Clin Chem Lab Med 2019; aop

Elisa Danese*, Martina Montagnana, Claudio Brentegani and Giuseppe Lippi

Short-term stability of free metanephrines in

plasma and whole blood
https://doi.org/10.1515/cclm-2019-0020
Received January 7, 2019; accepted April 3, 2019
Introduction
Abstract Reliable evidence and current guidelines indicate that
the initial screening of pheochromocytoma and paragan-
Background: Analysis of plasma metanephrine (MN) and
glioma (PPGL) should be based on the measurement of
normetanephrine (NMN) with liquid chromatography
plasma free or urine-fractionated metanephrines (MNs)
tandem mass spectrometry (LC-MS/MS) is the gold stand-
[1]. Among these two approaches, plasma free MNs have
ard for the screening of pheochromocytomas and para-
recently been preferred in view of higher sensitivity and
gangliomas (PPGLs). As scarce information is available on
specificity compared to the urinary counterparts (99%
the stability of MNs in diagnostic samples, this study was
vs. 95% and 96% vs. 89%, respectively) [2]. Nevertheless,
aimed at analyzing the short-term stability of plasma free
the advantages of using plasma rather than urine can
MNs in whole blood and plasma, using LC-MS/MS.
be offset by the inaccurate use of preanalytical, analyti-
Methods: The stability of plasma MNs was evaluated after
cal and postanalytical procedures. Some recommended
sample collection at 1, 2 and 3 h in whole blood, and at 2,
measures include, among others, blood drawing from
4 and 6 h in centrifuged samples. Both studies were per-
patients who have rested in the supine position for at least
formed while maintaining the samples at room tempera-
30  min [3, 4], the use of liquid chromatography tandem
ture (RT) and at 4 °C. The ClinMass Complete Kit (Recipe,
mass spectrometry (LC-MS/MS) as the reference technique
Munchen, Germany) was used for measuring MNs with
[1], and the use of age-adjusted reference intervals for
LC-MS/MS (Nexera X2 UHPLC-4500MD Sciex). Differences
achieving optimal diagnostic sensitivity and minimizing
from the baseline (T0) were assessed using repeated
­false-positive results [5].
measures one-way ANOVA, Students’ paired t-test and a
Beyond these widely recognized suggestions, no uni-
comparison of the mean percentage changes with the total
versally agreed recommendations have been endorsed for
change limit (TCL).
handling blood specimens after collection. The stability
Results: Statistically significant differences from T0 were
of MN and normetanephrine (NMN) in whole blood and
found for both MNs (p < 0.001) in whole blood stored at
plasma samples has been systematically evaluated only
RT, and for NMN (p = 0.028) but not MN (p = 0.220) at 4 °C.
three times in the present literature, to the best of our
The mean difference exceeded the TCL after 1 h and 3 h at
knowledge. The first study assessed short-term stability
RT for MN, and after 1 h at RT for NMN. Statistically signifi-
[6], the second assessed mid-term stability [7], whilst the
cant differences from T0 were only observed in the plasma
third evaluated long-term stability [8]. Two studies used
samples for NMN at RT (p = 0.012), but the variation was
an LC-based method, coupled with electrochemical detec-
within the TCL.
tion [7, 8], and one used a radioimmunoassay [6].
Conclusions: MN and NMN displayed different patterns of
Therefore, this study was aimed at evaluating the
stability before and after centrifugation. Even short-time
short-term stability of free MNs before and after centrif-
storage at RT in whole blood should hence be avoided.
ugation, using the reference LC-MS/MS technique. The
Keywords: free metanephrines; liquid-chromatography protocol was designed to identify a suitable preanalytical
tandem mass spectrometry; preanalytical phase; stability. procedure to be used in our routine practice.

