Optimized Method to Quantify Dopamine Turnover in the Mammalian Retina

• Published in: Analytical chemistry (Washington) 2017-11, Vol.89 (22), p.12276-12283
• Main Authors: Pérez-Fernández, Víctor, Harman, David G, Morley, John W, Cameron, Morven A
• Format: Article
• Language: English
• Subjects: Analytical chemistry; Anesthesia; Animals; Ascorbic acid; Chemistry; Chromatography, High Pressure Liquid; Dark adaptation; Dopamine; Dopamine - analysis; Dopamine - metabolism; Electrochemical analysis; Electrochemical Techniques; Electrochemistry; Female; Formic acid; Interrogation; Linearity; Liquid chromatography; Male; Mammals; Metabolites; Mice; Mice, Inbred C57BL; Molecular Structure; Neuromodulation; Optimization; Oxidation; Perchloric acid; Retina; Retina - chemistry; Retina - metabolism; Rodents; Tandem Mass Spectrometry; Tissues
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.7b03216

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-d 4 and DOPAC-d 5 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.