Measurement of plasma norepinephrine and 3,4-dihydroxyphenylglycol: method development for a translational research study
Denfeld et al. BMC Res Notes
Measurement of plasma norepinephrine and 3,4-dihydroxyphenylglycol: method development for a translational research study
Quin E. Denfeld 1 2 3
Beth A. Habecker 1 2
William R. Woodward 0 1
0 Department of Neurology, Oregon Health & Science University , Portland, OR , USA
1 Department of Physiology & Pharmacology, Oregon Health & Science University , Portland, OR , USA
2 Knight Cardiovascular Institute, Oregon Health & Science University , Portland, OR , USA
3 Present Address: School of Nursing, Oregon Health & Science University , 3455 S.W. U.S. Veterans Hospital Road, Mail code: SN-ORD, Portland, OR 97239-2941 , USA
Objective: Norepinephrine (NE), a sympathetic neurotransmitter, is often measured in plasma as an index of sympathetic activity. To better understand NE dynamics, it is important to measure its principal metabolite, 3,4-dihydroxyphenylglycol (DHPG), concurrently. Our aim was to present a method, developed in the course of a translational research study, to measure NE and DHPG in human plasma using high performance liquid chromatography with electrochemical detection (HPLC-ED). Results: After pre-purifying plasma samples by alumina extraction, we used HPLC-ED to separate and quantify NE and DHPG. In order to remove uric acid, which co-eluted with DHPG, a sodium bicarbonate wash was added to the alumina extraction procedure, and we oxidized the column eluates followed by reduction because catechols are reversibly oxidized whereas uric acid is irreversibly oxidized. Average recoveries of plasma NE and DHPG were 35.3 ± 1.0% and 16.3 ± 1.1%, respectively, and there was no detectable uric acid. Our estimated detection limits for NE and DHPG were approximately 85 pg/mL (0.5 pmol/mL) and 165 pg/mL (0.9 pmol/mL), respectively. The measurement of NE and DHPG in human plasma has wide applicability; thus, we describe a method to quantify plasma NE and DHPG in a laboratory setting as a useful tool for translational and clinical research.
Sympathetic nervous system; Norepinephrine; 3; 4-dihydroxyphenylglycol; High performance liquid chromatography; Electrochemical detection; Human plasma
As the principal sympathetic neurotransmitter,
norepinephrine (NE), plays a critical role in regulating
physiological processes [
] and is commonly used as an index
of sympathetic activity in healthy and diseased states
]. In some conditions (e.g. heart failure) there is a
noted increase in plasma NE  as a result of increases
in sympathetic activity and subsequent NE “spillover”
from synapses into the plasma [
] as well as reduced
reuptake of NE . Elevated plasma NE portends worse
outcomes such as worsening left ventricular function [
and mortality [
]. There is wide variation, however, in
approaches to measure plasma NE [
common approaches involve either radioenzymatic  or
enzyme immunoassay methods [
]. While these
methods are well validated, they either require significant
experimental considerations (e.g. use and disposal of
radioisotopes) or they are limited to the measurement of
NE alone. To gain a better sense of NE dynamics,
measuring its principal metabolite, 3,4-dihydroxyphenylglycol
(DHPG), provides insight into NE dynamics, offering an
index of NE turnover [
]. As such, the
measurement of both plasma NE and DHPG provides unique and
complementary information about sympathetic activity
in conjunction with other metrics. Our aim was to
present a method, developed in the course of a translational
research study, to measure NE and DHPG in human
plasma using high performance liquid chromatography
with electrochemical detection (HPLC-ED). This method
has the benefit of a simple sample preparation process
and concurrent measurement of both NE and DHPG, as
well as being high-throughput.
Materials and methods
Plasma sample and standard preparation
We processed samples that were previously collected
from heart failure patients as part of a National Institutes
of Health-funded study [
]. Whole blood was
centrifuged at 1000×g for 10 min at 5 °C to extract the plasma;
the plasma was then de-identified as part of a
biorepository and frozen at − 80 °C. When ready to process,
frozen plasma samples were thawed and then centrifuged at
8000×g for 3 min at 4 °C to remove insoluble material.
