Comparison of sample preparation methods, validation of an UPLC-MS/MS procedure for the quantification of tetrodotoxin present in marine gastropods and analysis of pufferfish

Tetrodotoxin (TTX) is one of the most potent marine neurotoxins reported. The global distribution of this toxin is spreading with the European Atlantic coastline now being affected. Climate change and increasing pollution have been suggested as underlying causes for this. In the present study, two different sample preparation techniques were used to extract TTX from Trumpet shells and pufferfish samples. Both extraction procedures (accelerated solvent extraction (ASE) and a simple solvent extraction) were shown to provide good recoveries (80-92%). A UPLC-MS/MS method was developed for the analysis of TTX and validated following the guidelines contained in the Commission Decision 2002/657/EC for chemical contaminant analysis. The performance of this procedure was demonstrated to be fit for purpose. This study is the first report on the use of ASE as a mean for TTX extraction, the use of UPLC-MS/MS for TTX analysis, and the validation of this method for TTX in gastropods.

Comparison of single and multi-analyte methods based on LC-MS/MS for mycotoxin biomarker determination in human urine

The performances of four LC-MS/MS methodologies for determination of up to eight mycotoxin biomarkers in human urines were compared by involving three laboratories that analysed common urine samples spiked at two levels of each biomarker. Each laboratory received a calibration solution, spiked urines and the corresponding unspiked urine. The two spiking levels for each biomarker were chosen by considering the levels naturally occurring in human urines and the limits of quantification of the LC-MS/MS methodologies used by the participating laboratories. The results of each laboratory were evaluated for their z-score values. The percentage of satisfactory z-scores (vertical bar z vertical bar 2) were obtained for fumonisin B-1 (7/12 results), ochratoxin A (4/8 results) and alpha-zearalenol (1/8 results). The percentage of satisfactory z-scores for fumonisin B-1 and ochratoxin A increased from 42 to 83% for fumonisin B-1 and from 50 to 62% for ochratoxin A when laboratories 1 and 2 used own calibrants. Factors that could explain the different results obtained for fumonisin B-1 and ochratoxin A with provided and own calibration solutions could not be identified in this study and should be carefully investigated in future studies.

Determination of the Mycotoxin Content in Distiller's Dried Grain with Solubles Using a Multianalyte UHPLC-MS/MS Method

There are more than 300 potential mycotoxins that can contaminate food and feed and cause adverse effects in humans and animals. The data on the co-occurrence of mycotoxins in novel animal feed materials, such as distiller's dried grain with solubles (DDGS), are limited. Thus, a UHPLC-MS/MS method for the quantitation of 77 mycotoxins and other fungal metabolites was used to analyze 169 DDGS samples produced from wheat, maize, and barley and 61 grain samples. All DDGS samples analyzed were contaminated with 13-34 different mycotoxins. Fumonisins were present in all 52 maize DDGS samples (81.0-6890 μg/kg for fumonisin B1), and deoxynivalenol was present in all 99 wheat DDGS samples (39.3-1120 μg/kg). A number of co-occurring mycotoxins were also identified. Due to the high co-occurrence of mycotoxins, routine screening of the animal feed ingredients is highly recommended to allow the highlighted risks to be effectively managed.

Detection of Tetrodotoxins in Puffer Fish by a Self-Assembled Monolayer-Based Immunoassay and Comparison with Surface Plasmon Resonance, LC-MS/MS, and Mouse Bioassay

The increasing occurrence of puffer fish containing tetrodotoxin (TTX) in the Mediterranean could represent a major food safety risk for European consumers and threaten the fishing industry. The work presented herein describes the development of a new enzyme linked immunosorbent assay (mELISA) based on the immobilization of TTX through dithiol monolayers self-assembled on maleimide plates, which provides an ordered and oriented antigen immobilization and favors the antigen-antibody affinity interaction. The mELISA was found to have a limit of detection (LOD) of TTX of 0.23 mg/kg of puffer fish matrix. The mELISA and a surface plasmon resonance (SPR) immunosensor previously developed were employed to establish the cross-reactivity factors (CRFs) of 5,6,11-trideoxy-TTX, 5,11-deoxy-TTX, 11-nor-TTX-6-ol, and 5,6,11-trideoxy-4-anhydro-TTX, as well as to determine TTX equivalent contents in puffer fish samples. Results obtained by both immunochemical tools were correlated (R(2) = 0.977). The puffer fish samples were also analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the corresponding CRFs were applied to the individual TTX contents. Results provided by the immunochemical tools, when compared with those obtained by LC-MS/MS, showed a good degree of correlation (R(2) = 0.991 and 0.979 for mELISA and SPR, respectively). The mouse bioassay (MBA) slightly overestimated the CRF adjusted TTX content of samples when compared with the data obtained from the other techniques. The mELISA has been demonstrated to be fit for the purpose for screening samples in monitoring programs and in research activities.

Wide-ranging alterations in the brain fatty acid complement of subjects with late Alzheimer’s disease as detected by GC-MS

Disturbed lipid metabolism is a well-established feature of human Alzheimer’s disease (AD). The present study used gas chromatography-mass spectrometry (GC-MS) analysis of fatty acid methyl esters (FAMES) to profile all detectable fatty acid (FA) species present in post-mortem neocortical tissue (Brodmann 7 region). Quantitative targeted analysis was undertaken from 29 subjects (n=15 age-matched controls; n=14 late-stage AD). GC-MS analysis of FAMES detected a total of 24 FAs and of these, 20 were fully quantifiable. The results showed significant and wide ranging elevations in AD brain FA concentrations. A total of 9 FAs were elevated in AD with cis-13,16-docosenoic acid increased most (170%; P=0.033). Intriguingly, docosahexanoic acid (DHA; C22:6) concentrations were elevated (47%; P=0.018) which conflicts with the findings of others (unaltered or decreased) in some brain regions after the onset of AD. Furthermore, our results appear to indicate that subject gender influences brain FA levels in AD subjects (but not in age-matched control subjects). Among AD subjects 7 FA species were significantly higher in males than in females. These preliminary findings pinpoint FA disturbances as potentially important in the pathology of AD. Further work is required to determine if such changes are influenced by disease severity or different types of dementia.