Application
Note: 417
Using Multiple Mass Defect Filters and Higher Energy
Collisional Dissociation on an LTQ Orbitrap XL
for Fast, Sensitive and Accurate Metabolite ID
Yingying Huang1, Shirley Liu2, Shichang Miao2, Patrick M. Jeanville1
1
Thermo Fisher Scientific, San Jose, CA, USA; 2ChemoCentryx, Mountain View, CA, USA
Overview
Key Words
• LTQ Orbitrap XL™
• MetWorks™
Software
• Accela™ High
Speed LC
• Hypersil GOLD™
Column
• Metabolite
Identification
• Multiple Mass
Defect Filters™
Purpose: To evaluate the use of Multiple Mass Defect
Filters (MMDFs)™ with LTQ Orbitrap XL data for
metabolite identification; to investigate the use of Higher
energy Collisional Dissociation (HCD) for structural
elucidation in metabolite identification experiments.
Methods: Rat hepatocyte incubation samples of Irinotecan
were analyzed using an LTQ Orbitrap XL with HCD
collision cell. Both Collision Induced Dissociation (CID)
MS/MS and HCD MS/MS were acquired for the potential
metabolites. MMDFs were then used to process the
acquired raw file.
Results: MMDFs were able to filter out the vast majority of
the background ions in the full scan spectra while preserving those related to the parent drug. Compared with the
results gathered from using only a single Mass Defect Filter
(MDF), results from MMDFs are more distinct and specific,
allowing users to do faster, more sensitive and more accurate analyses. HCD provides complementary fragmentation
pathways, in addition to the CID available in the ion trap,
and produces low mass diagnostic ions in MS/MS spectra
that are useful for metabolite structural elucidation.
Introduction
An integral part of drug discovery and development is the
identification of drug metabolites formed through phase I
and phase II metabolic reactions. These metabolites may
have either intrinsic pharmacological activity or display
specific toxicity. LC-MS has become the cornerstone in
drug metabolite identification because of its sensitivity
and ability to analyze complex mixtures. In particular,
ESI source
LC-MSn employing linear ion trap (LIT) technology has
become widely used because of its speed, sensitivity and
robustness in generating rich structure information.
However, challenges still remain in detecting and identifying metabolites in the presence of highly complex
biological matrices.
Coupling an orbitrap mass analyzer to the LIT greatly
facilitates the task of metabolite identification because it
not only enables parallel data acquisition with high mass
accuracy and resolution, but it also provides post-LIT ion
manipulations. High resolution and accurate mass help
resolve and identify metabolite peaks from background
matrix ions, and also allow the use of post-acquisition
data processing tools like MDF to reduce the number of
false positives by removing the vast majority of matrixrelated background ions.1,2 HCD was recently introduced
on the LTQ Orbitrap as an alternative dissociation
method by adding a new collision cell behind the C-trap
region (Figure 1). HCD can be used to generate low mass
diagnostic ions in MS/MS mode, and can also be used in
combination with CID in the LIT for MSn experiments.
Irinotecan (CPT-11; 7-ethyl-10-[4-(1-piperidino)-1piperidino] carbonyloxy-camptothecin) is a water-soluble
carbamate prodrug of camptothecin and is activated in
vivo to SN-38, a potent topoisomerase I inhibitor.3,4
Currently, Irinotecan, combined with 5-fluorouracil and
leucovorin, is approved by the U.S. Food and Drug
Administration as a first-line therapy in the treatment
of metastatic carcinoma of the colon or rectum.4 In this
study, we will utilize MMDF and HCD on an LTQ
Orbitrap XL to study the biotransformations of Irinotecan
in hepatocyte incubation and identify its metabolites.
Linear ion trap
C-trap
Orbitrap
Figure 1: Scheme diagram of the LTQ Orbitrap, highlighting the HCD collision cell behind the C-trap.
HCD
Collision Cell
Materials and Methods
Samples: Incubation was carried out using rat hepatocytes
pooled from 1 male and 1 female with a cell density of
0.5 million/mL and 10 µM of Irinotecan in the final 1 mL
incubation solution. The solution was shaken overnight
and quenched by cooling down on dry ice, followed by
the addition of 200 µL chilled Acetonitrile. The solution
was then vortexed and centrifuged. The supernatant
(~1 mL) was taken out, and 10 µL from such solution
was directly injected for each LC-MS/MS run.
