Simultaneous electrochemical measurement method of histamine and Nτ-methylhistamine by high-performance liquid chromatography-amperometry with o-phthalaldehyde-sodium sulfite derivatization

An electrochemical detection (ECD) method for analyzing sub-micro amounts of histamine (HA) and Nτ-methylhistamine (Nτ-MHA) in biological samples by high-performance liquid chromatography (HPLC)-amperometry has been developed. The method consists of a precolumn derivatization of the amines with o-phthalaldehyde (OPA) and sodium sulfite (Na2SO3) to N-alkyl-1-isoindole sulfonate and posterior separation with the HPLC system. Biological samples were pretreated by using a Vivapure sulfonic acid minifilter in which the reaction of the reagent with the amines took place during filtering. HA and Nτ-MHA retention times were 11.8 ± 0.02 and 18.3 ± 0.03 min, respectively (means ± standard deviations, n = 3). The lowest limit of amperometric detection at a signal-to-noise ratio of 3:1 was 0.125 pmol in both cases. HA and Nτ-MHA contents in hypothalamus, cortex, skin, and fundic gland, as well as histamine N-methyltransferase (HMT) activities of mast cell-deficient (Ws/Ws) and Wistar rats, were measured and compared with an HPLC-fluorometry system, among other experiments, in order to validate and demonstrate the usefulness of this assay system. Hence, this consequently confirms not only the sensitivity and specificity of the assay but also the potential and convenience it offers to laboratory work, especially in the analysis of the regulation of histaminergic neurons as well as enzymatic investigation of HA metabolism.