Simultaneous determination of histamine and serotonin in mast cells by high-performance liquid chromatography

A sensitive and rapid high-performance liquid chromatographic method for the simultaneous determination of histamine and 5-hydroxytryptamine (serotonin) in supernatants and cellular extracts from mouse bone marrow-derived mast cells was developed. The described method is based on a precolumn derivatization of histamine and serotonin with o-phthaldialdehyde (OPA) and subsequent separation of the amine-OPA adducts with an analytical reversed-phase C18 column combined with fluorometric detection (excitation wavelength 360 nm, emission at 455 nm). The mobile phase was 0.02 M sodiumacetate buffer (pH 8) including 5 mM 1-octanesulfonic acid and 50% methanol. The detection limits for histamine and serotonin were 3.3 pmol and 6.9 pmol, respectively (signal-to-noise ratio 2:1). The variations of peak areas of repeatedly injected low or high amounts of amine standards were 23.3% or 6.9% (for 0.5 ng or 12.5 ng histamine) and 26.4% or 4.0% (for 5.0 ng or 250 ng serotonin) during an experimental period of 4 weeks. The variation of retention times over 2 days was 1.1% for histamine and 1.7% for serotonin. A perfect linear relationship of amine concentrations and peak areas was documented by correlation coefficients of r > 0.999 for both histamine (0.1-125 ng) and serotonin (0.5-250 ng).