*Corresponding author: Dott.ssa Elisa Danese, PhD, Section of

Clinical Biochemistry, Department of Neurological, Biomedical and
Movement Sciences, University Hospital of Verona, Piazzale L.A
Scuro, 10, 37100 Verona, Italy, Phone: +39-045-8124308,
Fax: +39-045-8027484, E-mail: elisa.danese@univr.it
Martina Montagnana, Claudio Brentegani and Giuseppe Lippi: Section
of Clinical Biochemistry, Department of Neurological, Biomedical and
Movement Sciences, University Hospital of Verona, Verona, Italy
Materials and methods
The study was divided into two parts, the first aimed at assessing
pre-centrifugation stability and the second at evaluating post-cen-
trifugation stability. Whole blood from 10  healthy volunteers (mean
age 52 ± 8  years; six women and four men) was collected in the sit-
ting position into eight consecutive K2EDTA tubes (Vacutest, Vacutest

Brought to you by | Chalmers University of Technology

Authenticated
Download Date | 8/4/19 12:02 PM
 2      Danese et al.: Short-term stability of free metanephrines

Kima SRL, Padova, Italy). Two tubes were immediately centrifuged,
one each of the other three tubes was maintained for 1, 2 and 3 h at
room temperature (RT) before centrifugation whilst one each of the
remaining three tubes was maintained for 1, 2 and 3 h at 4 °C before
centrifugation. After each time period, the centrifuged plasma was
stored at −20 °C until measurement. From 10 other healthy volunteers
(mean age 35 ± 12 years; four women and six men), eight additional
consecutive K2EDTA tubes (Vacutest, Vacutest Kima SRL, Padova,
Italy) were collected. All tubes were immediately centrifuged and
then stored at RT (n = 4) and 4 °C (n = 4). These temperatures were
also maintained during plasma separation, which occurred at T0, 2, 4
and 6 h after collection. The centrifuged plasma was stored after these
time periods at −20 °C until measurement. A detailed description of
the study design is presented in the Supplementary Material, Table 1.
The quantification of plasma MN and NMN was performed in the
same analytical run for all samples, on thawed plasma, using the Clin-
Mass Complete Kit (Recipe, Munchen, Germany) with LC-MS/MS. The
LC-MS/MS system was composed of a Nexera X2 series UHPLC (Shi-
madzu, Kyoto, Japan) coupled with a 4500 MD triple quadrupole MS
detector (Sciex, Milan, Italy). Sample processing was carried out after
mixing with appropriate internal standards, via solid phase extrac-
tion (SPE), which provided sample purification and enrichment in
the MN content, according to the manufacturer’s specifications. Elec-
trospray ionization was carried out in the positive mode (ESI+). Data
were recorded in the multiple reaction monitoring mode (MRM). The
Analyst MD 1.6.2 and Multiquant MD 3.0.2 software (Sciex) were used
for data acquisition and quantification, respectively. This method
has a limit of quantitation (LOQ) of 20 pmol/L and was found to be
linear up to 100,000 pmol/L for both MNs [9]. The mean intra-assay
and inter-assay imprecision, as assessed using three plasma pools
with different analyte concentrations, was between 1.7 and 3.9% for
MN and between 3.2 and 6.2% for NMN, respectively. The recovery
was between 80 and 110%. Details on method validation have been
previously reported [9].
All data were normally distributed according to the Shapiro-
Wilk test.
The percentage variation of MN and NMN values at different
time points was calculated as follows: [(Tx − T0)/T0] × 100], where
“T0” is the baseline concentration, whilst “Tx” is the concentration
measured at each specific time point. The mean percentage devia-
tion (±SD) of the 10  healthy volunteers was calculated. Repeated
measures one-way ANOVA and Student’s paired t-test were used to
assess the significance of differences.
As a measure of acceptability, the total change limit (TCL) [10]
was also calculated using the formula [(2.77 × CVa)2] + [(0.5 × CVb)2]½,
where CVa is the analytical imprecision of the assay and CVb is the
within-subject variation. Considering CVa values of 1.7% and 3.2%
and CVb values of 8.4% and 13.4% for MN and NMN, respectively [11],
the calculated TCLs were 6.3% for MN and 11.1% for NMN. When the
mean percentage bias (±SD) was found to be higher than the TCL of
MN and NMN, the difference from the baseline was considered unac-
ceptable and the measurement was hence considered unreliable
[12]. Statistical analysis was carried out using the GraphPad Prism
5.0 (GraphPad Software, Inc., San Diego, CA, USA). Statistical signifi-
cance was set at p < 0.05. The study was performed in agreement with
the Declaration of Helsinki. All patients recruited to this study pro-
vided a written informed consent and the research was cleared by the
local Ethical Committee (University Hospital of Verona, n. 971CESC,
July 25, 2016).
Results
Stability of MNs before centrifugation
The MN concentration increased within the first hour
of storage at RT (from 207 ± 76 to 221 ± 79 pmol/L; +8%;
p = 0.039), and then rapidly decreased until values
approximately half lower than the baseline concentra-
tion were reached within 3  h (122 ± 49 pmol/L, −41%,
p < 0.001) (Figure 1A and Table 1). NMN increased rapidly
within the first hour of storage at RT (from 535 ± 182 to
614 ± 198 pmol/L, +16.7%; p < 0.001), and then returned
to the baseline values after 3  h (500 ± 166 pmol/L,
p = 0.148). Differences between the time points were sta-
tistically significant for both MNs (ANOVA p < 0.0001),
and were higher than the TCL after 1 and 3 h for MN and
after 1 h for NMN.

