Clinical Chemistry 45: 394-399, 1999;
(Clinical Chemistry. 1999;45:394-399.)
© 1999 American Association for Clinical Chemistry, Inc.
Evaluation of a New Method for the Analysis of Free Catecholamines in Plasma Using Automated Sample Trace Enrichment with Dialysis and HPLC
John Dutton1,
Andrew J. Hodgkinson1,
George Hutchinson2 and
Norman B. Roberts1,a
1
Department of Clinical Chemistry, Royal Liverpool and Broadgreen University, Hospital NHS Trust, Liverpool L7, 8XP, United Kingdom.
2
Anachem Ltd, Luton LU2 0EB, United Kingdom.
a Author for correspondence. Fax 44 (1)51 706 5813.
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Abstract
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Background: Analysis of urinary free catecholamines was automated
recently, but analysis of plasma samples posed special difficulties.
The present study was undertaken to evaluate a new method for the
automated analysis of plasma catecholamines.
Methods: The procedure is based on an improved sample handling
system that includes dialysis and sample clean-up on a strong cation
trace-enrichment cartridge. The catecholamines norepinephrine,
epinephrine, and dopamine are then separated by reversed-phase ion-pair
chromatography and quantified by electrochemical detection.
Results: Use of a 740-µL sample is required to give the
catecholamine detection limit of 0.05 nmol/L and analytical imprecision
(CV) between 1.1% and 9.3%. The assay can be run unattended, although
>12 h of analysis time is not recommended without cooling of the
autosampler rack. Comparison (n = 68) of the automated
cation-exchange clean-up with the well-established manual alumina
procedure gave excellent agreement (mean, 3.78 ± 2.76 and
3.8 ± 2.89 nmol/L for norepinephrine and 0.99 ± 1.72 and
1.08 ± 1.78 nmol/L for epinephrine). Hemodialysis had no clear
effect on plasma norepinephrine. Epinephrine concentrations were
similar (0.05 < P < 0.1) in chronic renal failure
patients (0.24 ± 0.3 nmol/L; n = 15) and healthy controls
(0.5 ± 0.24 nmol/L; n = 31). Dopamine was not quantified,
being usually <0.2 nmol/L.
Conclusion: The availability of such a fully automated procedure
should encourage the more widespread use of plasma catecholamine
estimation, e.g., after dialysis, exercise, or trauma/surgery and in
the investigation of catecholamine-secreting tumors, particularly
in the anuric patient. © 1999 American Association for
Clinical Chemistry
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Introduction
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The routine analysis of plasma free norepinephrine, epinephrine,
and dopamine is fraught with analytical problems, primarily associated
with low plasma concentrations and the relatively labor-intensive
manual extraction procedure required to remove interfering compounds
(1). The automation of such techniques using automated
sequential trace enrichment of
dialysates(ASTED)1
has been applied successfully to the
assay of urinary free catecholamines (2). The application to
plasma, however, presented specific problems; in particular, the
original ASTED system could not achieve regeneration and conditioning
of both the dialyzer and the trace-enrichment cartridge (TEC). This has
now been successfully resolved with the development of the second
generation ASTED.XL(TM), which utilizes two rheodyne valves and two
dilutors. The technique involves the dialysis of the plasma sample to
remove protein and other macromolecules. The catecholamines are then
trace-enriched onto a strong cation-exchange resin (3) and
eluted onto the analytical column. Separation is achieved by
reversed-phase ion-pair chromatography with coulometric detection of
the catecholamines (4), whereas the TEC and dialyzer are
regenerated for subsequent injections. Procedures reported
previously for automated plasma catecholamine analysis have
included a manual clean-up step or simple deproteinization
(5) and then solid-phase extraction (6) followed
by automated HPLC with electrochemical or fluorescence detection
(7).
We here report the analytical evaluation of the fully automated
analytical ASTED procedure for measuring plasma catecholamines. The
analysis of plasma norepinephrine (NE) and epinephrine (E) was compared
in healthy volunteers, patients being investigated for
catecholamine-secreting tumors, patients with chronic renal failure,
and in particular, assessment of the effect of regular hemodialysis in
patients with end-stage renal failure.
