engleski

Development of a HPLC-UV-FLD method for monoamine determination in lizard brain tissue samples

Lizards have simpler brains than mammals yet share common neural circuits in the brainstem responsible for motivational behavior including aggression. This simpler brain is favorable for studying mechanisms of behavioral control which could be translated to other species to clarify the axis that links aggressive behavior with the regulation of neurotransmitter levels. The aim of this study was to develop and validate a rapid HPLC method with UV and fluorescence detection for monoamine neurotransmitters, precursors and metabolites in lizard brain tissue. Samples of the left hemisphere were collected and homogenized in deproteinization solution, centrifuged and injected into the HPLC-UV. The RP-HPLC gradient method was modified in various ways: duration (20- 45 min), flow (0.45-1 mL/min), proportion of organic phase (0-30%), pH of the aqueous phase (2- 3) and injection volume (10-90 µL). We changed the composition of the mobile phase - organic phase (acetonitrile, methanol) and aqueous phase (formic acid, phosphoric acid). In the process, we changed three columns - two with C18 and one with phenyl as a stationary phase. A mix of known concentrations of 12 compounds - serotonin, dopamine hydrochloride, L-3, 4- dihydroxyphenylalanine, norepinephrine hydrochloride, epinephrine bitartrate, L- Tryptophan, L-Tyrosine hydrochloride, 5- Hydroxytryptophan, 5-hydroxyindole-3-acetic acid, vanillylmandelic acid, homovanilic acid and octopamine hydrochloride was used for identification of peaks in samples and for daily calibration curves. The peaks were identified by an UV detector (280 nm) and a fluorescence detector (270/320 nm). Monoamine levels in the brains of 200 coexisting lizard species (female and male of Podarcis siculus and Podarcis melisellensis) are presented.

HPLC-UV ; reverse-phase ; catecholamine ; monoamine metabolism ; lizard brain

nije evidentirano