Anion-exchange high-performance liquid chromatographic analysis of inositol phosphates

The analysis of inositol phosphates by anion-exchange HPLC is described. The method employs a citrate buffer gradient to resolve several inositol phosphates including inositol 1-phosphate, inositol 1,4-bisphosphate (IP2), and inositol 1,4,5-trisphosphate (IP3), as well as some of the isomers of these compounds. Since the buffer system does not contain any phosphate, we can use a phosphate assay to examine the chromatographic behavior of phosphate-containing compounds. The method shows good resolution and recovery (greater than 95% for IP2 and IP3). Total analysis time, including reequilibration, is about 90 min. In addition, an isocratic system that can rapidly (less than 10 min) measure IP3 is described. The HPLC system was used to characterize inositol phosphate turnover in thrombin-stimulated platelets and formylmethionyl-leucyl-phenylalanine-stimulated HL-60 cells.