Optimized method to quantify dopamine turnover in the mammalian retina

Victor Perez-Fernandez, David G. Harman, John W. Morley, Morven A. Cameron

Research output: Contribution to journalArticlepeer-review

14 Citations (Scopus)

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.
Original languageEnglish
Pages (from-to)12276-12283
Number of pages8
JournalAnalytical Chemistry
Volume89
Issue number22
DOIs
Publication statusPublished - 2017

Keywords

  • dopamine
  • mice
  • retina

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