Download Simultaneous qualitative and quantitative analysis using the Agilent

Simultaneous qualitative and
quantitative analysis using the
Agilent 6540 Accurate-Mass Q-TOF
Technical Overview
Authors
Abstract
Pat Perkins
Anabel Fandino
Lester Taylor
The utility of the Agilent 6540 Accurate-Mass Q-TOF LC/MS System for the
determination of metabolic stability, profiling, and identification in a single
experiment has been demonstrated in a buspirone metabolic study, thus maximizing the quantitative and qualitative information that can be obtained from one
experiment. The validity of the quantitative data was confirmed by analysis using
the Agilent 6460 Triple Quadrupole LC/MS System, operating in multiple reaction
monitoring (MRM) mode.
Agilent Technologies, Inc.
Santa Clara, CA USA
Introduction
Determining metabolic stability is an important aspect of early drug discovery, due
to its role in drug half-life and bioavailability. Metabolite identification is crucial in
order to understand drug safety and efficacy. In early drug discovery, samples are
typically analyzed by a variety of different LC/MS methods in order to generate
both metabolic stability (quantitative) and metabolite identification (qualitative)
data. The ability to obtain both quantitation and identification in a single analysis
makes metabolic stability, profiling, and identification studies much more efficient.
The study described here demonstrates the use of the high resolution and accurate
mass determination capabilities of the Agilent 6540 QTOF LC/MS to perform both
quantitation and identification of metabolites in the same experiment.
Experimental
UHPLC Run Conditions
Instruments
Column
Agilent ZORBAX Eclipse Plus C18, 2.1 mm x 100 mm, 1.8 μm
Injection volume
1 µL
Needle wash
Flush port (75:25 MeOH:H2O, 0.1% formic acid ,10 sec)
Mobile phase
A = H2O + 0.1% formic acid
B = acetonitrile + 0.1% formic acid
Flow rate
1.0 mL/min
Gradient
5% B to 95% B
Stop time
5.5 min
Metabolic stability, profiling, and
identification were performed using
an Agilent 1290 Infinity LC System
coupled to an Agilent 6540 AccurateMass Q-TOF System. The 6540 QTOF
was operated in two modes: an initial
MS-only mode at 5 Hz acquisition
rate, and an automatic MS/MS mode,
with the masses of the expected
metabolites on the inclusion list.
Quantification results were confirmed
using the same LC system and MRM
on the Agilent 6460 Triple Quadrupole
LC/MS System. The same conditions
(e.g. collision energy) were used for
both instruments because they use the
same hardware design from ion source
to collision cell. The instrument conditions are given in Tables 1 and 2.
Sample preparation
Buspirone was chosen as a model
compound for the metabolic stability
study. This drug was incubated at
1 μM in a rat liver S9 preparation,
using an NADPH regenerating system.
The incubation was carried out at 37°C
and 100 μL aliquots were taken at 0, 5,
10, 15, and 20 minutes. The reaction
was stopped by adding acetonitrile;
then the sample was centrifuged.
The supernatant was evaporated
to dryness using a gentle stream of
nitrogen and reconstituted in the
mobile phase for combined ultra high
performance liquid chromatography
(UHPLC) and MS.
Table 1. Ultra High Performance Liquid Chromatography (UHPLC) Conditions
QTOF and Triple Quadrupole MS Conditions
Ion Source Settings for Both Instruments
Ion mode
Positive, ESI
Drying gas temperature
350°C
Drying gas flow
10 L/min (nitrogen)
Sheath gas temperature
400°C
Sheath gas flow
12 L/min
Capillary voltage
4000 V
Nozzle voltage
0 V (+)
QTOF MS/MS Settings
Acquisition type
Auto MS/MS (data-dependent)
Mass range
100–1000 m/z for both MS and MS/MS
Transients
1557/spectrum for both MS and MS/MS
Acquisition rate
5 spectra/sec (combined MS and MS/MS)
Precursors/cycle
1
Active exclusion
Exclude after 2 spectra, release after 0.05 min
Collision energy
28 EV (same for Triple Quad)
Charge state
1+ and unknown
Preferred list
All expected metabolites
Mode
Extended dynamic range (2 GHz)
Triple Quadrupole MS/MS Settings
MS1/MS2 resolution
Unit
Delta EMV
200 V
Time filtering
0.03 min
MRM transitions
386.3 → 122.1 (Buspirone)
609.3 → 195.1 (Reserpine, internal standard)
