PO65277-EN 0518S
Flags all unknown
compounds that match
specified compound
class based on
fragmentation data.
Detection of expected
compounds using
biotransformation list.
Kate Comstock1, Shuguang Ma2, Caroline Ding1, Yan Chen1, Seema Sharma1 1Thermo Fisher Scientific, River Oaks Pkwy, San Jose, CA USA 2Genentech, DNA way, South San Francisco, CA USA
INSTRUMENT and METHODOrbitrap ID-X MS and AcquireX Acquisition
This study was conducted on the Thermo Scientific™ Orbitrap ID-X™ Tribrid™ mass spectrometer
which is a Tribrid instrument dedicated for small molecule research only. Orbitrap ID-X ready-made
method templates were used to generate the acquisition method, see Figure 1.
OVERVIEW
Purpose: AcquireX acquisition workflow for improved MSn data quality for metabolite identification.
Methods: Orbitrap Tribrid ID-X with AcquireX acquisition workflow
Results: AcquireX background exclusion workflow improves metabolite ID
INTRODUCTIONMetabolite Identification is a critical component through all stages of drug discovery and
development. High resolution mass spectrometry (HRMS) is an essential tool for metabolite
identification. HRMS with sophisticated data acquisition features can provide the critical information
for metabolite structure elucidation and alleviate the difficulties of matrix complexity.
Here we present a study for metabolite ID using Orbitrap Tribrid ID-X MS with AcquireX data
acquisition workflow which includes automatic background ions subtraction, inclusion/exclusion lists
generation, and real-time decision-making to optimize MSn data acquisition quality and speed.
The MSn data generated using the AcquireX data acquisition workflow was processed using structure
analysis and data mining software “Compound Discoverer 3.0” and “Mass Frontier 8.0”.
The results of using Data Dependent Acquisition (DDA) and AcquireX Background Exclusion
workflow were compared.
MATERIALS AND METHODS
Sample Preparation
Sample: Test compounds (5 μM): Amprenavir, Bosentan, Lopinavir, Tipranavir, Ritonavir
Control: Human liver microsomes (1 mg/mL) incubation in the presence of GSH and UDPGA without
test article at 37 degree for 1 hr
Liquid Chromatography
Thermo Vanquish Flex UHPLC system consisting of:
Vanquish Binary pump
Vanquish Autosampler
Vanquish Column Compartment
Vanquish Diode Array Detector
Column: Thermo Hypersil C18 100 x 2.1 mm, 1.9 µm Temperature: 45oC
Gradient: Mobile A: H2O/0.1% Formic Acid
B: ACN/0.1% Formic Acid
Flow rate: 400µl/min Injection Volume: 10 µl
LC gradient:
Time(min) 0 1.0 2.0 14.0 14.1 16.0 16.1 19
B% 5 5 15 70 95 95 5 5
Mass Spectrometry
The MS analyses were carried out on Thermo scientific Orbitrap ID-XTM TribridTM mass spectrometer
using electrospray ionization in positive mode. High resolution full-scan MS and top3 MS/MS data
were collected in a data-dependent fashion at resolving power of 120,000 and 30,000 at FWHM
m/z200 respectively, stepped HCD Collision Energy (%): 20, 40, 60. AcquireX data acquisition
Source parameters:
Positive Ion Spray Voltage (V): 3400
Sheath Gas (Arb): 40
Aux Gas (Arb): 5
Sweep Gas (Arb): 1
Ion Transfer Tube Temp (oC): 300
Vaporizer Temp (oC): 400
CONCLUSIONS
Orbitrap ID-X Tribrid MS delivers high quality data.
AcquireX data acquisition workflow overcomes the matrices interference in automated fashion
and enhances metabolites ID through increasing triggering MSn.
Compound Discoverer 3.0 and Mass Frontier software suite effective at data mining and
generates confident ID and structure characterization.
Orbitrap ID-X tribrid MS with the AcquireX data acquisition feature, coupled with Compound
Discoverer 3.0 and Mass Frontier software suite, provide a comprehensive workflow for confident
metabolite identification.
TRAEMARKSLICENSING
© 2018 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo
Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these
products in any manner that might infringe the intellectual property rights of others.