A B
*
* $
600 * $
MN and NMN, pmol/L
MN and NMN, pmol/L

NMN 400
NMN
400
MN
200
200 *
MN
* *
*
0 0
T0 +1 h +2 h +3 h T0 +2 h +4 h +6 h
Time points Time points

Figure 1: Short-term stability of free metanephrines before (A) and after (B) centrifugation.
The squares indicate NMN, and the circles indicate MN. The closed symbols indicate storage at room temperature, and the open symbols
indicate storage at 4 °C. (A) The mean (SE; error bars) concentrations of metanephrines vs. time before centrifugation (T0), reflecting
(in)stability in whole blood at room temperature and 4 °C. (B) The mean (SE; error bars) concentrations of metanephrines vs. time after
centrifugation (T0), reflecting (in)stability in plasma at room temperature and 4 °C. *Significant differences (p < 0.05) vs. initial concentrations
(T0) for tests at RT (closed symbols). $Significant differences (p < 0.05) vs. initial concentrations (T0) for tests at 4 °C (open symbols).

Brought to you by | Chalmers University of Technology

Authenticated
Download Date | 8/4/19 12:02 PM
 Danese et al.: Short-term stability of free metanephrines      3

Table 1: Stability of metanephrines at room temperature and 4 °C according to delay before centrifugation.

Parameter   T0  TCL  Mean % difference (±SD) at RT

 
1 h  2 h  3 h

MN, pmol/L   207 (98–332)  ±6.3  +8.4 (12.6)a  −5.4 (9.9)  −41.3 (6.3)a
NMN, pmol/L  535 (281–867)  ±11.1  +16.8 (13.7)a  +8.6 (12.5)  −4.8 (12.7)

      Mean % difference (±SD) at 4 °C

1 h  2 h  3 h

MN, pmol/L   248 (133–403)  ±6.3  +2.9 (5.0)  +2.5 (6.3)  +5.2 (6.7)
NMN, pmol/L  530 (333–845)  ±11.1  +0.7 (5.8)  +5.4 (9.2)  +6.2 (5.8)

Reference sample expressed as mean (min–max); MN, metanephrine; NMN, normetanephrine; RT, room temperature; TCL, total change
limit; amean percentage higher than the TCL value.

Statistically significant differences at 4 °C were
observed for NMN (p = 0.028) but not for MN (p = 0.220).
At 3 h, the NMN value increased by 6% (from 530 ± 156 to
561 ± 161 pmol/L, p = 0.007). Nevertheless, the mean varia-
tions did not exceed the TLC of both MNs at any time point
(Figure 1A and Table 1).
Stability of MNs after centrifugation
The MN concentration decreased by −4.4% after 4  h of
storage at RT (from 230 ± 81 to 220 ± 82 pmol/L, p = 0.0333).
The NMN concentration displayed a statistically signifi-
cant decrease compared to T0 after 6 h (from 399 ± 75 to
374 ± 69 pmol/L, −6%, p = 0.018) (Figure 1B and Table 2).
One-way ANOVA revealed that the differences between
the time points were statistically significant only for NMN
(p = 0.0117).
MN was stable up to 6  h after centrifugation and
plasma separation at 4 °C. Conversely, NMN displayed a
nearly 4% increase from the baseline at 6 h (from 362 ± 63
to 376 ± 65 pmol/L, p = 0.016) (Figure 1B and Table 2).
Differences between repeated measures did not reach
statistical significance. After plasma separation, the mean
variation at different time points did not exceed the TCL
for both MNs during storage at both temperatures.
Discussion
MNs have for long been considered stable metabolites
of their parental catecholamines [13]. The apparent
improved stability is thought to be due to methylation of
the 3-hydroxy group, which makes MNs less vulnerable
to oxidation than catecholamines. Therefore, as long as
the interest in biogenic amines for diagnosing PPGLs has
moved from plasma (or urinary) catecholamines to plasma
MNs, the scientific interest has been more focused on ana-
lytical rather than on preanalytical issues. The need for
developing stringent recommendations for preventing
degradation was hence mitigated, whilst the interest was
mostly focused on developing and validating LC-MS/MS-
based methods, sensitive enough to allow reliable, repeat-
able and accurate quantification [14, 15].
As originally pointed out by Willemsen et  al. [7],
MNs display pronounced and variably time-dependent

Table 2: Stability of metanephrines at room temperature and 4 °C according to delay after centrifugation.