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Materials and Methods
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Blood samples (10 mL) were collected into tubes containing lithium
heparin as the anticoagulant (Sarstedt) and transported to the
laboratory on ice within 30 min. Samples were centrifuged at 3500
rpm for 10 min at 4 °C. Plasma was transferred into 2-mL
polypropylene tubes (L.I.P.) and frozen immediately at
-25 °C. Analysis was then performed within 1 month of receipt of
the sample. For longer periods, storage was at -70 °C.
Samples were obtained from healthy volunteers (n = 31), patients
with chronic renal failure (n = 15), end-stage renal failure
patients on hemodialysis (n = 15), and hospital patients with
hypertension being investigated for catecholamine-secreting tumors
(n = 15). A group of healthy volunteer athletes (n = 10) was
sampled before and after exercise to exhaustion, i.e., 70% of maximum
O2 utilization on a cycle ergometer as part of a study on
the effects of exercise on glucose utilization and insulin production
(8). This allowed us to obtain plasma samples (n = 69)
with variable catecholamine concentrations within or greater than the
upper limit of the reference interval. The end-stage renal failure
patients were sampled before and after dialysis, and the hypertensive
patients were sampled in a supine position after a 30 min rest period.
The collection of samples complied with the ethics standards of the
Royal Liverpool University Hospital. The additional analyses were
performed anonymously, in accordance with the ethics guidelines of the
Hospital. The system used was the Gilson ASTED.XL (Anachem), a fully
automated sample processing system. Incorporated into this was a Gilson
307 pump (Anachem) with a 10WSC pump head, a dialyzer block volume of
370 µL with a 15 000 molecular weight cutoff cuprophan
dialysis membrane and a hema-SB cationic TEC (Anachem). The optimized
plasma sample volume was 740 µL, taken as two separate aliquots of
370 µL. The individual aliquots were dialyzed into 2 mL of 5 mmol/L
ammonium phosphate buffer, pH 8.6, and the dialysate was applied to the
TEC. To increase sensitivity, additional plasma aliquots could be used
simply by changing the program controlling the ASTED processor.
Electrochemical detection of the catecholamines was achieved using an
ESA Coulochem II (ESA Analytical) fitted with a guard cell and a 5011
analytical cell. Oxidation of the analytes was carried out at +300mV in
the guard cell followed by successively higher reduction potentials of
-100 mV and -300 mV in the analytical cell and a sensitivity set at
20 nA.
The mobile phase consisted of 50 mL/L in 125 mmol/L diammonium hydrogen
orthophosphate (BDH) containing 101 mg of heptane sulphonic acid (Sigma
Chemical Co.) and 73 mg of EDTA (Sigma) adjusted to pH 3.5. A 15.0
x 0.46 cm Ultratechsphere 5 µm ODS 2 column (HPLC Technology) was
used at a flow rate of 1.5 mL/min. The dialysis recipient solvent was 5
mmol/L diammonium hydrogen orthophosphate adjusted to pH 8.3.
Dihydroxybenzylamine was used as the internal standard (IS) at a
concentration of 60 nmol/L, 50 uL of which was added to 750 uL of
sample.
