Abstract
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.
Highlights
The separation of small strongly polar compounds is challenging for reverse-phase (RP)chromatography
The chromatographic separation of BMAA, diaminobutyric acid (DAB) and trimethylamine N-oxide (TMAO) is hindered by poor retention in RP mode, and Hydrophilic interaction liquid chromatography (HILIC) conditions have been used to overcome these problems
The isobaric BMAA and DAB are greatly retained by this stationary phase, their separation is not effective
Summary
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
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