Determination of Histamine and Leukotrienes from Basophils in Cell Supernatants by High-Performance Liquid Chromatography

A reversed-phase high-performance liquid chromatographic method with gradient elution for the separation of the mediator substances histamine and the leukotrienes C₄ (LTC₄), D₄ (LTD₄), and E₄ (LTE₄) is described. The detection occurs fluorimetrically after automated precolumn derivatization with o-phthaldialdehyde. All components are chromatographically separable. Because of the different excitation and emission wavelengths, only the most important biological active mediators histamine and LTC₄ are determinable in one parallel chromatographic run. The method is examined by linearity and precision tests and is applicable to biological sample matrices like cell supernatants of human basophils enriched by Percoll-density gradient centrifugation and stimulated for mediator release by anti-IgE. The established method is suitable to separate the mediators from other matrix components. The determination limit for histamine is 55.0 μg/L and that for LTC₄ 16.0 μg/L, referring to the reference solutions. Therefore, a fast, economical method for the common determination of the most important mediators histamine and LTC₄ is established. This method is also suitable for high sample amounts in routine medical analysis.