The effect of the serum matrix on dialysis was assessed by comparison
of peak areas after injection onto the complete system of an aqueous
calibrator, a serum blank, and the same serum to which 10, 5, 20, or 5
nmol/L NE, E, dopamine (D), and IS had been added. The mean ± 1
SD recoveries (n = 5) were 83% ± 2%, 75% ± 2.5%, 95%
± 3.8%, and 91% ± 3%, respectively, which indicated a small
loss because of the protein matrix effect. The relative recovery
through the TEC without dialysis was compared with loop injection only
using 100 µL of an aqueous solution containing 10 nmol/L NE, 5 nmol/L
E, 20 nmol/L D, and 5 nmol/L IS. The recoveries (mean ± SD;
n = 4) were 89% ± 1.5%, 77% ± 1.5%, 91% ± 2%, and 94% ±
2.1%, respectively, indicating some loss on the TEC, in particular for
E because of its reduced retention on the cation exchanger
(2). The overall recovery or efficiency of the dialysis was
assessed by comparison of the peak areas after injection of an aqueous
calibrator (through the loop only) and a serum blank supplemented with
10 nmol/L NE, 5 nmol/L E, 20 nmol/L D, and 5 nmol/L IS taken through
the complete system. The recoveries were 38% ± 2% for NE, 35% ±
1.5% for E, 19% ± 0.8% for D, and 34% ± 1.7% for IS (mean
± 1 SD; n = 4), indicating a relatively efficient dialysis.
The system was calibrated using standard additions of NE (Sigma) at 0,
5, 10, 15, and 20 nmol/L and E (Sigma) at 0, 4, 8, and 10 nmol/L to
drug-free serum (Bio-Rad). The individual catecholamine responses were
corrected to the IS response, and this ratio was related to known
concentrations of each analyte. The regression analysis of n = 6
observations for NE and E was: y = 11.1 x
105x + 10, r = 0.998; and
y = 12.5 x 105x + 8,
r = 0.988, respectively, where y is the
electronic signal in computed integration units (Gilson Unipoint
Software). In routine use, the calibration was confirmed with 0 and 10
nmol/L NE and 0 and 5 nmol/L E. Calibrators were run every tenth
sample, and controls were run every fifth sample. If the signal
response deviated >15%, then recalibration would be carried out to
ensure that the controls were always within 1 SD of the expected mean
value. The analytical performance (Table 1
), was monitored with commercially available Endocrine Controls
1 and 2 (Bio-Rad A and B). All solutions were prepared using doubly
deionized water (Elga Products Ltd).
The alumina extraction (9) was used as the comparative
reference procedure with 69 plasma samples obtained from the various
groups outlined above. The catecholamines from a 1.0-mL plasma sample
were desorbed from alumina with 400 µL of 0.1 mol/L phosphoric acid
(Sigma), and 370 µL was injected for separation by HPLC as outlined
for the ASTED procedure. The system was controlled and analytical
integrations were carried out by Gilson UniPoint Software with a Gilson
506C interface module (Anachem).
All data were processed using Microsoft Excel for Office 95, and data
were compared with standard linear regression analysis and differences
between groups with parametric Tukey and nonparametric Kruskal–Wallis
statistical analysis, using the ARCUS software as supplied by the
University of Liverpool, Liverpool, UK.
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Results
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Typical chromatograms obtained for automated plasma catecholamine
analysis are shown in Fig. 1
. The analytical performance given in Table 1
indicates good precision.
The detection limit, under the conditions outlined and determined as 3
SD above the mean background signal noise, was 0.05 nmol/L for both E
and NE with a 740-µL plasma volume injected. The commonly prescribed
beta blockers, angiotensin-converting enzyme (ACE) inhibitors,
L-dihydroxyphenylalanine, and metabolites from caffeine or
acetaminophen did not interfere in the assay.
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Figure 1. Chromatograms of the Bio-Rad B (top) and A
(bottom).
Concentration data are shown in Table 1
. D was detected in
controls A and B at 0.6 and 3.3 nmol/L, respectively, but was usually
<0.2 nmol/L in the patient samples. The IS is dihydroxybenzylamine.
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Stability of the catecholamines on the system (i.e., on the autosampler
rack at 20–22 °C) was assessed using various control solutions and
indicated a loss of activity of both NE and E of <1.0%/h in plasma
and 3.0%/h in an aqueous solution. Plasma samples stored >72 h at
4 °C decayed at 0.2%/h, over which time a 15% loss was considered
significant. The routine practice, therefore, is to calibrate with
serum-based calibrators and to perform the analysis in batches such
that samples remain on the autosampler for periods no longer than
10–12 h. Analytical comparison with the reference alumina extraction
procedure (Fig. 2
) gave excellent agreement for NE and E over a wide range of
concentrations, showing no significant difference between the two
analytical procedures.
