Influences of salts on high-performance liquid chromatography of leukotrienes.

The effects of concentrations and kinds of salts on the resolution of leukotrienes (LT) C4, D4, E4, and B4 were investigated by two kinds of reversed-phase high-performance liquid chromatography columns (muBondapak C18 and Novapak C18). When a mobile phase (acetonitrile/methanol/water) with a lower concentration of acetic acid (0.02-0.1%) at pH 5.6 was used, LTC4 and LTD4 were not eluted from the muBondapak column. On the Novapak column, LTC4 and LTD4 were eluted, but they were poorly resolved. When the concentration of acetic acid in the mobile phase was raised to 1.0% and adjusted to pH 5.6 with ammonium hydroxide or triethylamine, excellent resolution of LTs was obtained. Sodium hydroxide was, to some extent, useful for the pH adjustment of the mobile phase. Sodium chloride could not be substituted for acetic acid-ammonium hydroxide or -triethylamine salt. The resolution of LTC4, LTD4, and LTE4 was affected more strongly than that of LTB4 by changes of concentrations and kinds of salts. When the acetonitrile/methanol/water/acetic acid solvent system adjusted to pH 5.6 with triethylamine was applied to the analysis of the leukotrienes produced from rat peritoneal cells with stimulation of calcium ionophore A23187, de novo-synthesized LTC4, LTD4, LTB4, and isomers were clearly separated. This solvent system may be useful for the investigation of variations in the synthesis of subclasses of LTs with different stimuli and under different circumstances.