Determination of arginine-vasotocin and isotocin in fish plasma with solid-phase extraction and fluorescence derivatization followed by high-performance liquid chromatography.
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 r2=0.998 and 10-220 pmol ml(-1) for IT r2=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.
Gozdowska
Magdalena
M
Department of Genetics and Marine Biotechnology, Institute of Oceanology of Polish Academy of Sciences, Powstańców Warszawy 55 Str, 81-712 Sopot, Poland.
Kulczykowska
Ewa
E
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005432:Flounder; D005456:Fluorescent Dyes / Q000737:chemistry*; D017686:Oncorhynchus mykiss; D010121:Oxytocin / Q000031:analogs & derivatives* / Q000097:blood*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014668:Vasotocin / Q000097:blood*
D005456:Fluorescent Dyes; D010121:Oxytocin; C008215:isotocin; D014668:Vasotocin