High-performance liquid chromatographic separation of inositol phosphate isomers employing a reversed-phase column and a micellar mobile phase.
Surfactants have been employed in high-performance liquid chromatography (HPLC) for the separation of ionic and non-ionic compounds. We have developed a method employing a reversed-phase column and a mobile phase containing a surfactant, hexadecyltrimethylammonium hydroxide (HDTMA+OH-), for the separation of several inositol phosphate positional isomers. Various parameters were studied for their effect on the chromatographic capacity factor (k'). They included the concentration of HDTMA+OH-, the pH of the bulk micellar suspension and the addition of inorganic salts to the mobile phase. Resolution of the inositol monophosphates was controlled by a mixed mechanism, where the predominant elements were electrostatic forces and the formation of micelles. The elution of the inositol polyphosphate isomers was obtained by increasing the amount of a non-polar solvent, in agreement with an ion-pairing process. This method represents an alternative to ion-exchange HPLC. If offers a practical advantage when detection of radiolabeled samples by in-line radioactive flow detectors is required, because low-quenching solvents with good miscibility with scintillant fluids are employed. The analysis of various chromatographic conditions, the system reproducibility and its application to the analysis of biological samples are described.
Division of Blood and Blood Products, U.S. Food and Drug Administration, Bethesda, MD 20892.
C
C
Brando
T
T
Hoffman
E
E
Bonvini
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D007295:Inositol Phosphates / Q000032:analysis* / Q000097:blood; D007536:Isomerism; D007963:Leukocytes, Mononuclear / Q000737:chemistry
D007295:Inositol Phosphates