Analysis of histidine and urocanic acid isomers by reversed-phase high-performance liquid chromatography

Abstract

The qualitative separation performance of a C 18, C 8 and C 4 reversed-phase column was investigated for the separation of histidine and its metabolites histamine, 1-methyihistamine and trans- and cis-urocanic acid. Trans- and cis-urocanic acid were baseline separated from their precursor histidine on all three columns using isocratic elution with a mobile phase composed of 0.01 M aqueous TEAP pH 3.0 and acetonitrile at a ratio of 98:2 (v/v). However, histidine was not separated from histamine and 1-methyihistamine. Selecting the C 8 column and introducing 0.005 M of the ion pairing reagent 1-octanesulfonic acid sodium salt into the aqueous solution and acetonitrile at a ratio of 90:10 (v/v), significantly improved the separation. The separation was also followed by a change in the retention times and the order of elution. The sequence of elution was histidine, cis-urocanic acid, trans-urocanic acid, histamine and 1-methylhistamine with retention times of 5.58±0.07, 7.03±0.15, 7.92±0.18, 18.77±0.24 and 20.79±0.21 min (mean±SD; n=5). The separation on the C 8 column in the presence of the ion-pairing reagent was further improved with gradient elution that resulted in a reduction in the retention times and elution volumes of histamine and 1-methylhistamine. The detection limits of histidine and trans-urocanic acid at a wavelength of 210 nm and an injection volume of 0.05 ml were 5×10 −8 mol l −1 ( n=3). The kinetic of the in-vitro conversion of trans- into the cis-isomer after UV irradiation was depending on the time of exposure and the energy of the light source. UVB light induced a significantly faster conversion than UVA light. TUCA and cUCA samples kept at −25°C were stable for up to 50 weeks. Samples, eluted from human skin showed various concentrations of histidine and trans- and cis-urocanic acid with an average of 1.69±0.33×10 −5 mol l −1, 1.17±0.43×10 −5 mol l −1 and 1.67±0.33×10 −5 mol l −1, respectively ( n=8).