solution for extractables & leachables analysis · • chapter 87,88 (materials direct or...
TRANSCRIPT
The world leader in serving science
Kate Comstock Senior Marketing specialist Small molecule and E&L analysis Pharma/Biopharma Marketing, CMD
Solution for Extractables & Leachables Analysis - Instruments, Software, Database/Spectral Library
2
• Introduction • Definitions • Industries need Extractable & Leachable (E&L) analysis • Regulatory and industry groups
• Thermo Fisher Scientific solution for extractable & leachable analysis • Multi instrumentations and technologies • Data Analysis software for ID, structure elucidation, and quantitation • Database and spectral library
Outline
3
Plastics – A Complex Process
Courtesy of Doug Kiehl, Eli Lilly
4
The Broad Market of Extractable & Leachable Analysis
Medicine container/packaging, implant and diagnostic devices
Single-use systems for bioproduction and storage
Food package Wearable Consumer Electronic Products Printing ink and adhesives
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Food Contact Material Migration Test is Mandatory! This image cannot currently be displayed.
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Wearable Consumer Electronic Products
The overall market for wearable technology is expected to reach USD 31.27 Billion by 2020, at a CAGR of 17.8% between 2015 and 2020.
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• Extractable (E) • Compounds that migrate from the surface under more aggressive conditions
of exposure (solvent, time, and temperature). Controlled extraction study
• Leachable (L) • Compounds that migrate from the contact surface to drug formulation under
normal conditions of exposure. Formulated drug or simulants
Definitions
Extractables
Leachables
Additives Impurities Polymer Components Degradation Products Solvent-material Interactions
Known extractables Extractable – drug formulation interactions Extractable modified by process conditions
Material Safety Product Quality
Some leachables are subset of extractable, but not always the case.
Safety/risk assessment Submission to regulatory agency Market
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Safety • Extractables and Leachables are Chemical Impurities, they could contaminate medicine, food,
and drink, affect product quality, and cause adverse effects for consumers.
Regulations • E&L analyses are highly regulated and required for market approval of new products. • More regulations have been made along the way and will continue into the future.
E&L analyses are challenging and complex • It requires multiple instruments, expertise, data processing software, and database. • It needs both identification and quantitation. Unknown structure elucidation is challenging. • It must start at early stage to be ready for product launch.
The Importance and Challenges of E&L Analyses
9
E&L Regulatory and Method landscape
Industry Groups
National Regulators
Methods & Advisory Bodies
USP 1663 & 1664 665&1665 (drift)
Standardized Extractables Testing Protocol. Pharmaceutical Engineering 34 (2014)
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• FDA guidance • Container Closure Systems for Packaging Human Drugs and Biologics (May 1999) • 21 CFR 211.94
• EMA guideline • Guideline on plastic media packaging, EMEA/205/04
• ICH guidelines • Test procedures and acceptance criteria for new drug substances and drug products (Q6A,B) • Pharmaceutical development (ICH Q8)
• PQRI leachables and extractables working group (recommendations to the FDA) Safety thresholds and best practices for extractables & leachables in orally inhaled and nasal drugs
• USP Chapters • Chapter 87,88 (materials direct or indirect in contact with patients biological reactivity test in vitro, in vivo) • Chapter 661 (Containers – Plastics) • Chapter 1663 Extractables Associated with Pharmaceutical Packaging/Delivery Systems • Chapter 1664 Drug Product Leachables Associated with Pharmaceutical Packaging Delivery Systems • Chapter 1665_draft Plastic Components and Systems Used to Manufacture Pharmaceutical Drug Products.
• ISO 10993 parts (medical devices) • Japan, China, and other countries healthy authorities...
