Optimized Method to Quantify Dopamine Turnover in the Mammalian Retina.
Anal Chem
; 89(22): 12276-12283, 2017 11 21.
Article
em En
| MEDLINE
| ID: mdl-29057649
ABSTRACT
Measurement of dopamine (DA) release in the retina allows the interrogation of the complex neural circuits within this tissue. A number of previous methods have been used to quantify this neuromodulator, the most common of which is HPLC with electrochemical detection (HPLC-ECD). However, this technique can produce significant concentration uncertainties. In this present study, we report a sensitive and accurate UHPLC-MS/MS method for the quantification of DA and its primary metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in mouse retina. Internal standards DA-d4 and DOPAC-d5 result in standard curve linearity for DA from 0.05-100 ng/mL (LOD = 6 pg/mL) and DOPAC from 0.5-100 ng/mL (LOD = 162 pg/mL). A systematic study of tissue extraction conditions reveals that the use of formic acid (1%), in place of the more commonly used perchloric acid, combined with 0.5 mM ascorbic acid prevents significant oxidation of the analytes. When the method is applied to mouse retinae a significant increase in the DOPAC/DA ratio is observed following in vivo light stimulation. We additionally examined the effect of anesthesia on DA and DOPAC levels in the retina in vivo and find that basal dark-adapted concentrations are not affected. Light caused a similar increase in DOPAC/DA ratio but interindividual variation was significantly reduced. Together, we systematically describe the ideal conditions to accurately and reliably measure DA turnover in the mammalian retina.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Retina
/
Dopamina
/
Técnicas Eletroquímicas
Limite:
Animals
Idioma:
En
Revista:
Anal Chem
Ano de publicação:
2017
Tipo de documento:
Article
País de afiliação:
Austrália