Chromatographic Determination of Oxytocin via UV Irradiation-Induced Luminol Chemiluminescence of Its Tyrosine Residue

Abstract

A novel method for the determination of oxytocin using high-performance liquid chromatography (HPLC) with online ultraviolet (UV) irradiation and a chemiluminescence detection system was developed. This method is based on the discovery that tyrosine, a constituent amino acid of oxytocin, generates chemiluminescence when mixed with luminol and irradiated with UV light. The chemiluminescence reaction system was integrated into an HPLC system to facilitate the determination of oxytocin in samples containing complex matrices. The chromatographic separation was performed on a COSMOSIL PBr column using a mobile phase consisting of acetonitrile and 1.0 mM Tris-HNO₃ buffer (pH 7.4) in a ratio of 25/75 (v/v, %), respectively. The optimal conditions for UV irradiation and chemiluminescence reaction were investigated, and the method showed good linearity over the concentration range of 0.05-5.0 μM with a correlation coefficient of 0.994. The limit of detection (LOD, S/N = 3) was 14 nM, and the intra- and inter-day relative standard deviations (RSD) were less than 3.2 and 2.7 %, respectively. The proposed method was successfully applied to the determination of oxytocin in bovine milk samples, with good recoveries ranging from 85.5 % to 107 %. The developed method offers a highly sensitive and selective determination of oxytocin without the need for complicated pretreatment procedures, expensive reagents, or complex equipment, making it a promising tool for evaluating the biological effects of oxytocin in various samples.