Regulations & Guidelines
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Industry Group: BioPhorum Operations Group (BPOG) The Global Community
http://www.biophorum.com/
BPOG members are leading biopharmaceutical companies around the world to create an environment where the global biopharmaceutical industry can collaborate and accelerate their rate of progress, for the benefit of all. Many Technical Resources on their website http://www.biophorum.com/
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Industry Group: Bio-Process Systems Alliance BPSA Published Technical Guides
http://bpsalliance.org/
BPSA members are industry-led corporate member of single use manufactures. BPSA is encouraging and accelerating the adoption technologies used in the production of biopharmaceuticals and vaccines. Many Technical Guides on their website http://bpsalliance.org/
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Extractable & Leachable Analysis
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• ICP-MS • ICP-OES
• ESI & APCI • LC-UV or CAD • LC-MS/MS • LC-HRMS • Library
• GC-MS • GC-HRMS • EI & CI • Library
• GC-MS • GC-HRMS • Headspace • EI & CI • Library
Volatile Impurities
Semi-Volatile Impurities
Elemental Impurities
Non-Volatile
Impurities
Analysis of Extractables & Leachables
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Thermo Scientific Instruments and Software for Impurity ID and E&L Analysis
ASE
ICPMS
Benchtop HRMS Q Exactive
Charged Aerosol Detector
GCMS-Triple Quad GCMS -Single Quad
Software & Database
FTIR
IC
Hybrid HRMS
U3000 UHPLC
Orbitrap Fusion Tribrid MS
Q Exactive GC-MS/MS
Vanquish UHPLC
Triple Quad
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GC-MS – Volatile and Semi-Volatile
Thermo Scientific™ TSQ™ 8000 Evo Triple Quadrupole GC-MS/MS
Thermo Scientific™ Q Exactive™ GC Orbitrap GC-MS/MS and Thermo Scientific™ TRACE™ 1310 GC
Thermo Scientific™ ISQ™ Single Quadrupole GC-MS/MS
Solid Phase Microextraction (SPME)
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Thermo Scientific Q Exactive MS Models for E&L Analysis
Thermo Scientific™ Q Exactive Plus MS
Thermo Scientific™ Q Exactive™ MS
Thermo Scientific™ Q Exactive™ Focus
Resolving Power: up to 280K @ m/z 200 Scan Range: 50-6,000
Resolving Power: 70K @ m/z 200 Scan Range: m/z50-2,000
Resolving Power: 140K @ m/z 200 Scan Range: 50-6,000
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Elemental Analysis
Thermo Scientific™ iCAP™ RQ ICP-MS Series
Thermo Scientific™ iCAP™ 7000 Plus Series ICP-OES
Thermo Scientific™ iCAP Q™ ICP-MS
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Thermo Scientific iCAP RQ ICP-MS and Thermo Scientific Qtegra Software
Extract Automated dilution and standard preparation
ICP-MS Compliant data processing
Fully compliant Thermo Scientific™ Qtegra™ ISDS software Full USP 233 & ICH Q3D method capabilities The current compliance deadline for USP 232 and 233 is January 1, 2018
USP <232>/<233>: Be Prepared for the Changes to Come
Thermo Scientific™ iCAP™ RQ ICP-MS Series
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Data Analysis Software, Database and Spectral Library for E&L Analysis
Thermo Scientific™ Compound Discoverer™ 2.1
Thermo Scientific™ TraceFinderTM targeted screening and Quan
Thermo Scientific™ mzVault 2.0 Library Search and Manager
TM
HighChem LLC E&L Compound Database
Thermo Scientific™Mass Frontier™ spectral interpretation software
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High Resolution Mass Spectrometer for E&L Analysis
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The Power of High Resolution
222.1 222.2
222.1253
222.1463
222.1838
Carbofuran
17.5 K
222.1 222.2
222.1124
222.1467
