Simultaneous Profiling of Multiple Phosphorylated Metabolites in Typical Biological Matrices via Ion-Pair Reversed-Phase Ultrahigh-Performance Liquid Chromatography and Mass Spectrometry

Simultaneous analysis of multiple
phosphorylated metabolites (phosphorylated
metabolome) in biological samples is vital to reveal their physiological
and pathophysiological functions, which is extremely challenging due
to their low abundance in some biological matrices, high hydrophilicity,
and poor chromatographic behavior. Here, we developed a new method
with ion-pair reversed-phase ultrahigh-performance liquid chromatography
and mass spectrometry using BEH C18 columns modified with hybrid surface
technology. This method demonstrated good performances for various
phosphorylated metabolites, including phosphorylated sugars and amino
acids, nucleotides, NAD-based cofactors, and acyl-CoAs in a single
run using standard LC systems. Specifically, the method showed good
retention (capacity factor > 2) and reproducibility (ΔtR < 0.09 min, n = 6), peak
symmetry (tailing factor < 2), and sensitivity (limit-of-detection
< 238 fmol-on-column with QTOFMS) for all tested analytes especially
for the medium- and/or long-chain acyl-CoAs. The method demonstrated
reproducible applicability across numerous biological matrices, including
tissue (liver), human biofluids (urine, plasma), cells, and feces,
and revealed significant molecular phenotypic differences in phosphorylated
metabolite composition.