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Figure 2. Comparison between the ASTED-based cation-exchange
(x) and the alumina clean-up procedures (y).
(Top), plasma NE: y = 1.02x
- 0.1; r2 = 0.98; (bottom), plasma
E: y = 0.99x + 0.09,
r2 = 0.99.
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Plasma catecholamines varied little among the patient groups studied
(Fig. 3
). Plasma NE was significantly increased in the predialysis and
hospital patients compared with the healthy controls, P
<0.05, but pre- and postdialysis concentrations were not significantly
different. E was relatively higher in the healthy volunteers. In the
chronic renal failure patients, plasma NE was 2.0–5.8 nmol/L and E was
0.1–0.7 nmol/L; both were independent of creatinine clearance in the
range <5–120 mL/min. In patients investigated for a
catecholamine-secreting tumor, plasma NE and E concentrations similar
to healthy controls would suggest exclusion of such a diagnosis. One
patient (data not in Fig. 3
) with increased NE (11.8 and 9.6 nmol/L), a
strong clinical suspicion, and a negative clonidine suppression test
was subsequently investigated with selective venous sampling, which
identified a catecholamine-secreting tumor in the inferior vena fossa.
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Figure 3. Plasma NE and E in the different groups studied: pre
(n = 15) and post hemodialysis (n = 15); chronic renal
failure (CRF) patients not on dialysis (n = 15);
healthy volunteers (n = 31); and hospitalized patients (n =
15).
Values reported are in nmol/L as the mean ± 1 SD. Values for
NE and E were significantly different,
P <0.05, by both the Kruskal–Wallis and the Tukey
tests between predialysis and chronic renal failure patients, chronic
renal failure patients and controls, and controls and HT.
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In the volunteers studied after exercise, the plasma NE rose from
(mean ± 1 SD) 4.5 ± 2.5 nmol/L (range, 1.5–6.4 nmol/L) to
15.6 ± 5.6 nmol/L (range, 8.0–35.2 nmol/L), and E rose from
0.5 ± 0.3 nmol/L (range, 0.2–1.0 nmol/L) to 4.5 ± 1.5
nmol/L (range, 1.6–8.6 nmol/L).
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Discussion
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We have shown that the fully automated quantitative analysis of
the plasma free catecholamines NE, E, and D can be achieved without any
sample pretreatment, using the newly developed ASTED.XL system. D was
not usually measured under these conditions (being near the analytical
detection limit); however, it can be seen at higher concentrations, and
therefore more reliably quantified, after treatment with sulphatase
(7) or when patients are on
L-dihydroxyphenylalanine (10). This
methodological advance has been made possible because of the
incorporation of improved software to control two rheodyne valves that
allow access to the two dilutors to regenerate the TEC and recondition
the dialyzer block independently. In the ASTED configuration for urine
analysis (2), dialysis was not required and only one dilutor
was made available for regeneration of the TEC. However, when the
ASTED.XL was used, both dialysis and trace enrichment could be
controlled easily. The conditions were then optimized on 740 µL of
plasma. If measurement of concentrations below or near the detection
limit is required, more sample, e.g., 1000 µL of plasma or a longer
dialysis time could be used.
We have been performing the procedure routinely for several years,
having originally demonstrated that the system operates satisfactorily
for plasma catecholamine analysis (11). To maintain good
performance, reagents must be prepared fresh weekly, using pure water
as outlined. Any loss of sensitivity other than that caused by old
reagents can often be traced either to poor trace enrichment or to poor
dialysis, although the dialyzer should remain effective for up to 6
months. The TEC and the analytical column should be functional for a
similar period or a minimum of up to 2–3000 injections. The coulochem
analytical cells have expected working lives of at least 2 years or
6000 injections. However, this is very much dependent on the number of
samples analyzed, e.g., urine analysis should give a greater life
expectancy because of a reduced sensitivity requirement. If loss of
sensitivity is because of failure of the analytical cell, then
regeneration (see manufacturer's instructions) might be a possibility;
the best solution, however, is to invest in a new cell. The analytical
cost is approximately 1.5–2.0 (1 = $1.60) per test and compares
favorably with the reagent costs for new RIAs and enzyme immunoassays
(12) priced at 2 for each individual analyte.