Plasma samples (volumes ranging from 200 to 500 μL,
depending on availability) or standards (500 μL;
containing 0.1 μM NE and DHPG in distilled, deionized water
(ddH2O)) were mixed with 250 μL of 0.2 M perchloric
acid (PCA) containing 0.2 μM dihydroxybenzylamine
(DHBA; internal standard described below) and ddH2O,
if necessary, to make the final volume 1 mL. An aliquot
of the supernatant (700 μL) was combined with 300 μL of
3.0 M Tris, pH 8.5 containing 0.1 mM EDTA and 15 mg
of alumina (Activity Grade Super I; ICN Biomedicals).
The samples were tumbled for 15 min. The supernatant
was aspirated from the alumina, and the alumina was
washed once with 1 mL of 0.2 M sodium bicarbonate and
twice with 1 mL of ddH2O (with a vortex-mix and 10 s
centrifugation between washes). Following the final water
wash, 0.1 M PCA (150 μL) was added to the alumina to
desorb the catechols. A 50 μL aliquot of the sample was
injected onto the HPLC column for analysis.
High performance liquid chromatography
The catechols were separated by reversed-phase
chromatography on C18 column (Agilent Microsorb,
150 × 4.6 mm, 5 μm) using a filtered and degassed mobile
phase consisting of 75 mM sodium phosphate (pH 3.0),
1.7 mM sodium octane sulfonate, 1.5% acetonitrile with
a flow rate of 1.0 mL/min. The mobile phase was
maintained with a Shimadzu L10AD pump, and a Shimadzu
SIL-20AC HT autosampler was used to inject 50 μL
aliquots of either sample or standard.
An electrochemical detector (ESA Coulochem III;
Bedford, MA) was used to detect and quantify the
catechols. When using the oxidation protocol (detector set at
+180 mV) in test plasma runs, there was a large peak
that co-eluted with DHPG that we identified as uric
acid. Even though uric acid is poorly adsorbed onto
alumina, the high amounts of uric acid that are present
in the plasma of heart failure patients [
] result in
peaks that are much larger than those for DHPG. We
took advantage of the reversible oxidation of catechols
but irreversible oxidation of uric acid under our
conditions to analyze catechols in the plasma samples. Thus,
we used an oxidation–reduction protocol in which a
conditioning electrode was set at + 300 mV to oxidize
all analytes in the eluate, the first analytical electrode
was set at + 150 mV to insure complete oxidation of
all analytes, and the second analytical electrode set at
− 350 mV to reduce the analytes . The gain on the
reducing detector was set at 50 nA full-scale.
LabSolutions (Shimadzu) software was used to collect and
analyze the data (see Fig. 1 for flow diagram).
Fig. 1 Flow diagram for the oxidation–reduction protocol measuring
NE and DHPG in plasma. First, to each of the samples or standards, an
internal standard (i.e. DHBA) is added. Then, the catechols (NE, DHPG,
and DHBA) are adsorbed onto alumina. The alumina is washed first
with sodium bicarbonate and then twice with water to remove any
substances not bound to the alumina. The catechols are desorbed
from the alumina using 0.1 M PCA. An aliquot of the supernatant is
injected onto the HPLC, and the catechols are separated by liquid
chromatography on the HPLC. The catechols are first oxidized at
+ 300 and + 150 mV and then reduced at − 350 mV. The output of
the reduction signal is analyzed by the computer software. DHBA
dihydroxybenzylamine, DHPG 3,4-dihydroxyphenylglycol, HPLC
high performance liquid chromatography, NE norepinephrine, PCA
Calculation of results
The internal standard method is commonly
acknowledged to be the gold standard in quantifying analytes in
complex mixtures such as plasma [
]. Since the
internal standard, DHBA, behaves similarly to the plasma
catechols in the alumina extraction and
electrochemical detection process (but is not found in biological
samples), it is possible to quantify plasma catechols in
samples by referring them to standards, which both
contain a standard amount of DHBA. Using the ratio of
the plasma catechol peak area (i.e. NE or DHPG) to the
DHBA peak area, divided by a similar ratio for the
catechol standard, this ratio of ratios is then adjusted for
the fraction of the sample used in the assay [correction
factor (CF)] by the following equation to obtain the
catechol concentration per mL of plasma.
× CF = Catechol per mL plasma
Data were analyzed using means and standard
deviations. We used linear regression analysis to establish
the linearity and variability of the method. All analyses
were performed using GraphPad Prism version 7.02.
