optimized workflow for structure elucidation of pharmaceutically ...€¦ · seema sharma 1, kate...

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 [email protected] [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 [email protected] [email protected] [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


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


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


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


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


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


ID_01 #2884 RT: 10.76 AV: 1 NL: 8.79E6


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


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


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


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


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


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


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


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


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


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


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


ID_01 #2774 RT: 10.48 AV: 1 NL: 5.05E6


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


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


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


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



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



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



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

optimized workflow for structure elucidation of pharmaceutically ...€¦ · seema sharma 1, kate...

Documents