Phosphorus-specific, liquid chromatography inductively coupled plasma mass-spectrometry for analysis of inositol phosphate and inositol pyrophosphate metabolism.

Inositol phosphate (InsP) and diphosphoinositol phosphate (PP-InsP) analysis in tissues is plagued by multiple difficulties of sensitivity, regioisomer resolution and the need for radiolabeling with metabolic precursors. We describe a liquid chromatography (LC) inductively coupled plasma (ICP) mass spectrometry (MS) method (LC-ICP-MS) that addresses all such issues and use LC-ICP-MS to analyse InsPs in avian tissues. The highly sensitive technique tolerates complex matrices and, by powerful chromatography, resolves in a single run multiple non-enantiomeric myo-inositol tetrakisphosphates, myo-inositol pentakisphosphates and all inositol hexakisphosphates, including myo-inositol 1,2,3,4,5,6-hexakisphosphate (phytate), known in nature. It also separates and quantifies diphospho myo-inositol pentakisphosphate (PP-InsP5) isomers from their biological precursors and from 1,5-bis-diphospho myo-inositol 2,3,4,6 tetrakisphosphate (1,5-[PP]2-InsP4). Gut tissue inositol phosphates, belonging to a non-canonical, lipid-independent pathway, are shown to differ from phytate digestion products and to be responsive to diet.