## 3 Results and Discussion  
The chromatographic separation of BMAA, DAB and TMAO is hindered by poor retention in RP mode, and HILIC conditions have been used to overcome these problems. Figure 1A shows the extracted ion chromatogram (EIC, mass accuracy 10 ppm) of [M+H]\({}^{+}\) precursor ions at _m/z_ 119.0815, 76.0757 and 85.1322 for isobaric BMAA and DAB, TMAO and TMAO-d9, respectively, obtained from the analysis of a working standard solution at 2500 \(\upmu\)g/L in HILIC mode. Although the isobaric BMAA and DAB are greatly retained by this stationary phase (rt = 7.03 and 7.17 min, respectively), their separation is not effective. Conversely, TMAO and TMAO-d9 are not retained and are detectable at the column dead time. Although several attempts were made to increase the efficiency of the BMAA and DAB separation and the TMAO retention by varying both additive type and concentration (in particular, by changing FA concentration from 10 mM to 25 mM and by substituting FA with ammonium acetate, from 1 mM to 10 mM in both water and acetonitrile), no significant improvements were obtained. The effective separation of TMAO, and/or BMAA and DAB using HILIC chromatography are anyway reported in the literature, but the proposed methods are based on stationary phases that are quite different from the one herein described, e.g., ZIC-HILIC [8], ethylene-bridged hybrid (BEH) particles [12] or amide-based stationary phase [17]. Better results, in terms of peaks separation and retention, were obtained with a mixed cationic-RP column in reverse-phase conditions, as reported in Figure 1B. In this case, the isobars BMAA (rt = 5.00 min) and DAB (rt = 5.30 min) were retained and satisfactorily separated. Even for TMAO and TMAO-d9 (rt = 7.29 min) retention was considerably improved. Greater sensitivity and selectivity can be achieved by acquiring data in high-resolution MS/MS mode, in particular for BMAA and DAB. The comparison between the MS/MS spectra of BMAA and DAB (Figure 2, panel A), shows the presence of two significantly different fragment ions for DAB and BMAA: signal at _m/z_ 88.0399 was related to the loss of substituted amino moiety of BMAA, and signal a _m/z_ 101.0714 due to the loss of the terminal NH\({}_{3}\) of DAB. Once selected the precursor ions, the EIC (mass accuracy: 10 ppm) related to the fragment ions at _m/z_ 58.0880, _m/z_ 68.1302, _m/z_ 101.0714 and _m/z_ 88.0399 for TMAO, TMAO-d9, DAB and BMAA, respectively, were acquired in parallel reaction monitoring mode (Figure 2, panel B), thus improving the MS sensitivity and the selectivity obtained by the chromatographic separation. The obtained separation is anyway necessary when lower selective detection systems, such as LC coupled to single-stage or low-resolution MS are used. In the last cases, the shared fragment ions can produce inaccuracies or false-positive results. Figure 1: (**A**) Overlapped EIC chromatograms (accuracy 10 ppm) for BMAA ([M+H]\({}^{+}\), _m/z_ 119.0815, black, rt = 7.03 min), DAB ([M+H]\({}^{+}\), _m/z_ 119.0815, black, rt = 7.17 min), TMAO ([M+H]\({}^{+}\), _m/z_ 76.0757.0815, red, rt = 1.27 min) and TMAO-d9 (IS) ([M+H]\({}^{+}\), _m/z_ 85.1322, green, rt = 1.27 min) obtained with Luna HILIC. (**B**) Overlapped EIC chromatograms (accuracy 10 ppm) for BMAA ([M+H]\({}^{+}\), _m/z_ 119.0815, black, rt = 5.00 min), DAB ([M+H]\({}^{+}\), _m/z_ 119.0815, black, rt = 5.30 min), TMAO ([M+H]\({}^{+}\), _m/z_ 76.0757.0815, red, rt = 7.32 min) and TMAO-d9 (IS) ([M+H]\({}^{+}\), _m/z_ 85.1322, green, rt = 7.29 min) obtained with and Acclaim Mixed-Mode WCX-1. The concentration is 2500 \(\mu\)g/L for each analyte. Figure 2: _Cont._  
The method linearity, precision and accuracy, the last intended as the combined contribution of matrix effects and recoveries, were evaluated in plasma and urine to perform a preliminary evaluation  
Figure 2: (**A**) Comparison between the MS/MS spectra of BMAA and DAB, both obtained in HCD mode with a normalized collision energy (NCE) of 35.