Post on 23-Oct-2020
transcript
Seema Sharma1, Kate Comstock1, Doug Kiehl2, Graeme McAlister1, Ryo Komatsuzaki1, Caroline Ding1, Ralf Tautenhahn1, Derek Bailey1, Linda Lin1, Tim Stratton1, Shannon Eliuk1, Iman Mohtashemi1, Jonathan Josephs1, Vlad Zabrouskov1
1Thermo Fisher Scientific, San Jose, CA; 2Eli Lilly, Indianapolis, IN
INSTRUMENTS AND ACQUISITION METHODOrbitrap ID-X MS
This study was conducted on an Orbitrap ID-X mass spectrometer, a dedicated Tribrid instrument for
small molecule research. The MS methods were set up using predefined experimental method
templates in the Orbitrap ID-X method editor, see Figure 1.
ABSTRACT
Purpose: Optimized workflow for extractables and leachables (E&L) analysis.
Methods: Thermo Scientific™ Orbitrap ID-X™ Tribrid™ mass spectrometer with AcquireX
background exclusion data acquisition coupled with Thermo Scientific™ Compound Discoverer™ 3.0
software and Thermo Scientific™ Mass Frontier™ 8.0 software.
Results: Automatic background exclusion and MSn data improve overall E&L analysis quality and
confidence.
INTRODUCTION
The identification of unknown small molecules—such as impurities, metabolites, degradants,
extractables, and leachables—remains one of the most challenging workflows. Mass spectrometry
based chemical and structural characterization of small molecules greatly benefits from multistage
fragmentation (MSn) coupled with high resolution and high mass accuracy analysis. However,
applying these sophisticated methods to complex samples is challenging, owing to the slow precursor
interrogation rate and the large number of potential precursors. Herein we describe new data
acquisition approaches for the characterization of small molecule extractables and leachables.
Building upon sophisticated methods that employ multistage Orbitrap™ analysis (FTMSn), we
dynamically update inclusion and exclusion lists between LC analyses to enable efficient and deep
interrogation of all the features in a complex sample.
MATERIALS AND METHODS
Sample Preparation
Sample 1. Additive standard mixture was prepared at 10 ppm each in 1:1 IPA/H2O. The solution was
diluted 10-fold using drug excipients (USP).
Sample 2. Three different types of medical grade O-rings, A, B, and C, were extracted using
PH3 H2O, PH9 H2O, and IPA. The weight and volume ratio was 1/10: 2 g of O-ring material and 20 mL
of solvent. The extraction vials were placed in oven at 50 oC for 7 days. The extracts solutions were
analyzed directly by LC/MS.
Liquid Chromatography
Thermo Scientific™ Vanquish™ Flex UHPLC system consisting of: Vanquish Binary Pump, Vanquish
Autosampler, Vanquish Column Compartment, Vanquish Diode Array Detector
Column: Thermo Scientific™ Hypersil GOLD™ C18 100 x 2.1 mm, 1.9 µm
Temperature: 45 oC
Gradient: Mobile phase A: H2O/0.1% Formic acid; B: ACN/0.1% Formic acid
Flow Rate: 400 µL/min
Injection Volume: 5 µL
LC Gradient: Time (min) 0 1.0 4.0 15.0 20.0 20.1
B% 5 5 30 95 95 5
Mass Spectrometry
The MS analyses were carried out on an Orbitrap ID-X Tribrid mass spectrometer using electrospray
ionization in positive mode. The AcquireX data acquisition workflow was used for data acquisition.
High-resolution full-scan MS and MSn data, HCD MS2 and CID MS3, were collected in a data-
dependent fashion at resolving powers of 120,000, 30,000, and 15,000 at FWHM m/z 200,
respectively. Stepped HCD collision energy (%): 15, 35, 55 was used.
