Determination of histamine in microdialysis samples from rat brain by microbore column liquid chromatography following intramolecular excimer-forming derivatization with pyrene-labeling reagent.

This paper describes a sensitive and selective liquid chromatographic method with fluorescence detection for determination of histamine in brain microdialysis samples from awake rats. Samples containing histamine (10 microl) were derivatized with 20 microl of the reagent consisting of 3 mM 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), 3 mM potassium carbonate and acetonitrile (1:1:18, v/v), thereafter 20 microl volume was injected onto the microbore column packed with C18 silica gel. The histamine derivative contained two pyrene moieties, which generated intramolecular excimer fluorescence (450-540 nm) and allowed clear discrimination from the monomer fluorescence (360-420 nm) emitted by PSE itself. The separation of histamine-pyrene derivative was achieved within 25 min, the detection limit (S/N=3) was 0.3 fmol histamine in 20 microl injected. The basal extracellular levels of histamine collected in 10-min fractions (fmol per 10 microl, mean+/-S.D., not corrected for recovery, n=10 rats) were 35.45+/-4.56 (hypothalamus), 9.05+/-1.56 (prefrontal cortex), 7.83+/-0.86 (hippocampus) and 6.54+/-0.66 (striatum). The voltage-sensitive release of histamine was evaluated by perfusing the probes with high (100 mM) concentration of potassium ions or with sodium channel blocker tetrodotoxin (1 microM), and the calcium-dependent release was tested by perfusion with calcium-free Ringer solution. These data, together with physiologically induced increase of extracellular histamine in four examined brain regions during forced swimming demonstrate that this method is suitable for high-sensitive determination of neuronally released histamine under various pharmacological and physiological conditions.