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Article
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 is published by the American Chemical Society. 1155 Sixteenth
Street N.W., Washington, DC 20036
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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: m.cameron@westernsydney.edu.au; +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; <0.1 W/m2)
light from a head torch.
Retinal sample preparation.
The sample preparation process took place at 4°C. 30µL of a mixture containing 0.5 mM ascorbic acid (AA), 1% formic acid (FA) and
100 ng/mL of each deuterated standard was added to the 0.5mL tube
containing each retina prior to 1 min homogenization with a pellet
pestle cordless motor followed by 5 min of sonication. Each sample
was centrifuged for 10 min at 14000 rpm, 4°C and the supernatant
removed and stored at 4°C until analysis.
Statistical analysis
All graphical data is presented as mean ± SEM. One-way analysis
of variance (ANOVA) with Tukey’s post-hoc test (adjusted p value
reported) was used to test for significant differences. When stated,
unpaired Student’s t-test were utilized for pair-wise comparisons.
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Analytical Chemistry
A
B
C
D
E
Dopamine + ascorbic acid
F
DOPAC + ascorbic acid
Figure 2: Quantification of dopamine (DA) and DOPAC (100 ng/ml) by UHPLC-MS/MS in the presence of various acid/antioxidant
combinations. A&C, Absolute peak integration counts of DA and DOPAC standards (100 ng/ml) show significantly reduced signal
when dissolved in formic acid (1%) compared to water (***p<0.001), that can be prevented by addition of 0.5 mM ascorbic acid (orange/white stripes). While 0.01% sodium metabisulfite (SMBS; black/white stripes) prevents some signal loss (*p<0.05 formic vs
formic + SMBS), combination of antioxidants does not offer any advantage over ascorbic acid alone. B&D, Addition of perchloric acid
(0.1M) drastically reduces both DA and DOPAC signal in comparison to water and cannot be prevented by any combination of antioxidant (***p<0.001 all conditions compared to water). E&F, Ascorbic acid has a n-shaped protective effect on DA and DOPAC in formic acid with the least signal loss observed using 0.5 – 5 mM concentrations. Reduction in signal at higher concentration may reflect
ion suppression. *P values represent difference from 0.5mM (ANOVA, Tukey post-tests). All data mean ± SEM, n=3.
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Analytical Chemistry
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Results and discussion
By optimizing this technique, we aimed to produce a straightforward method that can accurately measure DA and DOPAC
concentrations in the mouse retina following rapid isolation
from the animal. To measure the concentration of these two
analytes in the retina, the tissue must be homogenized, centrifuged and the supernatant extracted. All previous methods
have involved acidification to pH ≲ 2, predominantly using
perchloric acid in which to homogenize the retina. Dropping
the pH causes precipitation of retinal proteins which aids purification of the sample, and, importantly, inactivates enzymes.
Since dopamine is metabolized to DOPAC by the enzyme
monoamine oxidase (MAO), this step is critical if the DOPAC
to DA ratio is to provide an accurate indication of endogenous
DA release. However, since catecholamines are known to be
easily oxidized,29 an antioxidant must be included to prevent
significant degradation of these species.
While perchloric acid is used almost exclusively when measuring DA and DOPAC in the retina by HPLC-ECD, it is not
used as extensively for MS/MS. Perchloric acid is not compatible with ESI-MS because it is a strong (pKa = -10) inorganic
acid of low volatility (bp = 203°C) which causes large ion
suppression effects. Additionally, it is a strong irritant and its
oxidising properties demand that care must be taken with disposal. For this reason, before measuring DA and DOPAC in
the retina, we compared the effect of formic acid (a commonly
used pH modifier in MS/MS) with perchloric acid and different combinations of antioxidants. Ascorbic acid and sodium
metabisulfite (SMBS) are the two most commonly used antioxidants when measuring DA and DOPAC, so we assessed
their protective abilities both alone, and together. We compared the absolute peak integration counts of DA and DOPAC
(100 ng/ml) in water, to various acid/antioxidant combinations. As shown in Fig 2 A&B, in comparison to DA in water
alone, both acids cause significant loss of DA signal
(***p<0.001). However, this loss could be completely pre-
A
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vented by the inclusion of 0.5 mM ascorbic acid, but only in
combination with formic acid (p=0.1773 compared to water).