Parameter   T0  TCL  Mean % difference (±SD) at RT

 
1 h  2 h  3 h

MN, pmol/L   230 (110–390)  ±6.3  −2.1 (6.2)  −4.4 (5.2)  −3.3 (9.3)
NMN, pmol/L  399 (290–548)  ±11.1  +0.2 (4.2)  −2.0 (6.8)  −6.0 (4.7)

      Mean % difference (±SD) at 4 °C

1 h  2 h  3 h

MN, pmol/L   248 (133–403)  ±6.3  +0.5 (6.2)  −0.2 (9.7)  −2.1 (9.1)
NMN, pmol/L  362 (281–501)  ±11.1  −1.3 (6.8)  +1.1 (3.9)  +4.1 (4.4)

Reference sample expressed as mean (min–max); MN, metanephrine; NMN, normetanephrine; RT, room temperature; TCL, total change limit.

Brought to you by | Chalmers University of Technology

Authenticated
Download Date | 8/4/19 12:02 PM
4      Danese et al.: Short-term stability of free metanephrines
variations in whole blood. Our results are in partial
agreement with these findings, at least for the part of the
study using samples stored at RT. More specifically, Wil-
lemsen et  al. showed that within the first 2  h of whole
blood storage at RT, the NMN concentration suddenly
increased, whilst the MN values began to decrease. In
the same study, MNs were found to be stable in whole
blood maintained at 4 °C for up to 6 h. Our data, obtained
on whole blood maintained at RT, revealed that both MN
and NMN displayed a mean percentage increase of 8%
and 17%, respectively, after only 1 h from sample collec-
tion. This increase is probably attributable to the ex vivo
activation of catechol-O-methyltransferase in red blood
cells and the consequent in vitro formation of MNs from
catecholamine metabolism [16]. Unlike these findings,
we found that the MN concentration was rather stable
throughout the entire study period when maintained at
4 °C, whilst NMN values increased by 6% within 3 h after
sample collection.
Interestingly, although the MN concentration was
found to be stable at both RT and 4 °C in centrifuged
samples, NMN values displayed a 6% reduction from base-
line at RT and a 4% increase at 4 °C after 6 h. At variance
with our experiments, Willemsen et al. performed a much
longer study, measuring MNs at 4, 24, 48 and 72  h after
sampling, and then observing a significant decrease in the
MN concentration (−9%) during the first 4 h at RT. MN at
4 °C, as well as NMN at both RT and 4 °C, were found to be
significantly decreased after 48 h.
According to our experimental findings, blood samples
for the assessment of plasma-free MNs shall always be
refrigerated before centrifugation, which should be pref-
erentially carried out within 3 h, thus earlier than the 6-h
period recommended in the study by Willemsen et al. [7].
Once centrifuged, MNs can then be maintained in separated
plasma (even at RT) for up to 6 h before being measured.
Although our recommendations on the stability of
MNs may appear stringent, and narrower than those sug-
gested by Willemsen et  al. [7], it is worthwhile mention-
ing here that even a slight change observed for NMN after
3 h of storage at 4 °C in whole blood, or for NMN after 6 h
of storage at both RT and 4 °C in separated plasma, are
probably irrelevant in the clinical perspective, as they did
not exceed the TCL. However, different conclusions would
have been possibly reached by analyzing values closer to
the upper limit of reference interval used for diagnosing
pheochromocytoma. Therefore, a practical take-home
message is avoiding the storage of whole blood at RT, as
this may generate a significant bias in NMN quantifica-
tion, thus potentially impairing the diagnostic efficiency
of this test.
Author contributions: All the authors have accepted
responsibility for the entire content of this submitted
manuscript and approved submission.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played
no role in the study design; in the collection, analysis, and