HPLC: Accela High Speed LC, Thermo Scientific
Column: Hypersil GOLD C18, 1 × 100 mm, 1.9 µm
particle size, Thermo Scientific
Gradient:
Time
A%
B%
µL/min
0
98
2
150
2
98
2
150
2.5
85
15
150
14
75
25
150
14.9
20
80
150
15
98
2
150
20
98
2
150
A = 0.1% Formic Acid in water
B = 0.1% Formic Acid in Acetonitrile
Mass Spectrometer: LTQ Orbitrap XL with
HCD collision cell.
Results and Discussions
MMDF is a new feature in the latest version of Thermo
Scientific MetWorks Software that combines the results
from up to six different MDFs. Through the use of
MMDF and the combination of HCD and CID MS/MS,
thirteen Irinotecan metabolites were identified from the
incubation sample using the LTQ Orbitrap XL with less
than 3 ppm mass accuracy. Table 1 summarizes the 13
metabolites identified, while Scheme 1 displays their
corresponding structures.
MDF
Template
Irinotican
MH+ m/z =
587.2864
SN-38
MH+ m/z =
393.1445
As shown in Table 1, all 13 metabolites were found
with peak areas less than 1% of that of the parent.
Figure 2 shows how the base peak chromatogram from
the same LC-MS/MS run changed after a single MDF
and after MMDF processing. Due to the low abundance
of the metabolites, in all these chromatograms the intensity
(y axis) for retention time 5.7-11.2 minutes and 12.5-16.1
minutes was expanded by 50 times to better illustrate the
effectiveness of MMDF. While all the peaks from the
Irinotecan metabolites were well buried in the original
chromatogram (Figure 2a), the most abundant metabolite
peaks (e.g. M2 @ 7.44 minute, M5 @ 8.84 minute,
M6 @ 9.92 minute) start to show up after applying a
single MDF (Figure 2b). However, even with a single
MDF, peaks from background matrix ions that are unrelated to the metabolite were still prominent (e.g. peaks at
6.55 minute, 7.85 minute, and 11.1 minute). This is due
to the fact that in order to use only one single MDF to
capture all the phase I and II metabolites, including those
from the hydrolysis product SN-38, a relatively wide mass
defect range needs to be used (-150 mmu, +70 mmu).
Therefore, a portion of the background ions remains after
the mass defect filtering.
When MMDFs were applied to the original data
(Figure 2c), four different mass defect filters were used,
whose corresponding metabolites identified were highlighted using different colors in Table 1: phase I metabolites of Irinotecan are shown with white background;
phase II metabolites of Irinotecan are shown in light blue;
phase I metabolites of SN-38 are shown in pink; phase II
metabolites of SN-38 are shown in yellow. Compared to the
results using a single MDF (Figure 2b), results from MMDF
(Figure 2c) are cleaner and more specific to the metabolites
related to Irinotecan, and are, therefore, easier to interpret.
#
R.T.
Metabolite Identity
Formula
Change
Integrated
Peak Area
% of
Parent
Theoretical
MH+ m/z
Measured
MH+ m/z
ppm
ΔMW (Da) from
Irinotecan
∆MD from
Irinotecan
∆MD from
Template
M1
7.19
Di-hydroxylation
+O2
4.0E+4
0.027
619.2762
619.2755
-1.1
31.9898
- 0.0102
- 0.0102
M3
8.45
Hydroxylation
+O
1.6E+5
0.107
603.2813
603.2805
-1.3
15.9949
- 0.0051
- 0.0051
8.7E+3
0.006
892.3546
892.3551
0.6
305.0682
+ 0.0682
+ 0.0682
M4
8.48
GSH conjugation
+C10H15
N3O6S
M5
8.84
Ring cleavage of
Piperidine
+H2O
9.8E+5
0.653
605.2970
605.2965
-0.8
18.0106
+ 0.0106
+ 0.0106
M6
9.92
Hydroxylation
+O
1.3E+6
0.867
603.2813
603.2800
-2.2
15.9949
- 0.0051
- 0.0051
M7
10.34
Oxidation to Ketone
-H2+O
9.7E+4
0.065
601.2657
601.2650
-1.2
13.9792
- 0.0208
- 0.0208
M8
10.64
Hydroxylation
+O
8.9E+4
0.059
603.2813
603.2805
-1.3
15.9949
- 0.0051
- 0.0051
M9
11.11
APC
+O2
1.7E+5
0.113
619.2762
619.2758
-0.6
31.9898
- 0.0102
- 0.0102
M10
11.55
Dehydrogenation
-H2
3.7E+5
0.247
585.2707
585.2699
-1.4
-2.0157
- 0.0157
- 0.0157
P
11.68
Irinotecan
N.A.