The loss of signal caused by the inclusion of perchloric acid
could not be prevented by any combination of antioxidants
(***p<0.001 for all). In the formic acid conditions, (Fig 2A),
while SMBS was effective at preventing some DA loss
(*p<0.01; formic vs formic + SMBS), it was not as effective
as ascorbic acid (*p<0.05 SMBS vs water), and no further
protection was provided by combining the two antioxidants
(p=0.217 formic + ascorbic vs formic, ascorbic + SMBS).
Similar results were obtained when measuring DOPAC where
formic acid, in combination with ascorbic acid, completely
prevented DOPAC loss (p=0.247 compared to water), and
perchloric acid significantly reduced DOPAC levels even in
combination with antioxidants (***P<0.001; Fig 2D).
We further assessed the effectiveness of ascorbic acid, in
combination with formic acid, at varying concentrations (Fig
2E&F). For DA, 50 µM – 5 mM concentrations of ascorbic
provided significant protection, with higher concentrations
causing signal loss (***p<0.001), presumably due to ion suppression effects. For DOPAC, 0.5 – 50 mM were the most
effective concentrations with less reduction in signal at higher
concentrations. Taken together, 0.5 – 5 mM ascorbic acid are
the most effective concentrations for protection of both DA
and DOPAC.
Since perchloric acid has been used for retinal DA and
DOPAC quantification so extensively, we examined the possible cause of the dramatic reduction in signal observed when
quantifying these analytes by MS/MS. Previous methods using perchloric acid involved electrochemical detection, rather
than MS, therefore we examined the possibility that the effects
of perchloric are specific to MS/MS detection. During recent
years, the phenomenon of ion suppression, specifically in electrospray ionisation MS/MS, has become a major concern.30-32
Ion suppression causes a reduced detector response as an effect of competition for ionisation efficiency in the ionisation
source, between the analyte of interest and other endogenous
or
exogenous
species.
B
Figure 3: Stability of DA and DOPAC over 72 hrs. A, Absolute peak integration counts for DA, DOPAC and their respective deuterated internal standards (all 100 ng/ml) sampled every 24 hrs, shows a progressive reduction in signal over time. The time course of signal loss of the standard analytes is mirrored by their respective deuterated analogues. B, Ratio of DA/DA-d4 and DOPAC/DOPAC-d5
was ~ 1:1 for DA over 72 hrs, whereas the DOPAC ratio began to deviate after 24 hrs.
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Analytical Chemistry
The putative ion suppressive effects of perchloric acid were
evaluated by quantifying DA and DOPAC concentration via
UV detection, rather than MS/MS (Fig S1). Both DA and
DOPAC can be readily quantified by UV light absorbance at
280 nm.33 The presence of perchloric acid has a much less
pronounced effect on DA and DOPAC peak size than with MS
detection (Fig S1). These data suggest that perchloric acid is
not greatly diminishing the actual quantity of DA and DOPAC
in the sample, but rather, the perchloric acid is having a significant ion suppressive effect. However, even with the addition
of ascorbic acid which significantly prevents the loss of DA
and DOPAC in both formic and perchloric conditions, the
formic acid condition yields a significantly larger signal
(***P<0.001). For this reason, irrespective of the method of
quantification, formic acid and 0.5 mM ascorbic acid will give
the most reliable measure of both DA and DOPAC.
Having determined the most appropriate acid/antioxidant conditions, we next examined the stability of the analytes over
time. Each sample takes ~ 20 min to run, usually run in triplicate, and several samples are often run at once. Given the existence of temperature-controlled sample autoloaders it would
be most convenient, and time saving, to load all the samples at
once and run them over an extended time. However, both DA
and DOPAC are oxidized in the presence of air. We ran 100
ng/ml DA and DOPAC samples in formic/ascorbic acid over
72 hr, measuring every 24 hrs, while holding in a sample autoloader at 4°C. We saw a reduction in both DA and DOPAC
A
MS/MS quantification
over 72 hrs, with DOPAC levels decreasing at a faster rate
than DA (Fig 3A). LC-MS runs of degraded DA and DOPAC
were obtained over the mass range 100-300 Th. Extracted ion
chromatograms showed the presence of degradation species
consistent with oxidation products of DA and DOPAC.
