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An optimized method to quantify dopamine turnover in the mammalian retina. Víctor Pérez-Fernández, David G Harman, John W. Morley, and Morven Alison Cameron Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03216 • Publication Date (Web): 23 Oct 2017 Downloaded from http://pubs.acs.org on October 26, 2017
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Analytical Chemistry
An optimized method to quantify dopamine turnover in the mammalian retina. Víctor Pérez-Fernández, David G Harman, John W Morley and Morven A Cameron* School of Medicine, Western Sydney University, Sydney, Australia. *Correspondence:
[email protected]; +61 (0) 2 4620 3739
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 anaesthesia 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 inter-individual variation was significantly reduced. Together, we systematically describe the ideal conditions to accurately, and reliably measure DA turnover in the mammalian retina.
Keywords: Dopamine, retina, UHPLC, DOPAC, mass spectrometry. Dopamine (DA) is one of the most influential neuromodulators in the mammalian central nervous system (CNS) controlling such disparate functions as locomotion, cognition, addiction and emotion. In the retina, it is thought to play a key role in retinal physiology including modulation of processes such as disc shedding,1-2 growth and development,3 cell death4 and light adaptation of retinal pathways.5-7 Release of DA in the retina is potently induced by light, and its effect on retinal function is considerable, including rearrangements in circuitry that optimizes the retina to the presenting light conditions.8-11 DA is thought to be released from only one cell type in the mammalian retina, dopaminergic amacrine cells,4 and therefore a light signal from photoreceptive cells must reach these amacrines to elicit DA release. The photoreceptors and circuitry contributing to this light input, have received significant attention in recent years.12-17 However, despite many methods examining DA-amacrine cell activation via electrophysiological assessment of membrane potential, or correlation of neuronal activation with c-fos expression, few recent studies have measured DA release directly. Furthermore, it has been suggested that changes in membrane potential, or c-fos expression, of these cells in response to light may not correlate directly with DA release.18-19 Therefore, accurate measurement of DA release from DA-amacrines by light is a necessary technique in this field. A commonly used method to measure dopamine release in the retina is to examine the ratio of DA to its primary metabolite DOPAC by high performance liquid chromatography using electrochemical detection (HPLC-ECD). This ratio is thought
to be a good indicator of recent DA release4 and shows a robust increase in response to light.18, 20 Dopamine is stored in vesicles in DA amacrine cells until it is released, after release the DA is taken back up by DA cells themselves, and others,21 by dopamine transporter (DAT) where it is then metabolized to DOPAC by monoamine oxidase (MAO; summarized in Fig 6D). Since dopamine vesicles are replenished following release, and generally the catecholamine content of whole retina is examined, the ratio of DOPAC to DA is thought to be a reliable in vivo indicator of dopamine turnover.4, 20, 22-24 While methods for the measurement of dopamine, and other catecholamines, in the brain and cerebrospinal fluid have been substantially revised over recent years to reflect updates in technologies,25-28 this has not been mirrored for retinal quantification. Indeed, we are not aware of any methods using UHPLC with mass spectrometry (MS) for retinal DA or DOPAC quantification. Interestingly, one of the first reports of dopamine quantification in the retina involved gaschromatography with MS quantification (GC-MS), however this method was not widely adopted in the field.8 LC-MS/MS is an advantageous analytical technique because there is no need for derivatization of analytes. There is a low incidence of false positive identifications due to the triple filters of retention time, parent mass and fragment masses of the MRM experiment. Additionally, the use of antioxidants to prevent catecholamine degradation is recommended, but a systematic analysis of which antioxidant provides the highest protection has not been published, and many different types and concentrations are used in the literature. Here we present an updated and optimized technique for measuring DA and DOPAC in the mammalian retina using ultra-high performance liquid chromatography (UHPLC) with
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Analytical Chemistry
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detection by tandem mass-spectrometry (MS/MS). We systematically address the impact of extraction parameters including the use of antioxidants, and describe a selective, stable, sensitive, and accurate method to quantify DA and DOPAC in the mammalian retina.