222.1843
222.1 222.20 0
222.1129
222.1465
222.1854
70 K 35 K
222.1237
222.1 222.20
222.1126
222.1466
140 K
Name Molecular Formula [M+H]+
Carbofuran C12H15NO3 222.1125
Formetanate C11H15N3O2 222.1237
O NHNHN
O
O
O O
NH
222.1238 222.1238
Carbofuran
Formetanate
Formetanate
not able to separate baseline separated partially separated completely baseline separated
∆M= 0.0109 Da
0.9 ppm
0.1 ppm
23
292.0 292.5 293.0 293.5 294.0 294.5 295.0 295.5 296.0 296.5m/z
0
20
40
60
80
1000
20
40
60
80
1000
20
40
60
80
1000
20
40
60
80
1000
20
40
60
80
100292.04031
293.04327 294.03862 295.04121 296.04098292.04031
293.04327294.03622 295.03952 296.04098
292.04031
293.04327 294.03612 295.03958 296.04127292.04031
293.04368 294.03610 295.03948 296.04035292.04031
293.04368 294.03610 295.03947 296.04035
Very High Resolution Shows Isotope Fine Structure
A1 A2 A0 A1 A2
Parathion C10H15O5NPS
[M+H]+ = 292.04031
35K
75K
140K
240K
500K
15N13C
34S 18O
213C
13C33S
2H
13C
33S 15N 2H13C
OPO
NO
O
S
O
24
• Mass Accuracy
• Isotopic Pattern Fidelity
• MSn Capabilities
MS Requirements for Characterization of Unknowns
Orbitrap MS High Resolution Accurate Mass (HRAM) data enable confident Component identification Elemental composition determination Structure characterization HR Quantitation
Instrument of Choice: Thermo Scientific™ Orbitrap™ MS
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Thermo Scientific Q Exactive MS - First Benchtop Orbitrap Mass Spectrometer
• Bench top • Tune Wizard • Method templates • Drag and drop • Robust • Plug and play
• Resolving power • Sensitivity • Acquisition speed • Mass accuracy • Dynamic range • Rapid polarity switching • Multiple ionization modes
Easy to use High Performance
26
Thermo Scientific Q Exactive MS Schematics
Hyperbolic Quadrupole Mass filter
HCD Cell
Orbitrap Mass Analyzer
C-Trap
RF-Lens
27
Thermo Scientific Orbitrap MS – Principle of Operation
1. Ions are injected through the source 2. …and trapped in the C-trap and squeezed into a smaller cloud 3. …then a voltage pulse across C-trap ejects ions towards the Orbitrap 4. …where they are trapped and detected
Collision cell C-Trap Octapole Split Lens
Flatapole
Skimmer
Tubelens
Capillary Orbitrap
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Thermo Scientific Q Exactive MS Specifications
• Max Resolution • 140,000 at m/z 200
• Max Scan Speed • Up to 12 Hz (at 17,500)
• Mass Accuracy • < 3 ppm external • < 1 ppm internal
• Mass Range: m/z 50 – 6,000 • Intra-scan Dynamic Range: > 5000:1 • Sensitivity
• Full MS: 500 fg Buspirone on column S/N 100:1 • SIM: 50 fg Buspirone on column S/N 100:1
• Polarity Switching • One full cycle in < 1 sec (one full scan positive mode and one full scan
negative mode at resolution setting of 35,000)
Resolution at m/z 200
Max. Scan Speed (Hz)
17,500 12 35,000 7 70,000 3 140,000 1.5
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Scan-to-Scan Mass Accuracy | Carbaryl, C12H11NO2
RMS = 0.62 ppm or 620 ppb across the peak
Resolution @ 70k 5 sec base peak
18 Pos scans
2.00 2.02 2.04 2.06 2.08 2.10 2.12 2.14 Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
0.84 ppm
0.62 ppm 0.62 ppm
0.69 ppm
0.84 ppm
0.62 ppm
0.47 ppm
0.77 ppm
0.54 ppm
0.32 ppm
0.24 ppm 0.16 ppm
0.54 ppm
0.69 ppm
0.69 ppm
0.69 ppm
0.24 ppm
0.84 ppm
Scan-to-Scan Mass Accuracy
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BPOG is Recommending Accurate MS for E&L Studies
31
Additives and Their Unknown Degradants are Common Extractables
O
O
O
O
O
O
O
O
O
O
O
O
OO
O
O
OH
OH
OH
OH
O
OH O
OS
O
O O
OH OH
N2H
O
N
NN
OH
O
O O
O
O
O
OO
OH
OH
OH
OH
OH
N
N
N
O
O
O
OH
OH
OH
O
OH
O
O
OH
OP
O
O
Antioxidant, plasticizer, slipping agent, light stabilizer… also monomer, oligomer.