Since we aim to use DOPAC/DA ratio as a measure of DA
release, it is imperative that the different rates of oxidation of
these two analytes are taken into account. To normalise the
effects of oxidation, we utilised deuterated analogues of both
DA and DOPAC as internal standards. Indeed, both DA-d4 and
DOPAC-d5 undergo comparable reduction in signal over time
(Fig 3A; dotted lines) relative to their unlabelled analogues.
Once the raw DA and DOPAC peak integration values are
normalised to these standards, the ratio of DA to DOPAC remains stable, up to 72 hrs for DA and 24 hrs for DOPAC (Fig
3B).
Using each deuterated internal standard, we then constructed
standard curves to determine the sensitivity of UHPLCMS/MS, and compared with standard curves measured using
UV detection. Standard solutions of DA and DOPAC from
0.05 ng/ml to 1000 ng/ml were combined with their corresponding internal standards (100 ng/ml) and the ratio of each
plotted against standard concentration. For both DA and
DOPAC quantification by MS/MS, a linear relationship between concentration and detector counts was obtained over
several log units (Fig 4; linear regression R2 > 0.99 for log
plots
of
both).
B
UV quantification
Figure 4: Standard curves for DA and DOPAC by two quantification methods: A, UHPLC-MS/MS quantification of DA (black) and
DOPAC (red) normalised to their respective deuterated internal standards (100 ng/ml) revealed a linear relationship for DA over >3 log
units (0.05 – 100 ng/ml; R2> 0.99) and DOPAC over >2 log units (0.5 – 100 ng/ml; R2> 0.99). Limit of detection (LOD) for each analyte is shown as red and black dotted lines. Concentration ranges of DA and DOPAC found in the retina using the described method
are shown in grey (40 µl homogenization volume per retina). B, UHPLC-UV quantification shows a similar linear relationship of the
two analytes from 0.1 – 1000 ng/ml, but R2 values are reduced (R2 = 0.95 DA; R2 = 0.97 DOPAC), LOD = 0.1 ng/ml for both. All data
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The limit of quantification for MS/MS (LOQ), calculated as
the lowest tested concentration in the linear phase, for DA was
50 pg/ml (326 pM) and DOPAC 500 pg/ml (3.0 nM), and both
analytes showed a linear relationship up to 100 ng/ml. The
limit of detection (LOD) for DA was 6 pg/ml (40 pM; 60 fg on
column) and DOPAC was 162 pg/ml (963 pM; 1.62 pg on
column). LOD was calculated as follows: LOD = meanblank +
1.645 (SDblank) + 1.654 (SDlow concentration sample)34 and is shown
for each analyte in Fig 4A (dotted lines). The intra-day variability was < 5% for all concentrations in the linear range for
DA, and <11% for all concentrations of DOPAC (coefficient
of variance). In comparison, while UV quantification also
showed a linear relationship, the accuracy was reduced (R2 =
0.95 DA; R2 = 0.97 DOPAC), LOQ = LOD = 0.1 ng/ml for
both. For illustration, an approximate range of retinal DA concentrations are show in grey (from Fig 6), demonstrating that
all retinal measurements will be well within the linear range
for this method. The LOQ for DA lies on, or below, that reported in the literature for previous quantification methods
including both HPLC-ECD and UHPLC-MS based detection
methods, however the LOQ for DOPAC lies significantly below previously reported values.35-38 Interestingly, the sensitivity was similar to GC-MS quantification reported in 1972.39
While the UV quantification method shows a relatively high
sensitivity, it is important to remember that this method will
be far less selective than MS/MS quantification as nonspecific species may also be detected
After optimising conditions for standard concentrations in
formic/ascorbic acid only, we applied this method to isolated
retinae. We further analysed the efficacy of deuterated DA and
DOPAC as internal standards when assessing the matrix ef-
Page 6 of 10
fects of retinal homogenate (Fig 5). Previously, 3,4dihydroxybenzylamine (DHBA), a homologue of DA, has
been used extensively as an internal standard when measuring
retinal DA and DOPAC by HPLC-ECD.40-42 Representative
chromatographs showing the retention times for DA, DOPAC,
DHBA, DA-d4 nd DOPAC-d5 are shown in Fig 5A. DA elutes
early in comparison to DOPAC and DHBA elutes early and is
much closer to DA than DOPAC. In contrast, the retention
times of DA-d4 and DOPAC-d5 deuterated standards are much
more similar to their respective analytes. In reverse phase liquid chromatography, ionised and highly polarised compounds
are poorly retained and elute close to the solvent front, causing
significant ion suppression. For this reason, the greater retention of DA-d4 over DHBA makes it a better internal standard
choice. To evaluate the matrix effect of retinal extract and to
determine the effectiveness of deuterated internal standards vs
DHBA, we spiked retinal extract (extraction described in
methods) with 1 µg/ml DA, DOPAC and DHBA and compared to water control. DA, DOPAC and DHBA levels are all
significantly reduced in retinal extract in comparison to water
control, indicating a significant matrix effect. These matrix
effects (count in retina/counts in water*100) are summarized
in Fig 5B. DA experiences the largest suppression, DOPAC
the least and DHBA an intermediate amount. For an internal
standard to be effective, its degree of matrix suppression must
mirror that of the analytes against which it is compared. In
contrast to DHBA, the DA-d4 and DOPAC-d5 showed the
same amount of matrix suppression as their respective analogues. This is illustrated in Fig 5C where retinal homogenate
was spiked with 1 µg/ml and the signal normalised either to
the deuterated internal standards, or to DHBA.
