Analysis of inositol mono- and diphosphate isomers using high-performance ion chromatography and pulsed amperometric detection

A rapid and sensitive high-performance ion chromatography method for separation and quantitative determination of inositol mono- and diphosphate (IP1-IP2) isomers using pulsed amperometric detection is described. The method involves extraction of samples with HCl, separation of inositol phosphates from the crude extract by anion-exchange chromatography, NaAc gradient elution in an NaOH environment to perform isomer separation on a high-performance anion-exchange column, and detection with pulsed amperometric detector. The applicability and sensitivity of the method is illustrated by measurement of the content of IP1 and IP2 in foods, human ileal contents, and enzymatic hydrolysis products of phytate (inositol hexaphosphate). The major IP1 and IP2 isomers formed during phytate hydrolysis with wheat phytase were shown to be Ins(2)P, DL-Ins(1)P, and DL-Ins(1,2)P-2 and with Aspergillus niger phytase, Ins(2)P and DL-Ins(1,2)P-2.