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Catecholamine Analysis: Evaluation of Method Optimization to Improve
Sensitivity and Reduce Limits of Quantitation using LC-MS/MS
Adam Senior1, Alan Edgington1, Lee Williams1, Rhys Jones1, Helen Lodder1, Geoff Davies1, Steve Jordan1, Claire Desbrow1, Victor Vandell2 & Elena Gairloch2
1
2
Biotage GB Limited, Distribution Way, Dyffryn Business Park, Ystrad Mynach, Cardiff, CF82 7TS, UK
Biotage, 10430 Harris Oaks Blvd., Suite C, Charlotte North Carolina 28269, USA
Introduction
Table 2. MRM Parameters
Catecholamines are biomarkers for the detection of diseases such as:
hypertension, pheochromocytoma and neuroblastoma.
OH
The main target analytes are epinephrine,
NH
HO
norepinephrine and dopamine (see Figure 1 for
epinephrine
details) which are traditionally analyzed using
HO
liquid chromatography with electrochemical
OH
detection. This poster investigates various parts of
NH2
HO
the method development process to evaluate the
norepinephrine
sensitivity of LC MS/MS analysis. A highly sensitive
HO
LC-MS/MS system, a Shimadzu Nexera UHPLC
coupled to an AB SCIEX Triple Quad 5500 MS was
used for analysis. Method sensitivity in terms of
precursor ion selection and MRM transitions,
chromatography and solid phase extraction
protocols were all evaluated for increased
sensitivity.
HO
HO
NH2
dopamine
Figure 1. Catecholamine Structures
Experimental
Reagents
Standards were obtained from LGC (Teddington, UK). Acetic acid (HAC), formic
acid, ammonium hydroxide, hydrochloric acid, ammonium acetate, ammonium
formate, ethylene glycol and LC MS grade chromatographic solvents were
obtained from Sigma-Aldrich Chemical Co. (Poole, UK). 18.2 MΩ.cm water was
drawn fresh daily from a Direct-Q 5 water purifier (Merck Millipore, Watford, UK).
Pooled human plasma was obtained from The Welsh Blood Service (Pontyclun,
UK).
Sample preparation
All extractions were performed using polymer-based SPE. EVOLUTE® EXPRESS WCX
was evaluated in the 30 mg 96 fixed well plate format.
Sample pre-treatment: 300 μL of plasma was pre-treated by diluting 1:1 with
various aqueous buffers and mixed thoroughly.
Sample extraction: EVOLUTE® EXPRESS WCX 30 mg 96 well plate; 602-0030-PX01.
600 μL of pre-treated sample was applied per well of the FWP, extraction
strategies are shown below in Table 1.
Table 1. Extraction Strategies
Step
Condition
Equilibration
Sample load
Wash 1
Wash 2
Elution
Volume
1 mL
1 mL
600 μL
1 mL
1 mL
1 mL
Strategy 1
MeOH
50 mM NH4OAC
plasma 1:1 50 mM NH4OAC
10% MeOH (aq)
propan-2-ol
5% formic acid in MeOH
Strategy 2
MeOH
50 mM NH4OAC
plasma 1:1 50 mM NH4OAC
50 mM NH4OAC
MeOH
5% formic acid in MeOH
Post extraction: 4 μL of ethylene glycol was added to each appropriate well of the
collection plate prior to analyte elution. The eluates were evaporated to dryness
at 40 °C under a stream of air and reconstituted in 200 μL 0.1% HCOOH in
2% MeOH .
Analyte
epinephrine
norepinephrine
dopamine
Transition
166.1>107.1
152.1>107.1
154.1>91.1
DP, V
148
25
50
EP, V
8
2
9
CE, V
24
22
29
CXP, V
16
25
13
Results
Mass Spectrometry Optimization
Source parameters were optimized to enhance the production of dehydrated
precursor ions
to increase
signal sensitivity
of epinephrine
and
norepinephrine.
Figure 2a
demonstrates at
least a two-fold
increase in
signal when
using a suitably
optimized MRM
transition for
b)
a)
epinephrine and
norepinephrine.
