## 4 Conclusions  
In this work, the effective chromatographic retention of selected highly polar metabolites was carried out by using a mixed cationic-RP column, simultaneously obtaining an efficient separation of  
Figure 3: EIC chromatograms obtained with an Acclaim Mixed-Mode WCX-1 in ESI(+)-HRMS. (**A**) Trimethylamine, [M+H]\({}^{+}\), EIC at _m\(\%\)_ 60.0808. (**B**) TMAO, [M+H]\({}^{+}\), EIC at _m\(\%\)_ 76.0757. (**C**) TMAO-49 (IS), [M+H]\({}^{+}\), EIC at _m\(\%\)_ 85.1322. (**D**) BMAA (left peak) and DAB (right peak), [M+H]\({}^{+}\), EIC at _m\(\%\)_ 119.0815. (**E**) Kynurecid, [M+H]\({}^{+}\), EIC at _m\(\%\)_ 190.0499. (**F**) Dopamine, [M+H]\({}^{+}\), EIC at _m\(\%\)_ 154.0863. (**G**) Homocysteine, [M+H]\({}^{+}\), EIC at _m\(\%\)_ 136.0427. (**H**) Carbidopa, [M+H]\({}^{+}\), EIC at _m\(\%\)_ 227.1026. (**I**) Picolinic acid, [M+H]\({}^{+}\), EIC at _m\(\%\)_ 124.0393. (**L**) L-DOPA, [M+H]\({}^{+}\), EIC at _m\(\%\)_ 198.0761. (**M**) 3-hydroxykynure, [M+H]\({}^{+}\), EIC at _m\(\%\)_ 225.0870. (**N**) Corticosterone, [M+H]\({}^{+}\), EIC at _m\(\%\)_ 347.2217. Mass accuracy: 10 ppm. The concentration is 2500 \(\upmu\)g/L for each analyte. the isobaric BMAA and DAB without derivatization and ion pairing. The selectivity of the method was increased by HR tandem MS, avoiding the contribution of the partial co-eluted peaks. A preliminary evaluation of the method performances showed good linearity, acceptable recoveries and matrix effects for all the analytes in urine, and DAB and TMAO in plasma. The full validation of the method, including the assessment of the LOD, LOQ, repeatability and reproducibility, is in progress. Further evaluation of the column retention and selectivity started on a larger panel of analytes with different chemical properties and related to the metabolism of tryptophan, the short-chain fatty acids, and other isomers of BMAA and molecules related to the Parkinson disease. The versatility of this alternative chromatographic method is of particular interest in the field of metabolomics, where it is essential to analyze simultaneously various classes of molecules with very different chemical properties, in terms of polarity and molecular weight. Conceptualization, M.R., S.B. and I.M.D.G. ; methodology, M.R., S.B. and I.M.D.G. ; validation, M.R. and I.M.D.G. ; formal analysis, M.R. and I.M.D.G. ; investigation, M.R., S.B. and I.M.D.G. ; resources, S.B. and P.P. ; data curation, M.R., G.F. and I.M.D.G. ; writing--original draft preparation, M.R., S.B. and G.F.; writing--review and editing, M.R., S.B. and P.P. ; visualization, S.B. and P.P. ; supervision, S.B. ; funding acquisition, S.B. and P.P. All authors have read and agreed to the published version of the manuscript. This research received no external funding. The authors are grateful to the research group of [PERSON] (University of Padua) for collaboration with the TMAO project. The authors declare they have no conflicts of interest. References  
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