Separation and Detection of Inositol Phosphates

Ion-exchange chromatography is used most frequently, because of its simplicity and low cost. Extracts usually containing [ HJinositol-labeled inositol phosphates are placed on a Dowex formate column from which GPI, IP, IP2 (inositol bisphosphate), and IP3 are eluted sequentially with buffers of ammonium formate of increasing molarity. 

High-performance liquid chromatography (HPLC) has much better resolution than the Dowex column, that is, it also resolves the individual isomers of the inositol phosphates (Button et al, 1994 Foster et al., 1994). Most HPLC procedures employ anion-exchange columns and an aqueous mobile phase of ammonium phosphate to elute the water-soluble inositols. Double-labeled (3H and 32p) inositol compounds can be used to follow by HPLC the fate of the inositol and phosphate residues during smooth muscle contraction. 

A metal-dye detection system has been developed that permits picomolar-range HPLC analysis of inositol phosphates from nonradioactively labeled tissues (Mayr, 1990), and was applied for the determination of masses of inositol phosphates in resting and stimulated skeletal muscles (Mayr and Thieleczek, 1991). 

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