Figure 2. XICs Showing Dopamine MRMs (blue: dehydrated precursor /
A concomitant
magenta: non-dehydrated precursor)
increase in the
dehydrated dopamine precursor was not observed suggesting only the alkyl OH
group is susceptible to dehydration. The accompanying blank chromatograms in
Figure 2b demonstrate the transitions are free of interferences.
Chromatographic Optimization
Chromatographic separation was optimized by comparison of 4 eluent systems
with low concentrations of either MeOH or MeCN. Target analyte peak shape and
height were maximized using 2 mM formate buffer pH 3.2; increasing the
concentration of formic acid to 0.1% was detrimental to chromatography, as was
use of acetate as a counter-ion. Increasing concentration of counter-ion resulted
in an increase in background noise (data not shown). Use of MeOH as an organic
modifier gave higher peaks and greater separation than with MeCN; a reduction in
peak width was observed using MeCN
Restek Pinnacle BiPh 100 x 2.1 mm 1.9 µm
ACE Excel 2 C18-NH2 100 x 2.1 mm 2 µm
Restek Raptor BiPh 50 x 2.1 mm 2.7 µm
ACE Excel 2 C18-PFP 100 x 2.1 mm 2 µm
UHPLC Conditions
Instrument: Shimadzu Nexera UHPLC (Shimadzu Europa GmbH, Duisburg,
Germany).
Column: ACE Excel 2 C18-PFP 100 x 2.1 mm, 2μ (Hichrom ltd., Theale, UK).
Mobile phase: A, 2 mM ammonium formate pH 3.2; B, MeOH; 0.4 mL min-1.
Gradient: 2% B isocratic hold for 2.2 min, step to 100% B, hold 0.5 min, step to
initial conditions, equilibrate 1.0 min.
Column temperature: 40 °C
Injection volume: 20 μL
Mass Spectrometry
Instrument: Triple Quad 5500 mass spectrometer (AB Sciex, Framingham, US). Ions
were acquired in the positive mode using a Turbo V ESI interface and either MRM
or Scheduled MRM transitions.
IS: 5500 V
Curtain: 35 psi
Temperature: 700 °C
Gas 1: 50 psi
Gas 2: 50 psi
Figure 3. Chromatographic Separation With 2% MeOH in 2 mM formate pH 3.2 (cyan: norepinephrine / blue:
epinephrine / red: dopamine)
(data not shown). The optimized chromatographic separation was applied to 4
HPLC or UHPLC columns as demonstrated in Figure 3. The C18-PFP column was
chosen for further work on the basis of its superior retention, separation, peak
shape and height.
Extraction Optimization
Sample pre-treatment optimization focused on control of pH below 8 to ensure
the amine functional group remained charged whilst maintaining the neutrality of
the hydroxyl groups. Additionally, pre-treatment should be at least 1 pH unit away
from the approximate pKa of the WCX polymer in order to maximize binding.
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The effect of
variation in pretreatment pH on
recovery is
80
demonstrated in
60
Figure 4.
40
Recoveries are
20
dependent on
0
extraction
epinephrine
norepinephrine
dopamine
strategy (see
50 mM NH4OAC strat1 50 mM NH4OAC strat2
table 2).
0.1% HCOOH strat1
0.1% HCOOH strat2
Where the
Figure 4. Catecholamine Recovery Comparing Sample Pre-treatment (100 pg
protocol is
mL-1 spike plus IS)
based on
strategy 2, 0.1%
HCOOH is detrimental to recovery; for strategy 1, 0.1% HCOOH pre-treatment
demonstrates improved recoveries. Sample pre-treatment with 50 mM ammonium
formate gave unreliable data with poor repeatability (data not shown).
Catecholamine Recovery with Differing
Pre-treatment (0.5 mL elute)
100
Several
Catecholamine Recovery Comparing
interference wash
Interference Washes with Other Conditions Fixed
strategies were
100
investigated,
90
80
modifying aqueous
70
interference
60
50
washes with either
40
buffer of organic
30
20
modifier and
10
modifying the
0
epinephrine
norepinephrine
dopamine
nature and
eluotropic strength
10% MeOH (aq)
50 mM NH4OAC
100% IPA
1:1 IPA:MeCN
100% MeOH
Figure 5. Catecholamine Recovery Comparing Interference Washes
of the organic
(100 pg mL-1)
interference wash.