To quantify linearity of the method, we analyzed a
range of concentrations of NE (0–0.12 μM), DHPG
(0–0.18 μM), and DHBA (0–0.12 μM) that reflected the
expected range of these compounds in plasma using
alumina extraction and an oxidation–reduction
protocol. The peak areas of NE and DHPG relative to DHBA
were compared (i.e. NE:DHBA and DHPG:DHBA,
respectively). The method was linear for NE (r2 = 0.997)
and DHPG (r2 = 0.983) over this range of
concentrations (Additional file 1: Figure S1).
To quantify recovery amounts, we processed known
concentrations of a standard mix (containing 0.5 μM
DHPG, 0.5 μM NE, and 0.5 μM DHBA) and uric acid
alone (50 μM). Using both oxidation and oxidation–
reduction protocols, we processed the standard mix
(n = 6) and uric acid (n = 6) with and without alumina
extraction. Average recoveries are reported in Table 1.
Despite removing > 99% of uric acid with alumina
extraction and an oxidation protocol, the remaining
uric acid still yielded a peak (recovery ~ 0.075 μM) that
overlaid the DHPG peak (recovery ~ 0.155 μM).
Alumina extraction coupled with the oxidation–reduction
Comparison of recoveries of processed samples using the oxidation and
oxidation–reduction protocols. Standard mixes (n = 6; each containing 0.5 μM
DHPG, 0.5 μM NE, and 0.5 μM DHBA) and uric acid (n = 6; each containing 50 μM)
were processed and subjected to either the oxidation protocol or the oxidation–
reduction protocol. We calculated recoveries of each compound based on
the starting amount without alumina extraction. Even though uric acid was
poorly adsorbed onto alumina, the concentration of uric acid in plasma is high
compared with the catechols and even a small percentage retained obscures the
DHPG peak. DHBA dihydroxybenzylamine, DHPG, 3,4-dihydroxyphenylglycol, M
mean, ND non-detectable, SD standard deviation
a Recoveries are expressed as a percentage of the starting amount
protocol eliminated the uric acid while maintaining
adequate recoveries of NE and DHPG.
Representative chromatograms from plasma samples
are shown in Fig. 2. In Fig. 2a, using oxidation alone, there
was a large uric acid peak that co-eluted with DHPG. By
including a sodium bicarbonate wash of the alumina and
an electrochemical protocol involving oxidation followed
by reduction, the co-eluting peak of uric acid was
eliminated (Fig. 2b), unmasking the underlying DHPG peak.
The peaks are negative due to the absorption of electrons
by the compounds as a result of reduction. The average
intra-assay coefficient of variation was 5.3% (n = 35–40),
and the inter-assay coefficient of variation was 4.6%
(n = 4). Based on a plasma sample of 500 μL, our
estimated detection limit was approximately 85 pg/mL
(0.5 pmol/mL) for NE and 165 pg/mL (0.9 pmol/mL) for
DHPG, using a signal/noise ratio of ≥ 3.
Building on previous methods [
], we describe a
method to measure plasma NE and its principal
metabolite, DHPG, that was developed as part of a translational
research study. Our main findings are: (1) alumina
extraction coupled with an electrochemical detection protocol
involving oxidation followed by reduction eliminated a
uric acid contaminant that co-eluted with DHPG, and (2)
we were able to quantify NE and DHPG in plasma
samples with reasonable detection limits.
An advantage of this method is the measurement of the
direct principal metabolite, DHPG, which yields
important information above and beyond the measurement
of NE. DHPG is closer to NE in the metabolic scheme
than the end metabolite, vanillylmandelic acid [
reflects the neuronal metabolism of NE compared with
the non-neuronal metabolite, normetanephrine [
Importantly, DHPG can be measured in the same assay as
NE. Our findings demonstrate that a simple set-up with
HPLC-ED, including an alumina extraction coupled with
a sodium bicarbonate wash and an oxidation–reduction
protocol, may rapidly increase the feasibility for clinical
laboratories to detect DHPG in addition to NE.
Further consideration relates to the quantity of
human plasma required for NE and DHPG detection.
Human plasma is often of limited quantity and must be
carefully allocated for various assays and experiments.
Complicating this circumstance, catecholamines exist
in small quantities in biological fluids, which demands
that bioanalytical methods must be specific and
sensitive enough to detect these small quantities. In our
application, we were able to detect and easily quantify
amounts of NE and DHPG in plasma samples as low as
Our intent with this paper was to demonstrate how
to measure plasma NE and DHPG with
applicability to both translational and clinical research studies.