Dwell time
100 ms per transition
Cycle time
203.5 ms
Table 2. 6540 QTOF and 6460 Triple Quadrupole LC/MS mass spectrometer conditions
2
Results and Discussion
Metabolic stability results were
plotted using the data from the QTOF
MS only analysis and the Triple Quad
MRM analysis. As shown in Figure 1,
there was good correlation of the
metabolic stability data obtained using
the 6540 QTOF full-spectrum analysis,
with the same data obtained using the
6460 Triple Quadrupole MRM analysis.
The semi-log plot of percent parent
versus time is a typical means used to
determine and compare metabolite half
lives. The 6540 QTOF is an extremely
sensitive instrument capable of good
quantitative measurements.
100
% Parent remaining
Reliable metabolic stability results
QTOF delivers accurate metabolic stability data
10
QQQ
1
Q-TOF
0.1
0.01
0
5
10
15
20
25
Incubation time (min)
Time
0 min
5 min
10 min
15 min
20 min
QTOF (%)
100
4.88
0.57
0.100
0.020
QQQ (%)
100
4.95
0.47
0.098
0.017
Simultaneous profiling of metabolites
The MS analysis using the 6540 QTOF
provided full spectra through the entire
run, allowing profiling of all metabolites, while the triple quadrupole analysis using the MRM mode provided
analysis only of the parent drug and
known, selected metabolites. Figure 2
shows extracted ion chromatograms of
the expected metabolites of buspirone,
detected in the QTOF analysis. This
sample was from a 5 min incubation
time, and the QTOF was operated in
high resolution MS scan mode.
Figure 1. The 6540 QTOF gave metabolic stability results for buspirone that were in good
agreement with those obtained using the 6460 Triple Quadrupole system. Results are
plotted using a semi-log scale (log %Parent remaining) to provide a linear relationship to
incubation time.
Simultaneous profiling of metabolites
5
x10
6
5
4
Buspirone
3
Monohydroxy
Monohydroxy
2
-C2H2
Dihydroxy
-C2H2 +O
Dihydroxy
1
Buspirone (parent)
N-oxide
0
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
Counts vs. Acquisition Time (min)
Figure 2. 6540 QTOF analysis EICs revealed the expected metabolites of buspirone.
3
1.5
1.6
Figure 3 shows representative spectra
from the QTOF analysis, acquired
in MS mode with a 5 Hz acquisition
rate. The spectra were taken from a
sample at a 10 minute incubation time,
when less than 1 percent of the parent
drug remained. The QTOF spectra
for buspirone and its monohydroxy
and dihydroxy metabolites showed
mass errors of less than 1 ppm, which
enabled confident identifications.
Isotope ratios provide additional
information that further increases
confidence of identification. The
insets in Figure 3 show the measured
isotope patterns versus theoretical
(the latter shown in rectangles), based
on the correct molecular formulae. The
isotopic fidelity is excellent, due in
part to the analog-to-digital converter
(ADC) detector employed in Agilent
TOF and QTOF systems. Time-to-digital
converter (TDC) detectors used in
some other TOF and QTOF systems are
known to have problems with isotope
ratios, which means that analysts
cannot rely on this information for
compound identification.
From these full scan MS mode
analyses, abundances of buspirone
and its metabolites were plotted as a
function of incubation time (Figure 4).
