The determination of histamine in challenged human leukocyte preparations by high-performance liquid chromatography

A highly sensitive and rapid method was developed for the determination of histamine in challenged human leukocyte preparations by high-performance liquid chromatography. The assay is based on the Shore's OPT-reaction of the unpurified sample and on a specific separation of the derivate with analytical reversed phase phenyl column combined with spectrofluorometric detection. The detection limit of histamine by this method was 0.07 pmol (signal to noise ratio 2:1) and the within-day variation for peak height was 3.6% and for retention time 0.8%. A good linear standard curve ranging from 12.5 pg to 500 pg (0.07 pmol-2.7 pmol) was obtained with correlation coefficient of 0.998. The histamine release from human basophils in mixed leukocyte preparation was induced by the calcium ionophore A 23187. A concentration of 0.4 micrograms/ml ionophore was required for 50% histamine release with a Ca2(+)-concentration of 1.8 mmol/l. The measured total histamine content was 1.5 pg/basophil.