While there are noted benefits and drawbacks to the
various methods to measure plasma NE [
14, 15, 24,
], researchers frequently do not have access to
extensive set-ups in laboratories nor the expertise to
perform complicated assays. Moreover, plasma may be the
only biological fluid available, as opposed to
multiplehour urine collection , for example. This described
method has the advantage of being simple and can be
set-up in any laboratory that has HPLC-ED. We
outline the necessary parameters, including the sample
preparation process and the chromatographic and
detector settings, which permit concentration,
separation, and quantification of the compounds of interest.
Finally, this method was high-throughput; we were able
to process about 40 samples in 2 days. In conclusion,
there are multiple applications within translational and
clinical research for HPLC-ED measurement of NE
and its principal metabolite, DHPG, yielding clinically
significant information on sympathetic activity and
contributing to translational knowledge regarding key
physiological processes in both health and disease.
There are few noted limitations to this method,
including the acknowledgment of well-documented analytical
1, 24, 28
]. First, even though we were able
to estimate the neuronal reuptake of NE with DHPG
levels, this method does not permit the estimation of
the non-neuronal clearance of plasma NE nor the
kinetics of NE. Second, we used samples collected from the
forearm, which does not necessarily reflect sympathetic
activity in the rest of the body because sympathetic
outflow varies among tissues and organs [
]. Finally, the
recovery of DHPG in the oxidation–reduction protocol
was lower than NE possibly due to the sodium
bicarbonate wash or less efficient reduction of DHPG. Future
work to improve this method should involve
developing techniques to increase the recovery of DHPG,
particularly when the absolute concentration is
necessary. Additionally, these methods should be compared
with other known methods using the same plasma
samples to quantify differences in reported concentrations.
Additional file 1: Figure S1. Linearity of the response for NE (A) and
DHPG (B) based on a range of physiological concentrations compared
with the ratio of NE or DHPG to the internal standard, DHBA. DHBA
dihydroxybenzylamine, DHPG 3,4-dihydroxyphenylglycol, NE norepinephrine.
CF: correction factor; ddH2O: distilled, deionized water; DHBA:
dihydroxybenzylamine; DHPG: 3,4-dihydroxyphenylglycol; HPLC-ED: high performance
liquid chromatography with electrochemical detection; NE: norepinephrine;
PCA: perchloric acid.
QED and BAH conceived of the initial design of the translational research
study. QED and WRW developed and tested the methods, collected the data,
and analyzed the results. BAH and WRW served as experts on neurochemistry
and HPLC-ED methods. QED took the lead in drafting the manuscript, and
BAH and WRW critically edited the manuscript. All authors read and approved
the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
Data related to the linearity, recovery, and coefficient of variation calculations
are available from the corresponding author upon request.
Consent for publication
Ethics approval and consent to participate
The parent study was approved by the Institutional Review Board at Oregon
Health & Science University (IRB#7907), and written informed consent was
obtained from all participants with the option to store their data and plasma
samples in a biorepository. This results reported in this paper were part of
an ancillary study that used a subset of de-identified plasma samples from
the biorepository (from the group of participants who provided written
informed consent to have their data and samples stored); this ancillary study
was exempted by the Institutional Review Board at Oregon Health & Science
University as non-human subjects research (IRB#16473).
The work reported in this paper was supported by the National Institutes
of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI) through
a post-doctoral fellowship (for Dr. Denfeld) at Oregon Health & Science
University Knight Cardiovascular Institute (T32HL094294). Dr. Denfeld is
currently supported as a Scholar of the Oregon Building Interdisciplinary
Research Careers in Women’s Health K12 Program funded by the Eunice
Kennedy Shriver National Institute of Child Health & Human Development of the
NIH under Award Number K12HD043488. Plasma samples were collected as
part of a parent study that was funded by the National Institutes of Health/
National Institute of Nursing Research (R01NR013492; Lee) and supported
by the National Center for Advancing Translational Sciences of the National
Institutes of Health (UL1TR000128). Drs. Habecker and Woodward are currently
funded by the NIH/NHLBI (R01HL093056; Habecker). The content is solely the
responsibility of the authors and does not necessarily represent the official
views of the NIH.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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