The graph demonstrates that the
6540 QTOF is capable of obtaining
both metabolic stability and metabolic
profiling in a single MS experiment.
x10 4
Reliable identification of metabolites
1.8
386.2548
C21 H32 N5 O2
1.6
1.4
x10 4
Buspirone
-0.62 ppm
1.2
1
121.0509
386.2548
C21 H32 N5 O2
922.0098
0.8
387.2589
C21 H32 N5 O2
0.6
0.4
2
273.1665
388.2533
C21 H32 N5 O2
0.2
0
386
387
388
Counts vs. Mass-to-Charge (m/z)
0
100
200
300
400
x10 4
500
600
6
700
650
402.2503
C21 H32 N5 O3
389
800
750
900
1000
Counts vs. Mass-to-Charge
(m/z)
5.5
5
4.5
4
Monohydroxy metabolite
x10
3.5
4
7.5
5
-0.90 ppm
3
402.2503
C21 H32 N5 O3
124.0869
2.5
403.2531
C21 H32 N5 O3
2
1.5
404.2497
C21 H32 N5 O3
1
0.5
922.0098
0
2.5
402
403
404
Counts vs. Mass-to-Charge (m/z)
0
405
x10 4
100
200
300
400
500
600
1.4
650
418.2448
C21 H32 N5 O4
700
750
800
900
1000
Counts vs. Mass-to-Charge (m/z)
1.2
x10 4
Dihydroxy metabolite
1
-0.10 ppm
0.8
124.0869
418.2448
C21 H32 N5 O4
2
0.6
419.2485
C21 H32 N5 O4
0.4
0.2
1
922.0098
420.2444
C21 H32 N5 O4
972.0061
0
418
419
420
Counts vs. Mass-to-CHarge (m/z)
0
100
200
300
400
500
600
700
800
900
1000
Counts vs. Mass-to-Charge (m/z)
Figure 3. Metabolite spectra taken with the 6540 QTOF demonstrated excellent mass accuracy and isotopic fidelity, which provided assurance of
correct identifications.
4
After successfully determining metabolic stability and metabolic profiling
using the 6540 QTOF, the possibility
of including MS/MS for more comprehensive metabolite identification
and structural characterization was
investigated. For this targeted MS/MS
analysis, the masses of the expected
metabolites were specified on an
inclusion list, to ensure that they
would be analyzed.
Metabolic stability and profiling in a single MS experiment
100
90
Relative amount (%)
Augmented metabolite identification
with QTOF MS/MS mode analysis
MS-only analysis, 5 Hz
80
70
buspirone (parent)
monohydroxy
dihydroxy
trihydroxy
N-oxide
-C2H2
-C2H2 +O
60
50
40
30
20
10
0
0
5
10
15
20
Incubation time (min)
Figure 4. Metabolic stability and metabolic profiling from the same
experiment (MS-only, 5 Hz acquisition rate) with the Agilent 6540
QTOF. Graph assumes all response factors were the same as for the
parent buspirone.
QTOF MS/MS mode enables more comprehensive metabolite
identification and structural characterization
100
90
MS data (2.5 Hz) from combined
MS and MS/MS analysis
80
Relative amount (%)
With the combined MS and MS/MS
acquisition, the MS acquisition rate
was 2.5 spectra/second and provided
sufficient data points across the peaks
to generate semi-quantitative results.
In fact, the resulting graph of drug and
metabolite concentrations over time,
shown in Figure 5, is almost identical
to that in Figure 4. When operated
in this mode, the Agilent 6540 QTOF
used half of the time to carry out full
scan MS data acquisition, and half of
the time for MS/MS data acquisition,
which was used for metabolite identifi cation, thus providing quantitative
and qualitative results in a single
analytical experiment.
70
buspirone (parent)
monohydroxy
dihydroxy
N-oxide
-C2H2
-C2H2 +O
60
50
40
30
20
10
0
0
5
10
15
20
Incubation time (min)
Figure 5. Metabolic stability and metabolic profiling from the same
experiment (combined MS and MS/MS, MS at 2.5 Hz acquisition rate)
with the Agilent 6540 QTOF. Graph assumes all response factors were
the same as for the parent buspirone.
5
The quality of the MS and MS/MS
spectra for metabolite identification was excellent, as shown in the
example for the buspirone monohydroxy metabolite (Figure 6). The
spectra are labeled with the results
produced by molecular formula generation (MFG), which show an excellent
match between the experimental data
and the calculated formula.
and the isotope abundances show
excellent agreement with the theoretical values (rectangles). Other
QTOF instruments that use a TDC
detector have difficulty achieving
these accurate isotope ratios, and
due to TDC dead time, may suffer from
poor mass accuracy for chromatographic peaks with high abundance.
