry. Additionally, bottled and tap water samples were tested for the presence of cyanotoxins. No cyanotoxins were detected in any of the collected water samples. However, quality controls and the results of a proficiency test show the validity of the method.3. Good health and well-bein

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

peer reviewedFood 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.PhD Wannes Van Hassel3. Good health and well-bein

A Summer of Cyanobacterial Blooms in Belgian Waterbodies: Microcystin Quantification and Molecular Characterizations

In the context of increasing occurrences of toxic cyanobacterial blooms worldwide, their monitoring in Belgium is currently performed by regional environmental agencies (in two of three regions) using different protocols and is restricted to some selected recreational ponds and lakes. Therefore, a global assessment based on the comparison of existing datasets is not possible. For this study, 79 water samples from a monitoring of five lakes in Wallonia and occasional blooms in Flanders and Brussels, including a canal, were analyzed. A Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) method allowed to detect and quantify eight microcystin congeners. The mcyE gene was detected using PCR, while dominant cyanobacterial species were identified using 16S RNA amplification and direct sequencing. The cyanobacterial diversity for two water samples was characterized with amplicon sequencing. Microcystins were detected above limit of quantification (LOQ) in 68 water samples, and the World Health Organization (WHO) recommended guideline value for microcystins in recreational water (24 µg L−1) was surpassed in 18 samples. The microcystin concentrations ranged from 0.11 µg L−1 to 2798.81 µg L−1 total microcystin. For 45 samples, the dominance of the genera Microcystis sp., Dolichospermum sp., Aphanizomenon sp., Cyanobium/Synechococcus sp., Planktothrix sp., Romeria sp., Cyanodictyon sp., and Phormidium sp. was shown. Moreover, the mcyE gene was detected in 75.71% of all the water samples

Targeted and untargeted lipidomics studies : towards a better understanding of the role of lipids in inflammation

Lipids, and bioactive lipids in particular, are key players during inflammation. The study of their implications in the physiopathological processes linked to inflammation requires analytical strategies allowing for their quantification in biological matrices. Thus this thesis explored two approaches used to quantify metabolites in biological systems (i) an untargeted approach and (ii) a targeted approach. Using an HPLC-LTQ-Orbitrap XL we set-up an HPLC-ESI-MS method to analyze several phospholipids, lysophospholipids, (dihydro-) ceramides, galactosylceramides, (hydroxylated-) sulfatides, and fatty acids from the same biological sample. We then applied this method to the characterization of the effect of J774 macrophage activation on lipid levels. The other topic of this work revolves around the study of lysophosphatidylinositols (LPI) in the context of inflammation. Indeed these lipids are produced, among other pathways, through the activity of the phospholipase A2, known to be activated during inflammation. We started our investigations by setting up and validating an original HPLC-ESI-MS method for the quantification of lysophosphatidylinositols and applied it to the determination of their levels in mice tissues. Then, using this method we studied how macrophage activation in vitro and inflammation in vivo would affect lysophosphatidylinositol levels. To try to understand the causes of the variations in lipid levels, we also investigated the expression of the enzymes involved in lysophosphatidylinositol metabolism. The changes in lysophosphatidylinositol levels observed in macrophages prompted us to investigate whether lysophosphatidylinositols would be able to affect lipopolysaccharides-induced macrophage activation. Using J774 cells overexpressing the GPR55 receptor, we were also able to suggest that part of the effect was indeed mediated by GPR55 activation. Taken together these studies illustrate how developing lipid quantification methods will advance our understanding of their role in inflammatory settings.(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 201

Development and validation of a specific and sensitive HPLC-ESI-MS method for quantification of lysophosphatidylinositols and evaluation of their levels in mice tissues

Increasing evidence suggests that lysophosphatidylinositols (LPIs), a subspecies of lysophospholipids, are important endogenous mediators. Although LPIs long remained among the less studied lysophospholipids, the identification of GPR55 as their molecular target sparked a renewed interest in the study of these bioactive lipids. Furthermore, increasing evidence points towards a role for LPIs in cancer development. However, a better understanding of the role and functions of LPIs in physiology and disease requires methods that allow for the quantification of LPI levels in cells and tissues. Because dedicated efficient methods for quantifying LPIs were missing, we decided to develop and validate an HPLC-ESI-MS method for the quantification of LPI species from tissues. LPIs are extracted from tissues by liquid/liquid extraction, pre-purified by solid-phase extraction, and finally analyzed by HPLC-ESI-MS. We determined the method's specificity and selectivity, we established calibration curves, determined the carry over ( 80%), intermediate precision (CV<20%) as well as the recovery from tissues. We then applied the method to determine the relative abundance of the LPI species in 15 different mouse tissues. Finally, we quantified the absolute LPI levels in six different mouse tissues. We found that while 18:0 LPI represents more than 60% of all the LPI species in the periphery (e.g. liver, gastrointestinal tract, lungs, spleen) it is much less abundant in the central nervous system where the levels of 20:4 LPI are significantly higher. Thus this validated HPLC-ESI-MS method for quantifying LPIs represents a powerful tool that will facilitate the comprehension of the pathophysiological roles of LPIs

