Mass assay for inositol 1-phosphate in rat brain by high-performance liquid chromatography and pulsed amperometric...

A high-performance liquid chromatographic method for direct mass measurement of inositol 1-phosphate (I(1)P) in rat brain is described. Separation of I(1)P from its isomers and from endogenous components is achieved by polymeric anion-exchange chromatography with a sodium hydroxide/sodium acetate mobile phase. Detection is performed at high pH by pulsed amperometric detection at a gold electrode. Sample preparation involves liquid-liquid extraction and ion-exchange solid-phase extraction, prior to HPLC. The method is sufficiently sensitive and selective to enable facile determination of basal levels of I(1)P in small amounts of brain tissue. The applicability of the method is demonstrated by the in vivo monitoring of I(1)P levels in rat brain after administration of the inositol monophosphatase inhibitor lithium and the cholinergic agonist pilocarpine. The method is a significant improvement over existing published mass assays for I(1)P by virtue of its simplicity, speed, sensitivity, and ruggedness.