The larger mass errors in TDC-based
instruments negatively affect both
identification and quantitation.
The ADC detector in the 6540 QTOF
does not have these problems.
In the MS spectrum at the top of
Figure 6, the measured mass matches
the calculated mass within 0.07 ppm,
High quality spectra enable accurate metabolite identification
In the MS/MS spectrum at the bottom
of Figure 6, the masses of the fragment ions agree very well with the
theoretical values. The collision cell in
the Agilent 6540 QTOF has a unique
design feature that utilizes high
pressure cooling, which means both
product and precursor ions have the
same relative energy. Therefore, the
same tuning parameters can be used
for both MS and MS/MS analysis,
resulting in excellent MS/MS mass
accuracy. Given the quality of the MS
and MS/MS data in Figure 6, the identification of a metabolite can be made
with a high degree of confidence.
x10 1
402.2500
C21 H32 N5 O3
7
6
0.07 ppm
5
4
Buspirone monohydroxy metabolite
3
x10 2
121.0509
1
403.2530
C21 H32 N5 O3
2
1
MS spectrum
0.8
402.2500
C21 H32 N5 O3
404.2532
0
0.6
0.4
0.2
109.0762
402
403
404
Counts (%) vs. Mass-to-Charge (m/z)
135.0443
194.1171
224.1282
327.2009
0
122.0705
C6 H8 N3
x10 2
1.2
219.1604
C12 H19 N4
-3.32 ppm
1
0.8
150.1022
C8 H12 N3
0.6
-2.36 ppm
0.4
-0.54 ppm
238.1424
C11 H18 N4 O2
178.1217
C9 H14 N4
402.2493
MS/MS spectrum
-0.28 ppm
2.07 ppm
0.2
0
100
120
140
160
180
200
220
240
260
280
300
320
340
360
380
400
420
Counts (%) vs. Mass-to-Charge (m/z)
Figure 6. The molecular formula generation algorithm identified the metabolites by taking advantage of both the excellent match of the precursor
isotope pattern and the mass agreement for precursor and product ions.
6
Identification of unexpected
metabolites with QTOF
MS/MS analysis
A compelling reason to use an accurate-mass MS/MS instrument for this
workflow is to identify un-targeted
metabolites in addition to the expected
metabolites. This may be done using
the standard data dependent acquisition parameters in combination with
appropriate data mining tools. The
Find by Auto MS/MS algorithm in
the Agilent MassHunter Qualitative
Analysis software was used to automatically discover metabolites which
may be present in the sample. This
processing feature revealed all the
known phase I metabolites detected in
this sample, which were automatically
extracted for verification, without prior
specification. This experiment demonstrates the feasibility of discovering
unexpected metabolites using this
QTOF MS/MS procedure.
Conclusions
This study clearly demonstrated the
feasibility of using the 6540 QTOF
for metabolic stability, profiling, and
identification in a single experiment.
It gave quantitation results that were
comparable to those obtained with
the triple-quadrupole instrument. The
Agilent Triple Quad and QTOF employ
common hardware from source to
collision cell, which makes it easy to
transfer methods and compare results.
While the QTOF does not provide the
ultimate detection limits of the most
advanced triple-quadrupole systems,
the sensitivity is more than sufficient
for early compound assessment in
drug discovery.
7
When operated in targeted MS/MS
mode, the 6540 QTOF enabled metabolite identification, a semi-quantitative
metabolic stability study, and metabolic profiling—all within the same
experiment. The 6540 QTOF produced
very high quality accurate mass MS
and MS/MS spectra for confident
metabolite identification, including
accurate isotope ratios. The Agilent
QTOF provided excellent data quality
in all dimensions, simultaneously,
for combined quantitative and
qualitative analyses.
www.agilent.com/chem/qtof
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© Agilent Technologies, Inc. 2010
Printed in the U.S.A. December 6, 2010
5990-6867EN