Source Parameters:
Positive Ion Spray Voltage: 3400 V
Sheath Gas (Arb): 40 Arb
Aux Gas (Arb): 5 Arb
Sweep Gas (Arb): 1 Arb
Ion Transfer Tube Temp: 300 oC
Vaporizer Temp: 400 oC
CONCLUSIONS
The results of this study demonstrate how the Orbitrap ID-X MS, equipped with the AcquireX
acquisition workflow, facilitates E&L structure elucidation. Coupled with the data processing suite
consisting of Compound Discoverer 3.0 and Mass Frontier 8.0 software, the Orbitrap ID-X instrument
improves the efficiency and increases the reliability of E&L identifications, and streamlines the E&L
analysis from data acquisition to data processing. This workflow is also well-suited to other small
molecule structure applications, such as Met ID, API impurity ID, degradants ID, and metabolomics
research.
ACKNOWLEDGEMENTS
The authors would like to thank the extraordinary efforts made by everyone who contributed to
Orbitrap ID-X MS and AcquireX.
TRADEMARKS/LICENSING
© 2018 Thermo Fisher Scientific Inc. All rights reserved. Mass Frontier is a trademark of HighChem
LLC. All other 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.
Optimized Workflow for Structure Elucidation of Pharmaceutically Relevant Extractables and Leachables
Figure 1. Orbitrap ID-X MS predefined experimental method templates for E&L analysis
Table 2. Alternate table style shown here.
AcquireX
The AcquireX acquisition workflow includes three ready-to-use workflow templates, see Figure 2:
1. BACKGROUND EXCLUSION. This workflow automatically creates and uses a background
exclusion list to reduce background fragmentation in sample ID runs.
2. BACKGROUND EXCLUSION & COMPONENT INCLUSION. Combines exclusion and inclusion
lists automatically and reliably acquires more relevant MSn data in a single injection.
3. DEEP SCAN: Combines a single exclusion and inclusion list with multiple ID injections to increase
fragments of ions of interest.
These three workflows are flexible and can be used for a variety of E&L analyses, such as different
loads, batches of bio-production bags, tubing, filters, etc., or one subject under different extraction
conditions, applications, and samples.
RESULTS
1. Additives Standard Spiked into Drug Excipients
Leachable studies investigate the possible leachables generated from the interaction of a single-use
system with a bio-production medium, or drug container/packaging with a formulated drug. Leachable
analysis is challenging because of the presence of complex biologic matrices.
In this study, a mixture of common additive standards was spiked into the drug formulation. Results
obtained using traditional data-dependent acquisition (DDA) and AcquireX were compared. The
results indicated that using AcquireX background exclusion yielded selection and fragmentation of
more ions of compounds of interest compared with the DDA method, as shown by Figure 3.
Figure 2. AcquireX Workflows
Figure 3. Additives spiked into drug formulation
Name Structure
DDA
Triggered
MSn
Acquire X
Triggered
MSn
Irganox 1076
X √
1,3,5-trimethyl-
2,4,6-tris(3,5-di-
t-butyl-4-
hydroxybenzyl)-
benzen
√ √
Irgafos 168
√ √
TINUVIN 328
√ √
OctabenzoneX √
Ethylene bis[3,3-
bis[3-(1,1-
dimethylethyl)-4-
hydroxyphenyl]
X X
Irganox 1010
X √
F:\SMurf_EnL_Excipient\01ppm_AX_2\ID_01 05/13/18 17:25:54 ID_01