Enhanced Metabolite Identification Using Orbitrap Tribrid Mass Spectrometer
Table 1. Metabolites Identified by DDA and AcquireX
Figure 9. Amprenavir Metabolites Detected
Figure 4. Model Compound Amprenavir in Bile
ANALYSIS RESULTS
Low-levels Model Compounds in Complex Matrices were Triggered MSn
A mixture of 5 model compounds was spiked into bile and human plasma at 0.1 uM level. The
samples were analyzed using data dependent acquisition (DDA) and AcquireX Background Exclusion
workflow. The results showed that with AcquireX Background Exclusion, the model compound was
detected with high mass accuracy and triggered MSn, see Figure 4, while DDA was not able to trigger
MSn for all five compounds due to the presence of complex biological matrices.
Parent Drug
O
ONHNS
OH
OO
O
N2H
Bile_Drug01uM_ID_1 04/16/18 04:41:22 Bile_Drug01uM_ID_1
Bile_Drug01uM_ID_1 #2787 RT: 9.49 AV: 1 NL: 1.63E5
T: FTMS + p ESI d Full ms2 [email protected] [100.0000-517.0000]
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
156.0113
245.1648
506.2916
108.0443 418.1792
129.0698
202.1589 261.1597392.1998146.0963 189.1021
351.2274 488.2792374.1898164.0707 470.2914
Bile_Drug01uM_ID_1 #2788 RT: 9.50 AV: 1 NL: 2.33E5
T: FTMS + p ESI d Full ms3 [email protected] [email protected] [50.0000-208.0000]
60 80 100 120 140 160
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
108.0442
92.0493
82.292553.8203 103.1688 120.0558
Bile_Drug01uM_ID_1 #2789 RT: 9.50 AV: 1 NL: 1.37E4
T: FTMS + p ESI d Full ms3 [email protected] [email protected] [62.0000-256.0000]
80 100 120 140 160 180 200 220 240
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
189.1022
202.1590129.0698
147.5185172.0747
153.1020103.4371236.2392
Bile_Drug01uM_ID_1 04/16/18 04:41:22 Bile_Drug01uM_ID_1
Bile_Drug01uM_ID_1 #3500 RT: 11.83 AV: 1 NL: 2.93E6
T: FTMS + p ESI d Full ms2 [email protected] [100.0000-732.0000]
100 150 200 250 300 350 400 450 500 550 600 650 700
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
140.0528
296.1426197.0742
268.1478
171.0950
426.1843
250.1589 507.2418117.0698 347.1576 533.2211 721.3197382.1946
311.1753 408.1738
217.0792 677.3297585.3033 653.3295
Bile_Drug01uM_ID_1 #3501 RT: 11.83 AV: 1 NL: 4.45E5
T: FTMS + p ESI d Full ms3 [email protected] [email protected] [50.0000-208.0000]
60 70 80 90 100 110 120 130 140
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
70.9949
107.0729
96.0808113.0418
98.0057 124.075982.064864.3719
Bile_Drug01uM_ID_1 #3502 RT: 11.83 AV: 1 NL: 3.39E4
T: FTMS + p ESI d Full ms3 [email protected] [email protected] [76.0000-307.0000]
100 150 200 250 300
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
268.1484
147.4975
113.9884
131.6344
87.6823
Bile_Drug01uM_ID_1 04/16/18 04:41:22 Bile_Drug01uM_ID_1
Bile_Drug01uM_ID_1 #4030 RT: 13.51 AV: 1 NL: 5.58E5
T: FTMS + p ESI d Full ms2 [email protected] [100.0000-614.0000]
100 150 200 250 300 350 400 450 500 550 600
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
585.2031
411.0625
190.0500 375.2196333.1726
346.1805270.1491
133.0886174.0915 279.1105244.1333219.0891 521.2410 603.2142481.1408385.0839
360.1964314.1551
427.2816 543.1918
Bile_Drug01uM_ID_1 #4036 RT: 13.53 AV: 1 NL: 1.31E4
T: FTMS + p ESI d Full ms3 [email protected] [email protected] [108.0000-422.0000]
150 200 250 300 350 400
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
