Determination of arginine–vasotocin and isotocin in fish plasma with solid-phase extraction and fluorescence derivatization followed by high-performance liquid chromatography

Abstract

A new HPLC assay for plasma arginine–vasotocin (AVT) and isotocin (IT) determination based on fluorescence detection preceded by combination of solid-phase extraction (SPE) and fluorescence derivatization is presented. Plasma samples retained on solid support were purified and then derivatized by the fluorescent compound 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The peptide derivatives were eluted from cartridges, pre-concentrated and analyzed by HPLC system with fluorescent detection. The separation was carried out on a reversed-phase column with solvent gradient system. The assay was linear in the range 15–220 pmol ml −1 for AVT ( r 2=0.998) and 10–220 pmol ml −1 for IT ( r 2=0.996). The detection limits for AVT and IT were 0.8 and 0.5 pmol ml −1 (3:1, signal-to-noise), respectively. The recoveries of derivatized hormones were in the range 89–93%. Both of the inter- and intra-day assay precision were below 5.5 and 9% for AVT and IT, respectively. The assay should be also applicable to plasma and tissue samples from other animals with only minor modification.