ID_01 #2043 RT: 8.48 AV: 1 NL: 3.14E6
T: FTMS + p ESI d Full ms2 327.1954@hcd40.00 [100.0000-338.0000]
100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
137.0232
327.1960
215.0704
105.0333
127.1116 249.1487
ID_01 #2046 RT: 8.49 AV: 1 NL: 2.56E4
T: FTMS + p ESI d Full ms3 327.1954@hcd40.00 105.0333@cid30.00 [50.0000-208.0000]
60 70 80 90 100
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
95.0490
105.0446
77.038551.5712102.313887.517466.3666
ID_01 #2044 RT: 8.48 AV: 1 NL: 2.72E4
T: FTMS + p ESI d Full ms3 327.1954@hcd40.00 137.0232@cid30.00 [50.0000-208.0000]
60 80 100 120 140
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
137.0232
81.0333
127.0388110.539762.5711 92.2209
ID_01 #2045 RT: 8.48 AV: 1 NL: 7.76E5
T: FTMS + p ESI d Full ms3 327.1954@hcd40.00 215.0704@cid30.00 [54.0000-226.0000]
60 80 100 120 140 160 180 200 220
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
137.0232
105.0334
215.070170.9625 91.1352 160.2015
F:\SMurf_EnL_Excipient\01ppm_AX_2\ID_01 05/13/18 17:25:54
ID_01 #2366 RT: 9.37 AV: 1 NL: 4.01E6
T: FTMS + p ESI d Full ms2 352.2385@hcd40.00 [100.0000-363.0000]
100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
352.2387
282.1602
212.0818
ID_01 #2368 RT: 9.38 AV: 1 NL: 3.30E5
T: FTMS + p ESI d Full ms3 352.2385@hcd40.00 212.0818@cid30.00 [53.0000-223.0000]
60 80 100 120 140 160 180 200 220
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
212.0818
129.0697 166.0650
183.0679156.0807
139.0539111.0440
147.5103
194.071569.0694
118.6640
216.588477.0745
ID_01 #2367 RT: 9.38 AV: 1 NL: 2.21E6
T: FTMS + p ESI d Full ms3 352.2385@hcd40.00 282.1602@cid30.00 [72.0000-293.0000]
80 100 120 140 160 180 200 220 240 260 280
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
212.0818
282.1601
252.1131 266.1288
184.0757 224.0819120.0556107.0602 166.0653 196.0754135.0805 238.097388.8691
F:\SMurf_EnL_Excipient\01ppm_AX_2\ID_01 05/13/18 17:25:54
ID_01 #2884 RT: 10.76 AV: 1 NL: 8.79E6
T: FTMS + p ESI d Full ms2 663.4540@hcd40.00 [100.0000-674.0000]
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
495.2660
607.3909
439.2033
551.3286
327.0781
383.1408
663.4538
251.0468
147.1167 233.0361
ID_01 #2887 RT: 10.77 AV: 1 NL: 2.66E7
T: FTMS + p ESI d Full ms3 663.4540@hcd40.00 439.2033@cid30.00 [115.0000-450.0000]
150 200 250 300 350 400 450
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
383.1411
327.0783
440.2068147.5109
405.0367
351.0247226.3417 281.2045
ID_01 #2885 RT: 10.77 AV: 1 NL: 2.24E7
T: FTMS + p ESI d Full ms3 663.4540@hcd40.00 495.2660@cid30.00 [131.0000-506.0000]
150 200 250 300 350 400 450 500
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
439.2039
383.1413
495.2668327.0785173.4332 404.8141272.7896215.2791
ID_01 #2891 RT: 10.78 AV: 1 NL: 3.56E4
T: FTMS + p ESI d Full ms3 663.4539@hcd40.00 607.3914@cid30.00 [162.0000-618.0000]
200 300 400 500 600
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
173.4213404.8765
241.5557 442.8021
192.4120
F:\SMurf_EnL_Excipient\10ppb_AX\ID_01 05/13/18 18:47:35 ID_01
ID_01 #2866 RT: 11.77 AV: 1 NL: 5.64E5
T: FTMS + p ESI d Full ms2 548.5038@hcd40.00 [100.0000-559.0000]
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540
m/z
0
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
Inte
nsity
107.0490
149.0596
