Hydrophilic interaction liquid chromatography-tandem mass spectrometry for the quantitative analysis of...

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.