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DHBA
140.16
105.03
0.106
8
21
DHBA
140.16
123.07
0.106
8
9
Experimental section Chemicals and Materials Fine chemicals and reagents were sourced as follows: 2-(3,4Dihydroxyphenyl)ethyl-1,1,2,2-d4-amine hydrochloride 98% (Dopamine-d4, CDN Isotopes), 3,4-Didroxyphenylacetic acid ring-d3, 2,2d2, 98% (DOPAC-d5, Cambridge Isotope Laboratories), 3,4Dihydroxybenzylamine hydrobromide 98% (DHBA, Sigma Aldrich), Dopamine hydrochloride 98% (Sigma Aldrich), 3,4Dihydroxyphenylacetic acid 98% (DOPAC, Sigma Aldrich), formic acid ≥95% (Sigma Aldrich), MS grade formic acid (Sigma-Aldrich), L-ascorbic acid (Sigma Aldrich), sodium metabisulfite 97% (ChemSupply), perchloric acid ACS reagent, 70% (Sigma-Aldrich) and LCMS grade methanol (Burdick and Jackson). Solutions of standards and reagents were prepared in ultra-pure water (Milli-Q, Millipore).
Instrumentation Mass spectrometry was performed using a Waters Xevo TQ-MS triple quadrupole mass spectrometer, fitted with an electrospray ionization source. The desolvation gas flow (N2) was 800 L/hr, desolvation temperature was 450°C, cone gas 0L/hr and collision gas (Ar) flow of 0.15 mL/min, which gave a collision cell pressure of 2.6 x 103 mbar. In positive ion mode the capillary voltage was set at 1.2 kV and in negative mode 1.0 kV. Waters MassLynx software was used for data analysis. Liquid chromatography was performed using a Waters Acquity UPLC, working at a flowrate of 0.20 mL/min. A Waters Acquity UPLC BEH C18 column of 1.7 µm particle size and dimensions 2.1 x 150 mm was used, operating at 40°C. Solvent A consisted of 0.1% formic acid in ultrapure water and solvent B was 0.1% formic acid in methanol. A 20-minute run was employed, commencing at 5% B for 1 min, increasing linearly to 100% B by 10 min, then returning immediately to 5% B at 15 min. The sample manager was kept at 4°C and injections of 10 µL were made in full loop mode from sample solutions contained in Total Recovery (Waters) glass vials. A Waters Acquity UPLC PDA was used for optical detection purposes and was placed between the UPLC and ESI source. Optimized MRM parameters are provided in the table below. Dopamine (RT=2.10 min), DHBA (RT=1.67) and dopamine-d4 (RT=2.04 min) were analyzed in positive ion mode, DOPAC (RT=5.09 min) and DOPAC-d5 (RT=5.01 min) in negative.
Table 1: MRM parameters for analytes and internal standards Parent (m/z)
Daughter (m/z)
Dwell (s)
Cone (V)
Collision energy (V)
Dopamine
154.22
91.01
0.078
16
22
Dopamine
154.22
137.03
0.078
16
10
Dopamine d4
158.22
94.94
0.078
16
25
Dopamine d4
158.22
141.03
0.078
16
12
DOPAC
167.1
123.04
0.161
18
10
DOPAC d5
172.10
128.05
0.161
16
10
DHBA
140.16
77.04
0.106
8
24
Figure 1: Structures of analytes, internal standards, and ascorbic acid.
Animals All procedures involving animals were performed in accordance with the Australian Code for the Care and Use of Animals for Scientific Purposes and were approved and monitored by the Western Sydney University Animal Care and Ethic Committee, project numbers: A10396 and A11900. Wild-type C57BL/6J mice were purchased from the Animal Resources Centre (ARC, Canning Vale, Australia). Mice were bred on site and only offspring (both male and female) >60 days were used. Animals were maintained under a 12hr light: dark cycle at ~300 lux illumination during the daytime.
Light pulsing and tissue extraction Animals were either light pulsed in their home cage (~1000 lux cage floor), or were anaesthetized prior to light pulsing with an intraperitoneal injection of ketamine (70 mg/kg) and xylazine (7 mg/kg) and their pupils dilated with 1% atropine. Following light pulse, or 1 hour dark control, eyes were enucleated and retinae quickly excised by making a slit along the ora serrata and squeezing the eye to ‘pop’ out the retina. Retinae were immediately frozen in liquid nitrogen in a 0.5 ml tube, and maintained in the dark until transfer to -80°C. Lightpulsed retinae were extracted under normal laboratory lighting (~300 lux) and dark-adapted controls under dim red (>650 nm;