419.3528
167.0702
219.1743121.0646 205.1224 475.4156
548.4941402.6186181.0864 340.9002143.9137
ID_01 #2868 RT: 11.78 AV: 1 NL: 1.40E5
T: FTMS + p ESI d Full ms3 548.5038@hcd40.00 149.0596@cid30.00 [50.0000-208.0000]
60 80 100 120 140 160 180 200
m/z
0
20000
40000
60000
80000
100000
120000
140000
Inte
nsity
107.0491
121.0647 183.488899.706467.5869
111.0964
197.1934159.9133
ID_01 #2869 RT: 11.78 AV: 1 NL: 5.04E3
T: FTMS + p ESI d Full ms3 548.5038@hcd40.00 419.3528@cid30.00 [110.0000-430.0000]
150 200 250 300 350 400
m/z
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
Inte
nsity
147.5113
403.5081
296.3241168.5112 194.8376
266.1133308.9206
116.2525
F:\SMurf_EnL_Excipient\01ppm_AX\ID_01 05/13/18 04:06:21 ID_01
ID_01 #2602 RT: 9.97 AV: 1 NL: 7.43E5
T: FTMS + p ESI d Full ms2 1194.8204@hcd40.00 [100.0000-1205.0000]
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200
m/z
0
100000
200000
300000
400000
500000
600000
700000
Inte
nsity
219.1743
729.2904563.2279
163.1116
675.3529785.3532
619.2903
841.4155
203.1430
899.6033147.0804307.2632 1194.8199
415.1754471.2380 711.2809527.3001 953.5413767.3437 823.4067 879.4663261.1851
231.1015
351.2899 581.2386 637.3013 1166.4503
ID_01 #2603 RT: 9.97 AV: 1 NL: 9.44E5
T: FTMS + p ESI d Full ms3 1194.8204@hcd40.00 219.1743@cid30.00 [55.0000-230.0000]
100 150 200
m/z
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
Inte
nsity
203.1431
219.1744
121.1011 161.1323 188.119686.4696
ID_01 #2605 RT: 9.98 AV: 1 NL: 1.23E5
T: FTMS + p ESI d Full ms3 1194.8204@hcd40.00 563.2279@cid30.00 [150.0000-574.0000]
150 200 250 300 350 400 450 500 550
m/z
0
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
110000
120000
Inte
nsity
457.1864
249.1122
231.1015 397.1655
213.0912 379.1543
349.1436297.1126
ID_01 #2604 RT: 9.97 AV: 1 NL: 4.63E3
T: FTMS + p ESI d Full ms3 1194.8204@hcd40.00 729.2904@cid30.00 [195.0000-740.0000]
200 300 400 500 600 700
m/z
0
500
1000
1500
2000
2500
3000
3500
4000
4500
Inte
nsity
615.3449
371.1924
434.9255295.5366 516.4063
M
S3
M
S3
F:\SMurf_EnL_Excipient\01ppm_AX_2\ID_01 05/13/18 17:25:54
ID_01 #2774 RT: 10.48 AV: 1 NL: 5.05E6
T: FTMS + p ESI d Full ms2 792.6291@hcd40.00 [100.0000-803.0000]
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
219.1742
569.4362307.2633203.1430
363.2686133.0857 513.3735163.1116 792.6304
ID_01 #2775 RT: 10.48 AV: 1 NL: 6.24E6
T: FTMS + p ESI d Full ms3 792.6291@hcd40.00 219.1742@cid30.00 [55.0000-230.0000]
60 80 100 120 140 160 180 200 220
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
203.1431
219.1744
175.1481121.1010
188.1197
145.101181.0697
ID_01 #2777 RT: 10.48 AV: 1 NL: 1.80E5
T: FTMS + p ESI d Full ms3 792.6291@hcd40.00 307.2633@cid30.00 [79.0000-318.0000]
100 150 200 250 300
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
251.1431
147.5122
236.1198307.2056
93.0697 159.1176 194.9034138.9437
ID_01 #2776 RT: 10.48 AV: 1 NL: 5.61E5
T: FTMS + p ESI d Full ms3 792.6291@hcd40.00 569.4362@cid30.00 [151.0000-580.0000]
200 250 300 350 400 450 500 550
m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
363.2689
513.3738307.2059
251.1433
173.4228404.6198 569.4346
MS2
MS3MS3 MS3
- Seven common additives spiked into complex drug formulation
- Traditional DDA only triggered MSn for three additives
