Use of a mixed cationic-reverse phase column for analyzing small highly polar metabolic markers in biological fluids for multiclass lc-hrms method

The determination of small highly polar metabolites at low concentrations is challenging when reverse-phase (RP) chromatography is used for multiclass analysis. A mixed cationic-RP column coupled to high-resolution tandem MS (HR-MS/MS) was tested for highly polar compounds in biological fluids, i.e., trimethylamine N-oxide (TMAO) and the isobaric molecules beta-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB). The efficient retention and separation of the above compounds were obtained with common and MS-friendly RP conditions, reaching high selectivity and sensitivity. The method was firstly assessed in plasma and urine, showing good linearity in the range 50–1000 µg/L and 500–10,000 µg/L for TMAO and both BMAA and DAB, respectively. Excellent precision (RDS < 3%) and good accuracies (71–85%) were observed except for BMAA in plasma, whose experimental conditions should be specifically optimized. Preliminary tests performed on compounds with biological relevance and a wider range of polarities proved the effectiveness of this chromatographic solution, allowing the simultaneous analysis of a larger panel of metabolites, from very small and polar compounds, like trimethylamine, to quite lipophilic molecules, such as corticosterone. The proposed LC-HRMS protocol is an excellent alternative to hydrophilic interaction liquid chromatography and ion-pairing RP chromatography, thus providing another friendly analytical tool for metabolomics.