32
RESTEK-STD-500ppb-APCI-MeOH-1 02/02/16 19:09:40APCI Accucore C18 2.1X100 2.6 um A: H2O/0.05% HAc/5mMNH4Ac B: MeOHRT: 8.4 - 35.6
10 12 14 16 18 20 22 24 26 28 30 32 34Time (min)
0
50
1000
50
1000
50
10027.1
27.1
27.022.7 27.325.3 28.326.5 29.5 30.724.321.48.5 34.69.5 16.69.8 10.4 16.111.2 22.712.0 17.712.9 13.5 14.5 30.820.4 33.719.0 32.9
27.1
27.1
27.3 28.3 29.5 30.724.321.4 26.122.716.616.1 17.7 24.620.4 33.713.8 31.115.2 19.0 34.623.8 32.98.6 13.010.6 12.411.810.222.7 27.2
25.3 26.5
8.5 34.68.7 9.4 10.3 11.2 12.1 30.713.4 14.0 25.614.8 23.921.916.1 29.524.416.5 17.3 21.418.0 19.0 29.328.019.6 33.731.7 32.3 35.2
NL: 3.49E8Base Peak MS RESTEK-STD-500ppb-APCI-MeOH-1
NL: 3.49E8Base Peak F: FTMS + p APCI corona Full ms [120.00-1600.00] MS RESTEK-STD-500ppb-APCI-MeOH-1
NL: 3.59E7Base Peak F: FTMS - p APCI corona Full ms [120.00-1600.00] MS RESTEK-STD-500ppb-APCI-MeOH-1
RESTEK-STD-500ppb-APCI-MeOH-1 #5755 RT: 30.68 AV: 1 NL: 1.52E7T: FTMS + p APCI corona Full ms [120.00-1600.00]
540 560 580 600 620 640 660 680 700 720 740 760 780m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
663.4532C 42 H64 O 4 P-0.6426 ppm
664.4565
665.4599607.3909608.3942533.5290 551.5036 666.4629575.5032
Irgafos 168 (Oxidation Product )
OP
O
O OP
O
O
O
(M+H)+
C42H63O3P (M+H)+ 647.45876
C42H63O4P (M+H)+ 663.4537
MS BP (+)
MS BP (-)
33
Irgafos 168 Degradant ID by Full Scan (+/-) and MS/MS Spectra
200 250 300 350 400 450 500 550 600 650 700 750 m/z
0 20 40
80 100
475.2969
519.2610 497.2787
540.5350 359.3151
200 250 300 350 400 450 500 550 600 650 700 750 m/z
0 20 40 60 80
100 473.2826
541.2703 609.2575
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 m/z
0 20 40 60 80
100 363.1713
251.0465
307.1089
57.0708 419.2344 95.0497 153.0697 233.0371 175.0153 73.0284
50 63 75 88 100 113 125 138 150 163 175 188 200 213 225 238 250 263 275 288 300 313 325 338 350 363 375 388 400 413 425 438 450 463 475
0
10
20
30
40
50
60
70
80
90
100363.1712
251.0463
307.1088
419.2340
57.0708
364.1755
308.1128252.0505153.0698
475.2978420.2371175.015695.0496 233.0350 337.5264282.2958
OPOH
OO
H
OHPOOH
O
O P OOH
O H
O P OOH
O H
OPOH
O
O H
OPOOH
O
C28H43O4P (M+H)+ 475.2971 (M-H)- 473.2826
FISh and auto annotation for structure elucidation of Irgafos 168 Degradant
Full Scan (+)
Full Scan (-)
MS/MS (+)
34
Quantitation of Octabenzone on Thermo Scientific Q Exactive Focus MS
327Y = -32228.1+812672*X R^2 = 0.9992 W: 1/X
0 200 400 600 800 1000ppb
0
100000000
200000000
300000000
400000000
500000000
600000000
700000000
800000000A
rea
327Y = -32228.1+812672*X R^2 = 0.9992 W: 1/X
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6ppb
0
200000
400000
600000
800000
1000000
1200000
Are
a
O O
OH
Octabenzone Cas# 1843-05-6 C21H26O3 (M+H)+ 327.18092
35
Quantitation of Tinuvin 328 on Thermo Scientific Q Exactive Focus MS
352 Y = 59802.9+851108*X R^2 = 0.9999 W: Equal
0 200 400 600 800 1000 ppb
0
100000000
200000000
300000000
400000000
500000000
600000000
700000000
800000000
900000000 A
rea
352Y = 59802.9+851108*X R^2 = 0.9999 W: Equal
0 2 4 6 8 10 12ppb