interpretation of data; in the writing of the report; or in the
decision to submit the report for publication.
References
1. Lenders JW, Duh QY, Eisenhofer G, Gimenez-Roqueplo AP, Grebe
SK, Murad MH, et al. Pheochromocytoma and paraganglioma:
an endocrine society clinical practice guideline. J Clin Endocrinol
Metab 2014;99:1915–42.
2. Lenders JW, Eisenhofer G. Update on modern management of
pheochromocytoma and paraganglioma. Endocrinol Metab
(Seoul) 2017;32:152–61.
3. Därr R, Kuhn M, Bode C, Bornstein SR, Pacak K, Lenders JW,
et al. Accuracy of recommended sampling and assay methods
for the determination of plasma-free and urinary fractionated
metanephrines in the diagnosis of pheochromocytoma and
paraganglioma: a systematic review. Endocrine 2017;56:
495–503.
4. Lenders JW, Willemsen JJ, Eisenhofer G, Ross HA, Pacak K,
Timmers HJ, et al. Is supine rest necessary before blood sam-
pling for plasma metanephrines? Clin Chem 2007;53:352–4.
5. Eisenhofer G, Peitzsch M, Kaden D, Langton K, Mangelis A,
Pamporaki C, et al. Reference intervals for LC-MS/MS measure-
ments of plasma free, urinary free and urinary acid-hydrolyzed
deconjugated normetanephrine, metanephrine and methoxyt-
yramine. Clin Chim Acta 2018;490:46–54.
6. Deutschbein T, Unger N, Jaeger A, Broecker-Preuss M, Mann K,
Petersenn S. Influence of various confounding variables and
storage conditions on metanephrine and normetanephrine
levels in plasma. Clin Endocrinol (Oxf) 2010;73:153–60.
7. Willemsen JJ, Sweep CG, Lenders JW, Ross HA. Stability of
plasma free metanephrines during collection and storage as
assessed by an optimized HPLC method with electrochemical
detection. Clin Chem 2003;49:1951–3.
8. Ross HA, Lenders JW, Sweep FC. A study of longer-time
stability of plasma free metanephrines. Ann Clin Biochem
2011;48:270–1.
9. Danese E, Tarperi C, Salvagno GL, Guzzo A, Sanchis-Gomar F,
Festa L, et al. Sympatho-adrenergic activation by endurance
exercise: effect on metanephrines spillover and its role in pre-
dicting athlete’s performance. Oncotarget 2018;9:15650–7.
10. Oddoze C, Lombard E, Portugal H. Stability study of 81 analytes
in human whole blood, in serum and in plasma. Clin Biochem
2012;45:464–9.
11. de Jong WH, Graham KS, van der Molen JC, Links TP, Morris MR,
Ross HA, et al. Plasma free metanephrine measurement using
automated online solid-phase extraction HPLC tandem mass
spectrometry. Clin Chem 2007;53:1684–93.
Brought to you by | Chalmers University of Technology
Authenticated
Download Date | 8/4/19 12:02 PM

12. Dupuy AM, Cristol JP, Vincent B, Bargnoux A, Mendes M,
Philibert P, et al. Stability of routine biochemical analytes in
whole blood and plasma/serum: focus on potassium stabil-
ity from lithium heparin. Clin Chem Lab Med 2018;56:
413–21.
13. Lenders JW, Pacak K, Eisenhofer G. New advances in
the biochemical diagnosis of pheochromocytoma: moving
beyond catecholamines. Ann NY Acad Sci 2002;970:
29–40.
14. Mullins F, O’Shea P, FitzGerald R, Tormey W. Enzyme-linked
immunoassay for plasma-free metanephrines in the ­biochemical
Danese et al.: Short-term stability of free metanephrines      5
diagnosis of phaeochromocytoma in adults is not ideal. Clin
Chem Lab Med 2011;50:105–10.
15. Eisenhofer G, Peitzsch M. Laboratory evaluation of pheochromo-
cytoma and paraganglioma. Clin Chem 2014;60:1486–99.
16. Floderus Y, Sääf J, Ross SB, Wetterberg L. Catechol-O-methyl-
transferase activity in human erythrocytes: methodological
aspects. Ups J Med Sci 1981;86:309–18.
Supplementary Material: The online version of this article offers
supplementary material (https://doi.org/10.1515/cclm-2019-0020).
Brought to you by | Chalmers University of Technology
Authenticated
Download Date | 8/4/19 12:02 PM