Rapid, simultaneous analysis of plasma catecholamines
and metanephrines by mixed mode SPE and HILIC
LC/MS/MS for clinical research
Sherri Naughton, Jonathan P. Danaceau, Erin E. Chambers, and Kenneth T. Fountain
Waters Corporation, 34 Maple Street Milford MA, 01757 USA
INTRODUCTION
Clinical researchers are often interested in measuring
elevated concentrations of urinary catecholamines and
their O-methylated metabolites (metanephrines).
However,
these
compounds
(in
particular,
norepinephrine, epinephrine, and dopamine) can be a
challenge to analyze via reversed-phase LC/MS/MS
due to their high polarity. As a result, many research
laboratories still analyze this panel using ion-pairing
reagents and ECD detection. This work describes a
single extraction and analysis method for monoamine
neurotransmitters and metanephrines from human
plasma. Samples are extracted from plasma using
mixed-mode weak cation exchange (WCX) SPE and
analyzed via HILIC chromatography using Waters’
UPLC BEH Amide column, combined with Waters’ Xevo
TQ-S mass spectrometer. This combination of sample
preparation, chromatography and mass spectrometry
results in a rapid, robust method with excellent
analytical
sensitivity,
linearity,
accuracy,
and
precision.
Table 1—UPLC Gradient
Time
Flow
%A
%B
0.0
0.6
0.0
100.0
1.0
0.6
0.0
100.0
2.0
0.6
10.0
90.0
2.1
1.0
10.0
90.0
2.5
1.0
30.0
70.0
2.6
1.0
0.0
100.0
3.9
1.0
0.0
100.0
4.0
0.6
0.0
100.0
Waters Xevo® TQ-S Conditions, ESI+
Capillary Voltage:
Desolvation Temp:
Cone Gas Flow:
Desolvation Gas Flow:
Source Temp:
MRM transitions, ESI+:
Sample Preparation:
Plasma samples (250 µL) were
pretreated with 250 µL of 50 mM NH4CH3COO and 50 µL of
internal standard working solution (2.5 ng/mL).
SPE Wells were conditioned with 200 µL MeOH and 200 µL
MilliQ water. The entire pretreated sample was then loaded in
individual wells. Wells were then washed with 200 µL of 20
mM NH4CH3COO and 200 µL of 50:50 ACN:IPA. All samples
were eluted with 2 x 25 µL of 85:15 ACN:H2O containing 2%
formic acid. 15 µL was injected onto the LC/MS/MS system.
Calibration curves were spiked from 10-2000 pg/mL for all
compounds except for norepinephrine and epinephrine, which
ranged from 50-10,000 pg/mL. After processing, calibration
curves were corrected for endogenous concentrations.
Figure 2. Recovery and matrix effects for urinary
R
Table 2
METHODS
Calibration standards were purchased from Sigma-Aldrich (St.
Louis, MO, USA). All deuterated internal standards were
purchased from Cerilliant (Round rock, TX).
0.5 kV
550 °C
150 L/Hr
900 L/Hr
150 °C
See Table 2
Compound
RT
3-Methoxytyramine
0.84
Metanephrine
0.91
Normetanephrine
1.17
Dopamine
1.25
Epinephrine
1.40
Norepinephrine
1.98
MRM
Transitions
151.1>91.16
151.1>119.2
180>165.1
180>148.1
166.1>134.1
166.1>149.1
137.1>91.1
154.1>137.2
184.1>166.1
166.1>107.1
152>135.2
152>79.2
Cone
(V)
17
17
35
35
50
50
50
29
15
15
46
20
Coll.
Energy
22
18
16
20
18
10
18
10
8
18
14
20
3-MT
Metanephrine
Normetanephrine
Dopamine
Epinephrine
Norepinephrine
2
0.999
1.000
1.000
0.999
0.999
1.000
Mean % Max %
Dev.
Dev.
0.25% 2.9%
0.00% 2.5%
0.00% 1.7%
-0.33% 4.6%
0.84% 11.8%
0.00% 2.6%
Endoge- Corrected
nous Calibration
(pg/mL)
Range
7.0
32
71
0
29
361
17-2007
42-2032
81-2071
10-2000
79-10029
411-10361
Table 3. Calibration summary for all analytes. The
corrected calibration range adds the endogenous
concentration to the spiked calibration levels.
3-MT
Metanephrine
Normet
Dopamine
RESULTS
SPE Procedure
Epinephrine
Norepinephrine
3-MT
Metanephrine
Normet
Dopamine
Epinephrine
Norepinephrine
QC spike concentration
40 pg/mL
100 pg/mL
Mean
S.D. %CV Mean S.D. %CV
99.9%
7.4% 7.4% 99.2% 3.0% 3.1%
99.9%
2.0% 2.0% 97.6% 0.8% 0.8%
99.8%
1.6% 1.6% 96.8% 1.7% 1.8%
97.0%
7.2% 7.4% 91.2% 3.4% 3.7%
200 pg/mL
500 pg/mL
Mean Mean %CV Mean S.D. %CV
97.3%
4.3% 4.4% 98.8% 2.2% 2.2%
105.1% 7.7% 7.4% 102.6% 8.2% 8.0%
QC spike concentration
400 pg/mL
800 pg/mL
Mean
S.D. %CV Mean S.D. %CV
105.9% 1.8% 1.7% 93.9% 2.6% 2.8%
107.3% 1.2% 1.1% 94.6% 1.7% 1.7%
104.6% 0.4% 0.4% 93.4% 1.0% 1.1%
103.7% 3.1% 3.0% 95.6% 2.7% 2.8%
2000 pg/mL
4000 pg/mL
Mean
S.D. %CV Mean S.D. %CV
100.8% 1.4% 1.4% 97.0% 2.6% 2.6%
96.7%
1.3% 1.3% 97.1% 4.2% 4.3%
Table 4.
Quality Control (QC) results for
catecholamines and metanephrines extracted from
urine by mixed-mode weak cation exchange.
CONCLUSIONS

Chromatographic Conditions
UPLC System:
ACQUITY UPLC®
MS:
XEVO TQ-S
Column:
ACQUITY UPLC® BEH Amide, 1.7
µm 2.1 x 100 mm
Mobile Phase A
(MPA):
95:5 Water:ACN with 30 mM
NH4COOH (pH 3.0)
Mobile Phase B
(MPB):
15:85 Water:ACN with 30 mM
NH4COOH (pH 3.0)
Column Temp:
30 ˚C
Sample Temp:
10 ˚C
Strong Needle Wash MPB
Weak Needle Wash
MPB




Figure 1. Chromatography of a 20 pg/mL calibration
standard
of
plasma
catecholamines
and
metanephrines.
Rapid,
simultaneous
analysis
of
plasma
catecholamines and metanephrines
Full baseline resolution of epinephrine and
normetanephrine
Excellent analytical sensitivity, linearity, accuracy
and precision for all compounds
Consistent recovery with minimal matrix effects
5x concentration without sample evaporation
For research use only– Not for use with diagnostic procedures
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