## 1 Introduction  
The separation of small strongly polar compounds is challenging for reverse-phase (RP) chromatography. The weak interaction between the hydrophobic stationary phase and analytes usually leads to short retention times, matrix interferences and poor selectivity [1]. Derivatization steps and ion-pairing chromatography are possible solutions. However, derivatization procedures are usually time-consuming, and both reagents and secondary reactions may interfere with the analysis. Ion pairing is another useful trick but may result in low sensitivity with LC-MS. Hydrophilic interaction liquid chromatography (HILIC) is nowadays a quite widespread approach for the analysis of highly polar molecules such as amino acids, saccharides, nucleic acids, and phosphate-containing molecules [2]. In this case, very common drawbacks are peak distortion, large consumption of organic solvents, long equilibration times, and lack of solubility of some polar compounds in organic solvents. Mixed-mode liquid chromatography is an interesting alternative to the RP and HILIC stationary phases. These phases, firstly described in 1984 by [PERSON] and [PERSON] [3], can be very effective for the simultaneous analysis of polar and non-polar compounds, due to the combined effects of different interaction mechanisms [4]. Beta-\(N\)-methylamino-t-alanine (BMAA) is a polar amino acid with a supposed neurotoxic activity. BMAA was described as a metabolite of several cyanobacterial strains, which can be bioaccumulated and biomagnified across the food chain [5]. Chronic exposure to BMAA has been associated with degenerative neurological conditions such as amyotrophic sclerosis, Parkinson's disease and dementia [6]. The risk assessment of BMAA is highly debated in the scientific literature since results are difficult to compare, because of the possible false-positives obtained by using different analytical techniques [7]. The main issue is the presence of several isomers of BMAA, such as 2,4-diaminobutyruc acid (DAB), so that the chromatographic separation and the correct quantification in biological and food samples remain challenging [8; 9]. Most of the existing analytical procedures are based on the derivatization of the analytes with 6-aminoquinolonyl-\(N\)-hydroxysuccinidyl prior to RP chromatography, or HILIC and ion-pairing RP separation without derivatization [5; 8; 9; 10; 11]. Solid-phase extraction performed using strong cation exchange sorbents is also reported for improving the sensitivity of the method and for samples clean up [12]. Recent works take advantage of tandem MS to improve the selectivity of the method and avoid false positives [13]. Trimethylamine \(N\)-oxide (TMAO) is a highly polar metabolite whose production is controlled by gut microbiota and liver enzymes. The production of TMAO is strictly related to the dietary consumption of t-carnitine and lectin rich food. Recently, the alteration of TMAO concentration in plasma was positively associated with an increased risk for cardiovascular diseases, heart failure, obesity, impaired glucose tolerance, diabetes, and colorectal cancer [14; 15]. The quantitative analysis of TMAO is usually performed with RP or HILIC chromatography coupled to electrospray (ESI) mass spectrometry [16; 17; 18]. BMAA and TMAO, such as other polar metabolites, are potentially useful biomarkers to be monitored in various biological fluids, but the experimental conditions conventionally used for RP mode can inhibit the simultaneous multiclass determination of very different compounds, which is a goal of the metabolomic approach. In this preliminary research work, a new separation approach, based on the use of a mixed cation-RP stationary phase, is evaluated to obtain the best retention and selectivity in the direct analysis of highly polar compounds such as TMAO, BMAA, and DAB. The analytical method is based on LC coupled to high-resolution tandem mass spectrometry (LC-HR-MS/MS) and applied to various biological matrices, such as plasma and urine, for clinical applications.