HIGH PERFORMANCE LIQUID CHROMATOGRAPHY FOR THE SEPARATION OF HISTAMINE, ITS PRECURSOR, AND METABOLITES - APPLICATION TO BIOLOGICAL SAMPLES

摘要

Histamine was separated from its precursor L-histidine and its metabolites 1-methylhistamine and methylimidazole acetic acid on a TSK SP-5 PW cation exchange column and gradient elution. A baseline separation of histamine, 1-methylhistamine and methylimidazole acetic acid was also achieved under isocratic elution conditions on a reversed phase C-8 column with a mobile phase of 15 % methanol and 85 % of an aqueous solution of 0.05 M NaH2PO4 pH 3.1 which contained 0.5 mM EDTA-Na-4 and 0.005 M octan-1-sulfonic acid sodium salt as an ion paring reagent. The most sensitive UV detection of histamine and 1-methylhistamine with the highest detector signal was obtained at a wavelength of 210 nm. In the absence of the ion pairing reagent and the organic modifier methanol histamine and 1-methylhistamine were not retained on a reversed phase C-18, C-8 or C-4 column. Octadecasilyl-silica cartridges were used to purify histamine fiom other constituents present in human urine and a commercially available heparin formulation. The analytical recovery of H-3-labeled histamine after the purification on octadecasilyl-silica cartridges was 95.16 +/- 0.92 % (mean +/- SEM, n=22). The concentration of the histamine-like material in urine samples from healthy volunteers was 46.28 +/- 15.42 mu g/24h (mean +/- SEM; n=7). In the heparin formulation the histamine concentrations were 81.93 ng/ml before and 279.24 ng/ml after the purification on octadecasilyl-silca cartridges. The histamine-immunoreactive material in urine samples could be characterized on a TSK SP-5 PW cation exchange column as 19.69 +/- 7.86 % histamine and 46.74 +/- 12.89 % 1-methylhistamine (mean +/- SEM, n=7). Rechromatography of the 1-methylhistamine peak from the ion exchange column on the reversed phase C-8 column disclosed a substance of unknown nature with a different retention time than 1-methylhistamine. Histamine purified from heparin eluted from the ion exchange column as a single peak with the same retention time as the histamine standard.