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
10000000
Are
a
N
NN
OH
Tinuvin 328 Cas# 21615-49-6 C22H29N3O (M+H)+ 352.23834
36
LC-HRMS Analysis for IV Bag Extractables
UHPLC-HRMS Thermo Scientific™
Compound Discoverer™ 2.1
Software
Result
Thermo Scientific™ Q Exactive™ Plus MS and Thermo Scientific™ Vanquish UHPLC
Extractables Identified . _________ . _________ . _________ . _________
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lativ
e A
bu
nd
an
ce
9.2
6.18.1
6.1
10.1 12.113.211.010.5 13.8 15.88.4
12.96.58.87.3 10.0 11.53.5 14.47.86.9
Urine-8-12hr-1 #4384 RT: 8.74 AV: 1 NL: 8.12E6F: FTMS + p ESI Full ms [120.0000-1200.0000]
150 200 250 300 350 400 450 500 550 600 650 700 750 800 850m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lativ
e A
bu
nd
an
ce
595.3498
310.2016
591.3188596.3531
311.2049267.1746219.0477 597.3562198.1280
E&L Mass List
HRAM data
37
General Workflow for Small Molecule Impurity ID and E&L Analysis
UHPLC-HRMS
Thermo Scientific™ Compound
Discoverer ™2.1 Software
Result
Impurities Identified . _________ . _________ . _________ . _________
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Time (min)
0
10
20
30
40
50
60
70
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90
100
Re
lativ
e A
bu
nd
an
ce
9.2
6.18.1
6.1
10.1 12.113.211.010.5 13.8 15.88.4
12.96.58.87.3 10.0 11.53.5 14.47.86.9
Urine-8-12hr-1 #4384 RT: 8.74 AV: 1 NL: 8.12E6F: FTMS + p ESI Full ms [120.0000-1200.0000]
150 200 250 300 350 400 450 500 550 600 650 700 750 800 850m/z
0
10
20
30
40
50
60
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80
90
100
Re
lativ
e A
bu
nd
an
ce
595.3498
310.2016
591.3188596.3531
311.2049267.1746219.0477 597.3562198.1280
Custom Database Library
HRAM data
General Workflow for API Impurity and Extractable & Leachable Analysis
38
• HRAM untargeted screening with polarity switching • ESI/APCI full scan MS and data-dependent top 3 MS/MS data with polarity switching
using 70K &17.5K resolution for FMS and HCD MS2 respectively. Stepped NCE: 30, 45, 60.
MS Method: High Resolution Accurate Mass Untargeted Screening
Positive Negative
39
IV Bag IPA-H2O Extract - MS Base Peak Chromatogram and UV
D:\SGS-E&L\SGS-IVBag\IPA_H2O_Extract_2 Accucore C18 2.1x100 2.6 um A: H2O/0.05% HAc/5 mM NH4AC B:ACN/0.05% HAc/5mM NH4Ac
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 Time (min)
0
10000
20000
30000
40000
50000
uAU
Rel
ativ
e A
bund
ance
22.7
20.9 22.9 13.1 22.7
24.0 25.3 30.9 22.1 17.6 27.2
19.4 28.3 17.4 15.4 18.7 15.1 9.9 10.8 28.5 32.5 33.7 9.8 16.4 34.0 4.8 9.0 6.0 4.1 3.2 1.6
21.0 18.0
25.3
15.4 28.1 22.4 19.7 15.1 31.8 36.2 33.6 30.9 10.4 17.7 10.9 9.8 8.6 7.1 1.1 6.1 4.5 3.8
13.1 31.5 24.0 30.9 34.7 33.5 30.0 25.3 22.9 20.8 19.7 19.3 17.9 16.6 12.6 11.4 8.8 8.0 2.0 6.9 3.8 5.7
NL: 1.00E9 Base Peak F: FTMS + p ESI Full ms MS IPA_H2O_Extrac t_2
NL: 1.00E9 Base Peak F: FTMS - p ESI Full ms MS IPA_H2O_Extrac t_2
NL: 5.00E4 Total Scan PDA IPA_H2O_Extrac t_2
X = present in the blank
X X
MS Base Peak (+)
MS Base Peak (-)
UV 200-400 nm