1.5E+8
100
587.2864
587.2860
-0.7
0
0
0
M12
12.69
Hydroxylation
+O
1.2E+5
0.080
603.2813
603.2803
-1.7
15.9949
- 0.0051
- 0.0051
M13
15.77
Decarboxylation
-CO2
3.2E+5
0.213
543.2966
543.2960
1.1
-43.9898
+ 0.0102
+ 0.0102
M2
7.44
SN-38 Glucuronide
+C6H8O6
4.0E+5
0.267
569.1766
569.1754
-2.1
176.0321
-0.1106
+ 0.0321
M11
11.95
SN-38
N.A.
6.1E+5
0.407
393.1445
393.1440
-1.3
0
-0.1415
0
Table 1. Summary of the putative metabolites identified from 10 µM Irinotecan rat hepatocyte incubation. Those highlighted in different colors were filtered
by different Mass Defect Filters.
Page 2 of 6
O H
O
O
O
O
O
O
O
N
N
O H
N
O
O
O H
O
N
O H
N
N
O
O H
N
M5
Ring cleavage of Piperidine
N H
M4
GSH Conjugation
M1
Di-hydroxylation
N
O
GS
N
N
O
O
N
O
O H
O
O
O
O
O
O
O
O
O
N
N
N
OH
N
O
O
N
O
O H
M3
Hydroxylation
N
O
O
N
N
O
OH
M7
Oxidation to Ketone
N
O
N
O H
N
O
O
O
N
O
N
O
Irinotecan
N
O
O H
N
O
HO
O
O
O
O
O
O H
N
O
M11
SN-38
O
N
O H
O
N
N
O
O
N
N
N
M8
Hydroxylation
N
O
O
N
O
O
O
O
O
N
OH
HO
O H
N
O H
N
OH
M2
SN-38 Glucuronide
M9
APC
N H
O
O
N
O
M13
Decaboxylation
O
OH
O
H O
O H
N
N
O H
N
O
M10
Dehydrogenation
N
O
OH
O
N
N
M6, M12
Hydroxylation
N
O
O
Scheme 1. Proposed structures and biotransformation pathways of Irinotecan metabolites.
x50
100
90
x50
11.68
6.28
(a)
13.28
8.33
5.74
8.64
13.87
9.45
6.92
80
13.24
11.73
10.12
15.32
15.02
15.36
Relative Abundance
70
60
15.71
50
40
15.92
16.05
30
Original
20
10
5.07
16.79
0
5
6
9
8
7
10
13
12
11
Time (min)
14
x50
x50
(b)
90
After Single MDF
80
11.73
M6
70
Relative Abundance
17
16
11.68
100
12.64
9.92
60
12.69
50
40
6.55
12.77
M5
7.85
15.86
8.84
12.92
M2
30
13.52
7.44
20
5.79
5.74
10
9.45
8.21
7.22
M13
13.87
14.03
10.84
15.77
15.92
16.05
14.41
16.28
5.39
0
5
6
7
9
8
10
11
Time (min)
12
13
14
15
x50
16
17
x50
11.68
100
(c)
90
P (Irinotecan)
After MMDF
80
11.73
M6
70
Relative Abundance
15
12.64
9.92
M12
60
12.69
50
M5
M2
30
7.44
M1
10
5.20
5
6.16
6
7.19
7
M8 M9
10.94
M7
10.34
M4
M3
20
8.45
9.82
7.61
12.77
M10
8.84
40
12.92
13.52
M11
M13
13.87
14.03
15.77
14.41
15.84
16.36
8
9
10
11
Time (min)
12
13
14
15
16
17
Figure 2: Base peak chromatograms of 10mM Irinotecan rat hepatocyte incubation: (a) Original; (b) After single Mass Defect
Filter; (c) After Multiple Mass Defect Filters (MMDF).
Page 3 of 6
Figure 3 further illustrates the power of MMDF
by showing an example of how the full MS spectrum
changed after the single MDF, and MMDFs were applied.
The peak at 603.2805 m/z is a hydroxylation metabolite
(M3) that elutes at 8.45 minute. The original full scan
MS is dominated by background ions and the M3 peak
only has less than 15% relative abundance (Figure 3a).
After a single MDF was applied, the M3 peak becomes
the base peak, however, there are still a lot of background
peaks remaining in the spectrum (Figure 3b). After
MMDFs were applied, only the M3 peak and trace of the
parent remain in the spectrum while almost all the background ions are gone (Figure 3c).
After MMDF processing, HCD and CID MS/MS
spectra of Irinotecan and its potential metabolites were
analyzed, and their corresponding structures were elucidated. Figure 4a shows the CID MS/MS of Irinotecan
100
from the LTQ, and Figure 4b shows the HCD MS/MS
acquired in the orbitrap. While all the major fragment
ions in the CID spectrum were also observed in the HCD
spectrum, the HCD spectrum contains additional fragment
ions including low m/z ions. A portion of the parent ions
still remains in the HCD spectrum. These are characteristics similar to those from a quadrupole collision cell.
