## 3 Results and Discussion
(**B**) EIC MS/MS chromatograms (accuracy: 10 ppm) obtained with Acclaim Mixed-Mode WCX-1: BMAA: EIC for _m/z_ 88.0394 (parent ion: _m/z_ 119.0815, NCE:35); DAB: EIC for _m/z_ 101.0709 (parent ion: _m/z_ 119.0815, NCE:35); TMAO: EIC for _m/z_ 58.0660 (parent ion: _m/z_ 85.1322, NCE:35); TMAO-q9 (IS): EIC for _m/z_ 68.1302 (parent ion: _m/z_ 85.1322, NCE:35). The concentration is 2500 ug/L for each analyte. of the performances. Matrix-matched calibration curves were performed in triplicate and were obtained by spiking blank samples at the same nominal concentrations of the external calibration curves. A very good linearity (R2 \(>\) 0.99) was observed for all the selected analytes in water, plasma and urine matrices. Precision, evaluated from the standard deviations of the regression slopes, was excellent and showed relative standard deviations (RSD%) \(<\) 3% for all the matrices taken into consideration. Matrix effects and recoveries were evaluated from the percent slope ratio of the matrix-matched and external standard calibration curves. Values within the range 70-110% are generally acceptable, as strong matrix effects and poor recoveries can be excluded. Considering the excellent precision of the method, it is possible to assume good recoveries and a limited matrix effect for BMAA and DAB in urine, as the values obtained for the percent slope ratio were 71% and 85%, respectively. Results obtained for plasma are acceptable for DAB (73%) and quite good for TMAO (81%). Such method performances were already proved to be reliable for the quantification of TMAO in mouse plasma (RSD of the IS \(<\) 15% for N = 77 samples) [19]. Nevertheless, the combined matrix effect, in terms of ESI signals variation and recovery, was not acceptable in plasma for BMAA, as values are lower than 50%. In this last case, probably due to the interfering compounds co-extracted from this complex matrix, it will be necessary to increase the dilution of the sample before analysis or modify the extraction procedure or the chromatographic conditions. The choice of a suitable IS could anyway improve its accuracy. LODs, assessed from the lowest point of the matrix-matched calibration curve, and corresponding to an S/N value of 3, were estimated to be 10 \(\mu\)g/L for BMAA and DAB and 2 \(\mu\)g/L for TMAO. Preliminary quantitative data in plasma and urine samples were obtained only for TMAO, which was present as an average (\(n=5\)) basal concentration of 121 \(\pm\) 8 \(\mu\)g/L in plasma and 193 \(\pm\) 9 \(\mu\)g/L in urine, respectively. BMAA and DAB were always lower than LODs in both plasma and urine samples, but the collection of specimens suspected to be positively correlated to BMAA has to be specifically planned, and it is beyond the focus of this work. Further tests are in progress in our laboratory using the chromatographic method herein reported in order to expand the panel of analytes potentially quantifiable using this mixed cationic-RP column. The novel set of analytes included: other isomers of BMAA, such as beta-amino-\(N\)-methylalanine and \(N\)-(2 aminoethyl) glycine; levodopa, carbidopa and dopamine, which are compounds involved in the [PERSON]'s disease, and may be useful for the assessment of the possible adverse effects of BMAA; trimethylamine, short-chain fatty acids, e.g., butyric, isobutyric, valeric, isovaleric, hexanoic and acetic acid are key compounds, together with TMAO, linked to the gut microbiota, whose alteration was recently associated with the development of type 2 diabetes and obesity [20]; metabolites such as picolinic and nicotinic acid, tryptophan, kynure acid, 3-hydroxykinurenine were selected as representative of other specific metabolic pathways, e.g., the tryptophan metabolism. Corticosterone, an important intermediate for the synthesis of glucocorticoid hormones in humans, was selected in order to evaluate the chromatographic interactions of the mixed-RP stationary phase with lipophilic substances.