190.0495
347.0998
147.5304
391.0055241.2287
121.4405
Bile_Drug01uM_ID_1 #4035 RT: 13.53 AV: 1 NL: 3.41E3
T: FTMS + p ESI d Full ms3 [email protected] [email protected] [156.0000-596.0000]
200 250 300 350 400 450 500 550
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
273.9205 571.6830
185.6382
Bile_Drug01uM_ID_1 04/16/18 04:41:22 Bile_Drug01uM_ID_1
Bile_Drug01uM_ID_1 #3587 RT: 12.10 AV: 1 NL: 2.51E6
T: FTMS + p ESI d Full ms2 [email protected] [100.0000-640.0000]
100 150 200 250 300 350 400 450 500 550 600
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
155.1178
183.1127
447.2640
120.0807
611.3584310.1801250.1589 400.3290131.0855 561.2828
418.3393
274.2244 453.2965215.1797
189.1641
508.4800
Bile_Drug01uM_ID_1 #3588 RT: 12.10 AV: 1 NL: 4.24E5
T: FTMS + p ESI d Full ms3 [email protected] [email protected] [50.0000-208.0000]
60 80 100 120 140 160
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
113.0708
101.0708
86.0962
155.1178
99.0915
84.0806
138.091272.0807 95.1945 118.616653.8232
Bile_Drug01uM_ID_1 #3589 RT: 12.11 AV: 1 NL: 1.60E6
T: FTMS + p ESI d Full ms3 [email protected] [email protected] [50.0000-208.0000]
60 80 100 120 140 160 180 200
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
155.1178
147.5205 189.069799.263779.638360.5147
Bile_Drug01uM_ID_1 04/16/18 04:41:22 Bile_Drug01uM_ID_1
Bile_Drug01uM_ID_1 #3257 RT: 11.04 AV: 1 NL: 2.39E6
T: FTMS + p ESI d Full ms2 [email protected] [100.0000-563.0000]
100 150 200 250 300 350 400 450 500 550
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
552.1915
280.0833
508.1653311.1017106.0400 175.0616149.0459 202.0725 294.0990241.0896
264.0883324.1097 400.1442355.1273 516.3152
Bile_Drug01uM_ID_1 #3254 RT: 11.03 AV: 1 NL: 2.63E5
T: FTMS + p ESI d Full ms3 [email protected] [email protected] [72.0000-291.0000]
100 150 200 250
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
280.0832
147.0551
252.0881120.0440
175.0502100.5523 205.9448
Bile_Drug01uM_ID_1 #3255 RT: 11.03 AV: 1 NL: 4.13E4
T: FTMS + p ESI d Full ms3 [email protected] [email protected] [134.0000-519.0000]
150 200 250 300 350 400 450 500
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
311.1018
280.0848202.0739 400.1422147.5026 256.4470
Bosentan
Tipranavir
Lopinavir
Ritonavir
Amprenavir
DATA ACQUISITIONModel Compounds HLM Metabolite ID Using AcquireX
The model compounds HLM digest were analyzed on Orbitrap ID-X™ Tribrid™ mass spectrometer
using AcquireX Background Extraction workflow. The results showed that AcquireX acquisition
identified more metabolites by effectively triggering more MSn compared with data-dependent
acquisition (DDA), see Figure 8. The MSn spectra were critical for metabolites structure elucidation,
which is especially useful for identifying structures for isomeric metabolites.
Figure 3. AcquireX Acquisition Sequence for Metabolite ID
Figure 2. AcquireX Workflows
Figure 1. Orbitrap ID-X Ready-Made Method Template
AcquireX Data Acquisition workflow
AcquireX is a data acquisition workflow which can generate background exclusion list and sample
component inclusion list in an automated fashion to improve quality and efficiency of analysis. There
are three AcquireX workflows with ready-to-use acquisition templates, see Figure 2. The Background
Exclusion workflow was used for this study to reduce the biologic matrices, see Figure 3.