Figure 5
demonstrates the effect of variation in interference wash steps.
The effect on
recovery by
modification of
100
the strength and
80
choice of elution
60
solvent acid
40
modifier was
20
0
investigated. The
epinephrine
norepinephrine
dopamine
effect on analyte
5% HCOOH in MeOH 2% HCOOH in MeOH
recovery is
2% HCOOH in MeCN 0.5% HCl in MeOH
shown in
Figure 6. Recovery Comparing Elution Solvent / pH Modifier (100 pg mL-1)
Figure 6. The
chart demonstrates 5% formic acid in MeOH to be the most efficient recovery
solvent. There may be the potential to reduce this to 2% by modification of the
interference wash regime to replace 10% MeOH as an interference wash solvent
with 10 mM to 50 mM ammonium acetate (data not shown).
lower using strategy 1 and higher using strategy 2. However, as demonstrated
elsewhere in the poster relative recoveries were lower even if absolute recoveries
were higher.
Due to the structure of
the analytes shown in
Figure 1 there was
concern the extracted
100.0
analytes may
80.0
60.0
demonstrate volatility
40.0
under certain conditions.
20.0
0.0
Two strategies to reduce
epinephrine
norepinephrine
dopamine
this effect were
5% FA / 2µL Et glycol
2% FA / 2µL Et glycol
investigated: addition of
5% FA / no treatment
2% FA / no treatment
HCl to the collection plate
5% FA / 50µL 50mM HCl in MeOH 2% FA / 50µL50 mM HCl in MeOH
to promote formation of
Figure 9. Recovery Comparing Analyte Blow-down/Recovery (50 ng
hydrochloride salts or
epinephrine/dopamine, 250 ng epinephrine)
addition of small volumes
of ethylene glycol to act
as a ‘keeper solvent’. The results are shown in Figure 9. The chart demonstrates
that there is analyte loss during extract blowdown and reconstitution. Addition of
HCl has a detrimental effect on evaporative losses. The use of glycol as a keeper
solvent gives the most beneficial effect to analyte recoveries.
Blow-down / Recovery of Catecholamines With Differing
Collection Plate Eluate Modifiers
The procedure gives linear recoveries from 100 to 2000 pg mL-1 when spiked into
pooled human plasma as demonstrated in Figure 10. Coefficients of
determination were greater than 0.99 for all analytes. S/N ratios at 100 pg mL-1
were demonstrated >10:1 (data not shown).
epinephrine
norepinephrine
Catecholamine Recovery Comparing
Elution Solvent and Modifier
Of the two principal extraction strategies investigated, a protocol based on
strategy 1
Normalized Catecholamine Area Response with
demonstrated
Differing Pre-treatment (0.5 mL elute)
the greatest peak
100
areas. In
80
comparison, a
60
protocol based
on strategy 2
40
gave rise to
20
lower analyte
0
peak areas, see
epinephrine
norepinephrine
dopamine
Figure 7 for
50 mM NH4OAC strat1 50 mM NH4OAC strat2
details. Matrix
0.1% HCOOH strat1
0.1% HCOOH strat2
Figure 7. Relative Catecholmine Area Responses
interference is
dopamine
Figure 10. Calibration Curves, Plasma
Extraction 10 to 2000 pg mL-1
Conclusions
» We demonstrate that the use of EVOLUTE EXPRESS WCX 96 fixed well plates to
extract polar catecholamines from pooled human plasma can be used to
perform a sensitive, linear assay.
» LOQs were demonstrated to be 100 pg mL-1 for each analyte (per mL of sample
loaded) based on an observable peak at 10:1 S/N.
» An extraction protocol based on strategy 2 demonstrates reduced signal
suppression when compared to a protocol based on strategy 1. Work is
ongoing to fully optimize the extraction protocol.
» Reducing elution volume to 0.5 mL for a protocol based on strategy 1 gave rise
to increased recoveries compared to strategy 2. This could be due to increased
co-extractives with increasing elution volume.
» Work is ongoing to increase assay recoveries, reduce matrix interference and
improve assay reliability, e.g. with the use of high pH extraction, neutralized on
collection.
MSACL 2015, San Diego
Part Number: P121