Zoom-in View
40
Full Scan and HCD MS/MS Data for Component ID and Structure Elucidation
G:\Old Passport\...\IPA_H2O_Extract_1 01/18/16 07:08:13Accucore C18 2.1x100 2.6 um A: H2O/0.05% HAc/5 mM NH4AC B:ACN/0.05% HAc/5mM NH4Ac C24 H39 O6: C24 H39 O6 pa Chrg 1
200 300 400 500 600 700 800m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
422.2899
405.2634
427.2451
149.0233
463.3162321.2057279.1589
343.1877
C24 H39 O6: C24 H39 O6 pa Chrg 1
200 300 400 500 600 700 800m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
405.2634
149.0234
427.2452
463.3163
389.2683335.2213
O
OO
O
O
50 63 75 88 100 113 125 138 150 163 175 188 200 213 225 238 250 263 275 288 300 313 325 338 350 363 375 388 400
0
13
25
38
50
63
75
88
100149.02341
57.0708371.08633 109.10157
83.08629 167.03340 276.19998202.07068
O
OH
O
OH
O
OH
O
OH
O
O
50 63 75 88 100 113 125 138 150 163 175 188 200 213 225 238 250 263 275 288 300 313 325 338 350 363 375 388 400
0
13
25
38
50
63
75
88
100149.02348
109.10166
127.11199
71.08636
83.08618
180.52640 256.29141
OH
O
OH
O
O
OHO
OHO
O
OO
O
O
O
(M+H)+
(M+H)+
422.2898
(M+NH4)+
(M+NH4)+
(M+Na)+
(M+Na)+
Full Scan Full Scan
HCD MS/MS HCD MS/MS
41
Pos/Neg Switching Maintain High Mass Accurate Mass and Fine Isotope Pattern
200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 m/z
0
50
100
Rel
ativ
e Ab
unda
nce
251.1214 C 13 H 19 O N 2 S 0.7336 ppm
253.1169 233.1108
200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 m/z
0
50
100
Rel
ativ
e Ab
unda
nce
249.1066 C 13 H 17 O N 2 S -0.3776 ppm
304.9139 251.1023
253.11 253.12 253.13 m/z
253.1171
253.1272
253.1169
253.1272
Experimental
Theoretical
A+2 isotope pattern show separation of 34S and 213C at 70K
A+2
S
NNH
OH
Proposed Structure From O-Ring WFI Extract
42
Ultra High Resolution MS Reveals Fine Isotope Structure
Accurate Mass
High Isotope Fidelity
MS/MS Spectrum
Fine Isotope Structure
HRAM Data
Structure Elucidation ------------ ------------ ------------ ------------
mzCloud
ChemSpider
Thermo E&L DB
CD 2.1 Result - Predicted Compositions for a unknown component in cosmetic product IPA/H2O extract
Thermo Scientific™ Fusion™ Lumos™ Tribrid™ MS 1 Million Resolution
#6 and #7
43
A2 Isotope Fine Structure Validates Correct Elemental Composition
549.240 549.245 549.250 549.255 549.260m/z
0
10
20
30
40
50
60
70
80
90
1000
10
20
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40
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100
Rel
ativ
e A
bund
ance
549.2536549.2429
549.2515
549.2475549.2500
549.2564
549.2536
549.2511549.2473
549.2565549.2409 549.2594
549.240 549.245 549.250 549.255m/z
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
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100
Rel
ativ
e Ab
unda
nce
549.2536549.2429
549.2515
549.2475 549.2500 549.2564
549.2430 549.2540
549.2515
549.2500549.2476 549.2569549.2412
Experimental Experimental
Simulation C30H32N3O4P
Simulation C28H35NO7S
The correct formula matches the ana fine isotope structure.
15N13C
18O
33S13C
The incorrect formula doesn’t match the result.
