Using HILIC-MS/MS Method to Study Inositol Pyrophosphate

The direct detection of InsP₇ (diphosphoinositol pentakisphosphate) and InsP₈ (bis-diphosphoinositol tetrakisphosphate), known as inositol pyrophosphates (PP-InsPs), in mammalian specimens faces technical difficulties owing to their characteristic chemical properties and minute quantities in mammalian tissues. We developed an analytical protocol to sensitively and directly detect PP-InsPs and their precursor, inositol hexakisphosphate (InsP₆), using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS). This analytical protocol, coupled with high-purity synthetic standards, allows for the absolute quantitation of these analytes in cellular samples as well as in various organs and tissues obtained by autopsies of animals and humans, offering an effective option to study PP-InsP functions in mammalian physiology and pathology. Herein, we describe this protocol in detail, from the sample preparation procedure throughout the column regeneration after analysis, along with several notable cautionary points.