A
3,4-DHBA
B
d4
d5
DA
DOPAC
C
Figure 5: Retinal extract matrix effect. A, Representative chromatographs of DA, DOPAC, 3,4-dihydroxybenzylamine (DHBA),
DA-d4 and DOPAC-d5 (1µg/ml). B, Quantification of the % matrix effect (counts in matrix/counts in water) reveals DA experiences
the greatest reduction, DOPAC the least and DHBA an intermediate amount. C, Retinal extract spiked with 1µg/ml DA and DOPAC
with either deuterated standards, or DHBA as an internal standard. Calculated concentration, with respect to deuterated or DHBA
standards, yields ~ 1µg/ml using the deuterated, but normalisation to DHBA underestimates DA, and overestimates DOPAC concentration. All data mean ± SEM, n ≥3.
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While standardization against deuterated analogues provided
an accurate measure of the spiked concentration, standardization against DHBA underestimated the DA concentration, and
greatly overestimated the DOPAC concentration. Therefore, it
is imperative that appropriate deuterated internal standards are
used when quantifying DA and DOPAC with MS/MS due to
the differential matrix suppression of these two species. The
further advantage of using deuterated standards is that they are
added at the beginning of the homogenisation and each individual sample is normalised. Therefore, any differential effects
of the homogenization and extraction process will be normalized, greatly reducing intra-sample and inter-day variability.
Being able to utilise deuterated standards is therefore a further
advantage of the MS/MS quantification method as they can
only be used with MS given that their chromatographic retention times (and redox potentials) are identical to DA and
DOPAC .
Once we had optimized the homogenization conditions, internal standards, sensitivity, and stability of DA and DOPAC
quantification, we applied these methods to measure lightinduced dopamine release in the wild-type C57BL/6 mouse
retina. Mice were maintained in the dark overnight and retinae
were extracted either in the dark, or following a 1 hr lightpulse (free-moving in home cage; ~1000 lux) at approximately
midday (± 2hrs) as described previously.43 Retinal homogenization and supernatant extraction (described in methods) was
performed in 40 µl formic acid/ascorbic acid with 100 ng/ml
deuterated standards. DA and DOPAC absolute peak integration counts were divided by their respective deuterated standards and concentrations were calculated using the fitted lines
in Fig 4. As DA-HCl salt was used as a standard, free-DA
concentration in the retina was calculated by mathematically
adjusting
for
the
difference
in
mass.
A
B
C
D
Figure 6: Light-induced dopamine turnover in the mouse retina. A, Dopamine (DA) concentration in the mouse retina is unaffected by
a 1 hr light pulse (~1000 lux) in both free-moving and anaesthetised mice. B, DOPAC concentration is significantly increased in both
free-moving (*p<0.05) and anaesthetised mice (***p<0.001) after a 1 hr light pulse. C, DOPAC/DA ratio is significantly increased in
both free-moving (**p<0.01) and anaesthetised mice (***p<0.001) after a 1 hr light pulse. All data mean ± SEM, unpaired t-test, n
=5. D, Schematic of DA release, reuptake, and metabolism to DOPAC. TH – tyrosine hydroxylase; MAO – monoamine oxidase; AD –
aldehyde dehydrogenase; DD – dopa decarboxylase; DAT – dopamine transporter.