The ASTED system can easily be adapted to run urinary catecholamines,
the organic acids vanillylmandelic acid, 5-hydroxyindoleacetic acid,
and homovanillic acid, the methylated catecholamine derivatives, and
serotonin (13). The serotonin assay can, if required, be
included in the plasma catecholamine analysis because its retention
time on the chromatography system is ~20 min. However, because the
system outlined is programmed to re-inject every 20 min, this would
need to be changed to 30 min.
The ASTED procedure gives good analytical precision for the routine
assay of plasma catecholamines, although previous studies using manual
alumina clean-up procedures on 1–2 mL of plasma have reported equally
good sensitivity and analytical precision (14). However, we
have found that the ASTED system is much more sympathetic to general
routine clinical applications, a sentiment confirmed by the UK external
quality-control assessment scheme for urine catecholamines
(15). We have been able to carry out several large studies,
with up to 4000 analyses, involving the serial changes in plasma
catecholamines after hip operations and in healthy individuals after
strenuous exercise (8). Alternative automated procedures
have been developed that require some manual pretreatment involving
deproteinization followed by either individual solid-phase extraction
with an automated processing device or precolumn
o-phthaldehyde derivatization. Subsequent separation can use
standard ion-pair chromatography with an electrochemical detection or
ion-pair soap chromatography with fluorescence detection
(6)(7). The latter procedure suffers from glutathione
interference with the internal standard dihydroxybenzylamine and has
thus caused difficulties in plasma assay reliability. The latter was
also adapted to postcolumn derivatization with a fluorogenic agent,
diphenylethylenediamine, but again without addition of an IS
(16).
Of particular interest are the data now presented in patients with
end-stage renal failure; the effect of dialysis on NE and E, D was not
usually detected, i.e., the concentrations were generally <0.1 nmol/L
(5). Previous reports have shown inconsistent findings, with
either increased catecholamine concentrations or concentrations within
the reference interval (17)(18); however, the number of
studies has been limited presumably because of difficulties in
catecholamine analysis in renal failure. We have now shown that plasma
NE was significantly increased in both patients on regular dialysis and
those in varying stages of renal failure but not on dialysis compared
with healthy controls. Although the range of results was similar, some
of the end-stage renal failure patients would appear to be more
stressed than others. Clearly, additional studies are required to
explain the mechanism of this phenomenon and whether the stressed
patients are more likely to have some form of autonomic nerve
dysfunction associated with renal disease (19). E
concentrations were generally the same in all groups and were not
affected by dialysis, although concentrations were shown to be
increased in the healthy volunteers. A particular benefit of the plasma
assay for patients in end-stage renal failure is the ability to
identify catecholamine-secreting tumors. This is highlighted by the
recent report of an anuric patient presenting with paroxysmal
hypertension suspected to be as a result of a dopamine-secreting tumor
(20).
In conclusion, a procedure has been developed for the automated
assay of plasma catecholamines. The assay is suitable for routine
clinical applications, is relatively inexpensive to perform, and is
sensitive and precise. This procedure will facilitate routine
measurement of catecholamines and therefore, additional studies of
changes in health and disease, in particular, renal disease and the
investigation of catecholamine-secreting tumors in patients who are
anuric.
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Acknowledgments
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We thank the medical staff of the renal unit of the Royal
Liverpool and Broadgreen University Hospital, Liverpool, UK for data on
patients and collection of blood samples during hemodialysis sessions.
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Footnotes
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1 Nonstandard abbreviations: ASTED, automated sequential trace enrichment of dialysates; TEC, trace-enrichment cartridge; NE, norepinephrine; E, epinephrine; IS, internal standard; and D, dopamine. 
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Abstract
Introduction