Bile_5Drugs_01uM #2991-3058 RT: 9.43-9.62 AV: 17 NL: 4.09E7F: FTMS + p ESI Full ms [100.0000-1000.0000]
100 200 300 400 500 600 700 800 900
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
500.30407
316.24824
556.33020384.27455
464.28298 578.31208
125.98622 669.41404 900.50668758.56906239.14892
Bile_5Drugs_01uM #2991-3058 RT: 9.43-9.62 AV: 17 NL: 4.09E7
F: FTMS + p ESI Full ms [100.0000-1000.0000]
480 490 500 510 520 530 540 550
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
500.30407
517.33052
522.28599
482.29350 538.25095
502.29957
542.28943
524.37097
484.36327
496.33975
514.34874
506.23193
Amprenavir spiked
into bile at 0.1 M
Figure 5. AcquireX Workflow Triggered MSn of All 5 Model Compounds in Bile
Figure 8. AcquireX Workflow Triggered MSn of All 5 Model Compounds in Bile
Figure 6. Detect Expected and Unexpected Metabolites Using Biotransformation List and
Compound Class
Amprenavir
Transformation shifted
fragment ion annotations
help localize the site of
biotransformation.
[ ] +O
[ ] +O
Figure 7. Auto Annotations of Fragment Ion Search (FISh) – Localize the Site of Biotransformation
DATA PROCESSData Processing Using Compound Discoverer 3.0 and Mass Frontier 8.0
The data was processed using “Compound Discoverer 3.0” and “Mass Frontier 8.0” software.
Compound Discoverer 3.0 (CD 3.0) features automatic dealkylation prediction, and nodes enable the
detection of targeted metabolites through biotransformation list, as well as detection of unexpected
using “Compound Class Scoring” node. Parent compound fragments were used to generate “Class
Coverage” list, and user can also add other fragments based on parent compound structure. This
feature effectively enhances the detection of unknown or unexpected metabolites other than common
phase I and II biotransformation. See Figure 6.
CD 3.0 Fragment Ion Search (FISh) features automatically annotated fragment ions. The blue color-
coded peaks indicate the mass shift of the fragment, which facilitates localizing the site of
biotransformation, see Figure 7.
DDA triggered
8 metabolites
Acquire X triggered
21 metabolites
RT [min] Molecular Weight Formula Transformations DDA AcquireX
2.29 236.1889 C14H24N2O Sufonamide hydrolysis + amide hydrolysis Y
4.84 407.18788 C20 H29 N3 O4 S Amide hydrolysis + oxidation Y
5.02 350.22056 C19 H30 N2 O4 Sufonamide hydrolysis Y Y
5.14 364.23621 C20 H32 N2 O4 Sufonamide hydrolysis + methylation Y
5.25 407.18788 C20 H29 N3 O4 S Amide hydrolysis + Oxidation Y Y
6.09 407.18788 C20 H29 N3 O4 S Amide hydrolysis + Oxidation Y Y
6.41 697.24513 C30 H43 N5 O10 S2 Oxidation + Cysteine + Glycine conjugation Y
6.50 391.19296 C20 H29 N3 O3 S Amide hydrolysis Y Y
6.61 519.20392 C25 H33 N3 O7 S Oxidation (+O-2H) Y Y
6.88 826.28773 C35 H50 N6 O13 S2 Oxidation + GSH Conjugation Y
7.19 624.22876 C28 H40 N4 O8 S2 Cysteine Conjugation Y
7.32 521.21957 C25 H35 N3 O7 S Oxidation Y Y
7.62 405.20861 C21 H31 N3 O3 S Amide hydrolysis + methylation Y
7.81 681.25674 C31 H43 N3 O12 S Glucuronidation y Y
7.84 521.21957 C25 H35 N3 O7 S Oxidation Y Y
8.01 537.21449 C25 H35 N3 O8 S Oxidation Y
8.28 503.20901 C25 H33 N3 O6 S Desaturation Y
8.37 503.20901 C25 H33 N3 O6 S Desaturation Y
9.02 521.21957 C25 H35 N3 O7 S Oxidation Y
9.11 503.20901 C25 H33 N3 O6 S Desaturation Y
9.52 505.2246 C25 H35 N3 O6 S Amprenavir Parent Y Y
10.61 519.24031 C26 H37 N3 O6 S Methylation Y
