Polarity-extended liquid chromatography-triple quadrupole mass spectrometry for simultaneous hydrophilic and...

Although various metabolomic methods have been reported in recent years, simultaneous detection of hydrophilic and hydrophobic metabolites in a single analysis remains a technical challenge. In this study, based on the combination of hydrophilic interaction liquid chromatography (HILIC) and reversed phase liquid chromatography (RPLC), an online two-dimensional liquid chromatography/triple quadrupole mass spectrometry method (2D-LC/TQMS) was developed for the simultaneous analysis of hydrophilic and hydrophobic metabolites of various biological samples. The method can measure 417 biologically important metabolites (e.g., amino acids and peptides, pyrimidines, purines, monosaccharides, fatty acids and conjugates, organic dicarboxylic acids, and others) with logP values ranging from -10.3 to 21.9. The metabolites are involved in a variety of metabolic pathways (e.g., purine metabolism, pyrimidine metabolism, tyrosine metabolism, galactose metabolism, gluconeogenesis, and TCA cycle). The developed method has good intra- and inter-day reproducibility (RSD of retention time <2%, RSD of peak area <30%), good linearity (R² > 0.9) and wide linear range (from 0.0025 μg/mL to 5 μg/mL). The applicability of the method was tested using different biological samples (i.e., plasma, serum, urine, fecal, seminal plasma and liver) and it was found that 208 (out of 417) identical metabolites were detected in all biological samples. Furthermore, the metabolomic method was applied to a case/control study of urinary of bladder cancer. Thirty differential metabolites were identified that were involved in carbohydrate and amino acid metabolism.