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No significant effect of light on DA concentration was observed (Fig 6A “free-moving”), but DOPAC showed a significant increase following light pulsing (unpaired t-test *P<0.05;
Fig 6B). As we are measuring total retinal dopamine levels
which would include both released DA and that stored in intracellular vesicles it is unsurprising that total DA content
would be unchanged by light-pulsing and has been previously
described.20 The increase in DOPAC concentration mirrors
previous reports23 and reflects the metabolism of DA by MAO
after re-uptake by the dopamine transporter (DAT; Fig 6D).
As these concentrations were not normalized to protein levels
(as we focus on DOPAC/DA ratio) they reflect variation in the
retinal extraction method. The ratio of DOPAC/DA or simply
DOPAC release has been used ubiquitously in the literature to
measure DA turnover.18, 20, 23-24, 44 As described previously,
DOPAC/DA ratio revealed a significant increase in response
to the light-pulse, as variation in the extraction method is normalized, the increase in DOPAC/DA ratio shows reduced
standard error (unpaired t-test **P<0.01; Fig 6C). While the
pattern of this increase mirrors published data,43 the ratios are
significantly larger for both dark and light pulsed conditions.
This is likely due to our method more accurately reporting DA
and DOPAC concentrations, and could be related to the faster
oxidation of DOPAC than DA that we observed (Fig 3). Our
method accounts for any differences in oxidation rate following extraction by normalization to the deuterated standards
(which undergo oxidation at the same rate as their unlabeled
analogues).
Given that mice are free-moving in their home cage when
light-pulsed, a large variation in the light intensity each animal
receives could occur depending on the behavior of each animal. To standardize the amount of light these animals received, we repeated these experiments on anaesthetized mice
(ketamine 70 mg/kg, 7 mg/kg xylazine). Once light stimuli
were standardized we saw the same pattern of results for DA
and DOPAC as the free-moving mice, however the coefficient
of variance (CV) of the data in light conditions was reduced:
free-moving CV=29.74%, anaesthetized CV=20.65% for
DOPAC/DA ratio. Furthermore, anesthesia had no effect on
the basal DA and DOPAC concentrations measured in the
retina (Fig 6A&B). This indicates, firstly, that anaesthesia
does not interfere with basal or light-induced DA turnover,
and secondly, that it may improve the accuracy of this method
to assess DA release in the mammalian retina.
Conclusion
We have described an optimized method to accurately measure DA and DOPAC concentrations in the mammalian retina.
UHPLC-MS/MS quantification has several advantages over
previous HPLC-ECD methods. First and foremost, MS quantification allows the utilization of deuterated internal standards
for each analyte. The use of these standards means that any
variation in the quantification of analytes is significantly reduced as the deuterated standard will undergo the same fate as
its respective unlabeled analyte. This is particularly important
here, where the ratio of two analytes may dramatically change
if the oxidation rate/column interactions/ion suppression of
each analyte is not the same. Furthermore, MS/MS quantification is a far more species selective method of quantifying the
analyte in question. This is critical for retinal samples as a
large array of changes in the release of neurotransmitters/modulators occurs in response to retinal illumination. We
Page 8 of 10
have described ideal retinal homogenization conditions for
extracting DA and DOPAC with minimal oxidization of these
analytes, as well as optimal performance for MS/MS quantification. Moreover, these homogenization conditions involve
very simple, cheap and quick preparation techniques and solvents, making the biological sample preparation an accessible
option for non-chemists.
AUTHOR INFORMATION
Corresponding Author
* Morven Cameron, e-mail: m.cameron@westernsydney.edu.au
Author Contributions
MC and DH devised the experiments, VPF completed the experimental work. Manuscript was written by MC with contributions
from all authors. All authors have given approval to the final version of the manuscript.
ACKNOWLEDGMENT
We would like to acknowledge the support of the Mass Spectrometry Facility at Western Sydney University for providing use of
the instruments, equipment, and LC solvents to complete these
experiments.
SUPPORTING INFORMATION
Supplementary figure 1 (S1): Dopamine (DA) and DOPAC quantification by UHPLC-UV using various acids and antioxidants.
Effect of perchloric acid on DA and DOPAC levels is not as pronounced when UV quantification is utilized.
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Std curve 0.05-100ng/mL
Retinal
conc
Retina
Dopamine
3,4-DHBA
Dopamine D4
DOPAC D5
DOPAC
For TOC only
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