Hydrophilic interaction liquid chromatography-tandem mass spectrometry for the quantitative analysis of mammalian-derived inositol poly/pyrophosphates

Although myo-inositol pyrophosphates such as diphosphoinositol pentakisphosphate (InsP7) are important in biology, little quantitative information is available regarding their presence in mammalian organisms owing to the technical difficulties associated with accurately detecting these materials in biological samples. We have developed an analytical method whereby InsP7 and its precursor inositol hexakisphosphate (InsP6) are determined directly and sensitively using tandem mass spectrometry coupled with hydrophilic interaction liquid chromatography (HILIC). InsP6 and InsP7 peak symmetry is influenced greatly by the buffer salt composition and pH of the mobile phase used in HILIC analysis. The use of 300 mM ammonium carbonate (pH 10.5) as an aqueous mobile phase resolves InsP6 and InsP7 on a polymer-based amino HILIC column with minimal peak tailing. Method validation shows that InsP6 and InsP7 can be quantitated from 20-500 pmol with minimal intra-day/inter-day variance in peak area and retention time. The concentration of InsP6 in C57BL/6J mouse brain (40.68 ± 3.84 pmol/mg wet weight) is successfully determined. HILIC‒MS/MS analysis using HEK293 culture cells confirms previous observations that InsP7 is induced by NaF treatment and ectopic expression of InsP6K2, a primary kinase for InsP7 synthesis. Furthermore, this analysis reveals the abundance of InsP6 (50.46 ± 18.57 pmol/106 cells) and scarcity of InsP7 in human blood cells. The results demonstrate that HILIC‒MS/MS analysis can quantitate endogenous InsP6 and InsP7 in mouse and human samples, and we expect that the method will contribute to further understanding of InsP7 functions in mammalian pathobiology.