Lysophosphatidylinositols, from Cell Membrane Constituents to GPR55 Ligands

Lysophosphatidylinositols (LPIs) are membrane constituents that alter the properties of said membranes. However, recent data showing that the once orphan receptor, GPR55, can act as a receptor for LPIs has sparked a renewed interest in LPIs as bioactive lipids. As evidence supporting the importance of LPIs and/or GPR55 is continuously accumulating and because LPI levels are altered in a number of pathologies such as obesity and cancer, the coming years should bring new, exciting discoveries to this field. In this review, we discuss the recent work on LPIs and on their molecular target, the GPR55 receptor. First, we summarize the metabolism of LPIs before outlining the cellular pathways activated by GPR55. Then, we review the actions of LPIs and GPR55 that could have potential pharmacological or therapeutic applications in several pathophysiological settings, such as cancer, obesity, pain, and inflammation

Controlling 2-arachidonoylglycerol metabolism as an anti-inflammatory strategy

The endocannabinoid system is implicated in, and regulates, several physiological processes, ranging from food intake and energy balance to pain and inflammation. 2-Arachidonoylglycerol (2-AG) is a full agonist at the cannabinoid receptors which classically mediate its effects. The activity of this bioactive lipid is dependent on its endogenous levels, which are tightly controlled by several hydrolases, monoacylglycerol lipase and a/b-hydrolase domain 6 and 12. Moreover, 2-AG is also a substrate of cyclooxygenase-2, and this reaction leads to the formation of prostaglandin glycerol esters, the effects of which remain to be fully elucidated. In this review we discuss the multiple mechanisms by which 2-AG controls inflammation and the therapeutic potential of 2-AG metabolism inhibitors. 2-Arachidonoylglycerol metabolism: where the endocannabinoid and the eicosanoid systems meet The endocannabinoid 2-arachidonoylglycerol (2-AG) is a bioactive lipid activating the CB1 and CB2 cannabinoid receptors, following on-demand production (Box 1). As for many bioactive lipids, the activity of 2-AG is controlled by its tissue levels, and therefore by the balance between its production and degradation. For years, 2-AG metabolism was considered to be straightforward. Its primary biosynthetic route consists of an activity-dependent phospholipase C-b (PLCb) that releases diacylglycerol (DAG) from phosphatidylinositol- 4,5-bisphosphate, which in turn is metabolized by diacylglycerol lipases (DAGLs) – with DAGLa and DAGLb having prevalent roles in the brain and in several peripheral tissues, respectively – to produce 2-AG [1–4]. 2-AG is then inactivated through hydrolysis into arachidonic acid by monoacylglycerol lipase (MAGL). It is now clear that this description is over simplistic. Several elements need to be considered to obtain a full picture of 2-AG metabolism [5]. For instance, although MAGL is the main enzyme controlling 2-AG levels in numerous tissues, two additional a/b hydrolases – a/b-hydrolase domain (ABHD)6 and ABHD12 – can similarly hydrolyze 2-AG [6–8]. Beside these hydrolases, recent report

Analysis of Patulin in Apple Products Marketed in Belgium: Intra-Laboratory Validation Study and Occurrence

Apple and apple derivatives (e.g., juices, puree) are the most important foodstuffs contaminated with patulin (PAT) in the human diet. To routinely monitor these foodstuffs and ensure that the PAT levels are below the maximum permitted levels, a method using liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) has been developed. Afterwards, the method was successfully validated, reaching quantification limits of 1.2 μg/L for apple juice and cider, and 2.1 μg/kg for puree. Recovery experiments were performed with samples fortified with PAT in the range of 25–75 μg/L for juice/cider and 25–75 μg/kg for puree. The results show overall average recovery rates of 85% (RSDr = 13.1%) and 86% (RSDr = 2.6%) with maximum extended uncertainty (Umax, k = 2) of 34 and 35% for apple juice/cider and puree, respectively. Next, the validated method was applied to 103 juices, 42 purees and 10 ciders purchased on the Belgian market in 2021. PAT was not found in the cider samples, but it was present in 54.4% of the tested apple juices (up to 191.1 μg/L) and 7.1% of the puree samples (up to 35.9 μg/kg). When comparing the results to the maximum levels set by Regulation EC n° 1881/2006 (i.e., 50 μg/L for juices and 25 μg/kg for puree for adults, and 10 μg/kg for infants and young children), exceedances were observed in five apple juices and one puree sample, for infants and young children. Using these data, a potential risk assessment for consumers can be suggested, and it is found that the quality of apple juices and purees sold in Belgium needs further regular surveillance

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.</p

Towards a better quantification of cyanotoxins in fruits and vegetables : validation and application of an UHPLC-MS/MS-based method on Belgian products

Vegetables and fruits can potentially accumulate cyanotoxins after water contaminated with cyanobacteria is used for irrigation. We developed and validated an analytical method to quantify eight microcystin congeners (MCs) and nodularin (NOD) using ultra high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) in three different matrices. Strawberries, carrots and lettuce are selected as model matrices to represent the fruits/berries, leafy and root vegetables, sequentially. The validation of a UHPLC-MS/MS method in the strawberry matrix is novel. Matrix effects are observed in all three matrices. Our methodology uses matrix-matched calibration curves to compensate for the matrix effect. The implementation of our method on 103 samples, containing nine different sorts of fruits and vegetables from the Belgian market, showed no presence of MCs or NOD. However, the recoveries of our quality controls showed the effectiveness of our method, illustrating that the use of this method in future research or monitoring as well as in official food controls in fruit and vegetable matrices is valid