Quantification of histidine-containing dipeptides in dolphin serum using a reversed-phase ion-pair high-performance liquid chromatography method

Abstract

The quantification of histidine-containing dipeptides (anserine, carnosine, and balenine) in serum might be a diagnostic tool to assess the health condition of animals. In this study, an exist-ing reversed-phase ion-pair high-performance liquid chromatography (HPLC)–ultraviolet detection method was improved and validated to quantify serum anserine, carnosine, and balenine levels in the dolphin. The serum was deproteinized with trichloroacetic acid and directly injected into the HPLC system. Chromatographic separation of the three histidine-containing dipeptides was achieved on a TSK–gel ODS-80Ts (4.6 mm × 150 mm, 5 µm) analytical column using a mobile phase of 50 mmol/L potassium dihydrogen phosphate (pH 3.4) containing 6 mmol/L 1-heptanesulfonic acid and acetonitrile (96:4). The standard curve ranged from 0.1 µmol/L to 250 µmol/L. The average accuracy of the intra-and inter-analysis of anserine, carnosine, and balenine was 97–106%. The rel-ative standard deviations of total precision (RSDr) of anserine, carnosine, and balenine in dolphin serum were 5.9%, 4.1%, and 2.6%, respectively. The lower limit of quantification of these compounds was 0.11–0.21 µmol/L. These results indicate that the improved method is reliable and con-cise for the simultaneous determination of anserine, carnosine, and balenine in dolphin serum, and may be useful for evaluation of health conditions in dolphins. Furthermore, this method can also be applied to other biological samples.