Better than 2 ppm mass accuracy and high resolution
were obtained on the fragment ions in the HCD MS/MS
spectrum because it was acquired in the orbitrap. Mass
FrontierTM software, with its accurate mass capability, was
used to assist spectrum interpretation. Structures of the
fragment ions were assigned accordingly. The fact that
HCD spectra display rich fragment ions, especially in the
low mass region, as well as high mass accuracy on these
product ions, greatly facilitates the MS/MS interpretation.
131.1176
R=115501
(a)
Relative Abundance
80
Original
60
40
20
159.1125
R=105204
226.9512
R=88404
603.2805
R=54201
445.1193
R=64101
684.2020
R=48301
0
900.5536
R=40904
603.2805
R=54201
100
(b)
536.1645
R=58201
Relative Abundance
80
462.1458
R=62101
171.1489
R=102401
After Single MDF
60
279.1588
R=79401
40
391.2838
R=68101
20
0
684.2020
R=48301
827.1611
R=48204
991.3182
R=35204
603.2805
R=54201
100
(c)
Relative Abundance
80
After MMDF
60
40
20
587.2862
R=47901
0
200
400
600
m/z
800
1000
Figure 3: Full MS spectrum at 8.45 minute: (a) original, (b) after single Mass Defect Filter, (c) after Multiple Mass Defect Filters (MMDF).
Page 4 of 6
543.3
100
CID
90
(a)
80
Relative Abundance
70
60
50
40
502.2
30
195.2
20
10
167.1
50
150
100
250
200
350
m/z
565.1
450
400
500
600
550
195.1493
1 00
O
O
0.7 ppm
N
(b)
90
HCD
O
O
N
N
N
N
O
0.1 ppm
124.1121
O
OH
N
N
N
70
H+
O
N
O
80
Relative Abundance
300
458.3
375.2
331.2
0
N
O
N
0.8 ppm
H+
O
N
O
N
167.1180
O
O
60
O
N
N
50
O
OH
N
O
O
N
O
40
N
O
O
0.9 ppm
587.2869
O
N
O
N
O
N
30
0.5 ppm
N
O
-0.005 ppm
20
N
110.0600
1.9 ppm
196.1531
98.0964
-0.9 ppm
-0.6 ppm
331.1447
502.1975
O
OH
N
-0.2 ppm
10
O
N
-0.4 ppm
543.2964
458.2083
503.2019
375.1337
0
50
100
150
200
250
350
300
400
450
500
550
600
m/z
Figure 4: MS/MS spectra of Irinotecan: (a) CID MS/MS acquired in the LTQ. (b) HCD MS/MS acquired in the orbitrap.
Conclusions
With the help of MMDF and the combination of HCD and
CID MS/MS, 13 Irinotecan metabolites whose peak areas
were less than 1% of that of the parent were identified on
an LTQ Orbitrap XL coupled to an Accela High Speed LC.
This report demonstrates that MMDF is more effective
than a single MDF to uncover phase I and II metabolites
specifically and concurrently. It also allows the detection of
metabolites from hydrolysis or N-dealkylation, even when
the products from such processes have mass defects that
are significantly different from the parent. MMDF allows
users to use low threshold values during data processing
so that metabolites at very low levels can be easily identified. The resulting chromatogram from MMDF is accurate
and specific because it is based on exact mass and mass
deficiencies, which are highly specific to the parent drug
compound. It provides speed, sensitivity and accuracy to
facilitate the identification of drug metabolites in drug
discovery and development.
Page 5 of 6
HCD provides an alternative fragmentation method
on the LTQ Orbitrap XL in addition to the CID in the
linear ion trap. HCD spectra display characteristics similar
to those from a quadrupole collision cell: rich in product
ions, has no low mass cut off, and typically a portion of
the parent ions still remains. The fragment ions in HCD
spectra have high mass accuracy and resolution. These
characteristics of HCD spectra complement the power
of ion trap MSn and allow easy spectrum interpretation
and high confidence in structural elucidation.
References
1
Zhang et al., J. Mass Spectrom. 2003, 38 (10), 1110-1112.
2
Zhu et al., Drug Metabolism and Disposition, 2006, 34 (10), 1722-1733
3
Haaz et al., Drug Metabolism and Disposition, 1998, 26 (8), 769-774.
4
Sanghani et al., Drug Metabolism and Disposition, 2004, 32 (5), 505-511.
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