- AcquireX with background exclusion triggered MSn for six additives
- AcquireX triggered MSn for twice as many additives, producing a two-fold increase in
leachable IDs
Figure 4. Positive ion chromatogram of IPA extraction of samples A, B, and C
IPA_Black_ID_01 05/27/18 12:11:37 IPA_Black_ID_01
RT: 0.0 - 23.0
0 2 4 6 8 10 12 14 16 18 20 22
Time (min)
0
500000000
1000000000
1500000000
2000000000
Inte
nsi
ty
0
500000000
1000000000
1500000000
2000000000
Inte
nsi
ty
0
500000000
1000000000
1500000000
2000000000
Inte
nsi
ty
14.5 18.9 21.018.5 19.412.5 21.116.41.0 8.4 11.3 13.99.66.8 7.71.5 3.93.1 4.6 6.2
10.8
18.611.3 20.1
14.913.9 15.713.37.77.1 8.5 17.5 20.219.0 21.010.36.55.44.03.60.7 2.81.1
14.913.9
15.7
13.6
4.410.3 12.811.36.6 16.46.5 8.64.74.1 6.4 20.620.018.616.83.8 20.92.8 9.41.0
NL: 2.00E9
Base Peak F: FTMS + p ESI Full ms [125.0000-1250.0000] MS IPA_White_ID_01
NL: 2.00E9
Base Peak F: FTMS + p ESI Full ms [125.0000-1250.0000] MS IPA_Red_ID_01
NL: 2.00E9
Base Peak F: FTMS + p ESI Full ms [125.0000-1250.0000] MS IPA_Black_ID_01
Sample A
Sample B
Sample C
The constant presence of solvent background often masks the low-abundant additives in the extracts
solution. By using the AcquireX background exclusion workflow, several sulfur-containing additives
were readily identified, which are substances of concern.
IPA_Black_ID_01 IPA_Black_ID_01
IPA_Black_ID_01 #4441 RT: 14.92 AV: 1 NL: 9.96E8
F: FTMS + p ESI Full ms
150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250
m/z
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
448.3634
431.3368199.1693
474.3790392.3737338.3414283.1580 633.8976 950.3265592.8785 667.1442243.5625
IPA_Black_ID_01 #4426 RT: 14.88 AV: 1 NL: 3.33E7
T: FTMS + p ESI d Full ms2 431.3367@hcd30.00 [70.0000-442.0000]
80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440
m/z
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
199.1692171.1379
95.085485.1011 109.1011 127.1117 155.1427 189.4347 317.4189207.0167
IPA_Black_ID_01 #4544 RT: 15.24 AV: 1 NL: 4.04E4
T: FTMS + p ESI d Full ms3 431.3369@hcd30.00 171.1378@cid30.00 [50.0000-182.0000]
60 80 100 120 140 160 180
m/z
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
57.0696
109.1008 171.1378
67.0539
73.0280147.5304127.111391.9066 177.6199
IPA_Black_ID_01 #4428 RT: 14.88 AV: 1 NL: 7.45E6
T: FTMS + p ESI d Full ms3 431.3367@hcd30.00 199.1692@cid30.00 [50.0000-210.0000]
60 80 100 120 140 160 180 200
m/z
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
199.1690
95.0850
81.0694
71.0852
137.1320 155.142757.0696 97.1006 120.5772
Figure 5. MSn Spectra of identified additive
DATA PROCESSING
Compound Discoverer 3.0 software and Mass Frontier 8.0 software were used for data processing
and structure characterization.
Figure 6. Compound Discoverer 3.0 software processing workflow
Figure 7. Compound Discoverer 3.0 software processing result
(M+H)+
MS2
MS3 MS3
(M+NH4)+
(M+Na)+O
OO
O
O
O
Figure 8. Mass Frontier 8.0 software – Ion tree for structure elucidation and library generation
File MS1 #4491
453.3187
448.3634
338.3418199.1693
File MS2 #4474
199.1693171.1379
File MS3 #4476
199.1690
95.0851
137.1321
File MS3 #4544
57.0696
109.1008
171.1378
67.0539 147.5304
S
N
NHS
N
NH
N
S
NHN
S
NH
O
N
NH
Figure 9. Partial list of compounds identified containing sulfur
PO65277-EN 0518S