#7 #6
The above results demonstrate that the fine isotope structure made available by ultra high resolution mass spectrometer greatly enhances the ability to confidently determine the correct unknown elemental composition.
44
mzCloud - HR Mass Spectral Database
https://www.mzcloud.org
mzCloudTM - Advanced high resolution mass spectral database (Free) - Very high quality data: standardized acquisition, highly curated data - Search through spectrum, structure, substructure, m/z, … - Identifying compounds even when they are not present in the library through substructure search
Free and cloud-based
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mzCloud Spectral Library
Spectral tree Reference library
Search by different terms
Metadata available for selected library entry
Recalibrated spectra fragment ions annotation
Permanent citable links for entry, tree, and spectra
Breakdown curves
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Thermo Scientific mzVault 2.0
Exact Mass-Enabled Library Search and Management - local mzCloud - Creating customer library - Searching library
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E&L Compound Database the “E&L Mass List” in CD 2.1
This Excel sheet database contains ~2000 common E&L related compounds. This is a “living document” and new E&L related compounds are added periodically.
This database has been added to Thermo Scientific™ Compound Discoverer™ as “E&L Mass List”.
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Thermo Scientific Compound Discoverer 2.1 Software for Compound ID and Structure Elucidation
Component extraction and elemental composition prediction • HRAM data • Isotope pattern • fine isotope structure • MS/MS fragments
Unknown identification through multiple databases searching in parallel Known unknown compound ID
Unknown compound structure elucidation using “Structure Proposals” • Predicted Compositions • MS/MS fragment ions • The similarity search identify the fragment ions or substructures in mzCloud, which facilitated unknown structure elucidation.
De Novo ID Most Challenge Task Need Chemistry knowledge and expertise
Validity check of the proposed structure • using “FISh Scoring” feature by searching the embedded “HighChem Fragmentation Library”.
Differential Analysis for different lots, batch, or type samples analyses.
FISh stands for “Fragment Ion Search”
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Thermo Scientific Compound Discoverer 2.1 Software Study and Analysis Wizard
Creating a study and analysis use the guided “New Study and Analysis wizard”
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Pharma Label Extractable Study - Nodes-based Processing Workflow
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Result View – Data Interpretation
sub-tables
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469.32846 472.24991462.14740464.28354
479.31470461.28552460.28329 478.31226 482.26532
477.31000459.27982[M+H]+1481.26184[M+Na]+1
476.30664[M+NH4]+1
460 465 470 475 480 485m/z
0
10
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30
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50
Inte
nsity
[cou
nts]
(10^
6)
LF_IPA_Extrac-1-PN-1 (F2) #1230, RT=4.258 min, MS1, FTMS (+) C20 H42 O11 as [M+NH4]+1
Component Detection with Adducts Grouping
Resolution aware Mass accuracy Isotope pattern are color coded.
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Result Filters - Flexible Result Review
Filtering the result based on your need
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Multiple Database Searching in Parallel to Identify Known Unknowns
Mirror plot for each mzVault hit: observed MS2 vs. library MS2 • Matches collision energy between raw file MS/MS and reference MS/MS
Sub table show detailed info for compound
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Add Chemspider Search Result to “Structure Proposal and Apply FISh” for Unknown ID
FISh stands for “Fragment Ion Search”
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FISh Scoring Result: Fragment Ion Matching and Fragment Structure Annotation
95.08617C7 H11 [M-e]+1
225.23193C14 H29 N2 [M-e]+1309.3262
71.08630C5 H11 [M-e]+1
57.07080C4 H9 [M-e]+1
310.31003C20 H40 N O [M-e]+1226.21648C14 H28 N O [M-e]+1
282.27905C18 H36 N O [M-e]+1
254.24771C16 H32 N O [M-e]+1
100 200 300 400 500m/z
0.0
0.5
1.0
1.5
2.0In
tens
ity [c
ount
s] (1
0^6)
IPA_H2O_Extract_2 (F1) #9364, RT=35.375 min, MS2, FTMS (+), (HCD, DDF, 509.5041@(30;45;60), +1) N,N'-1,8-Octanediyldidodecanamide, C32 H64 N2 O2 FISh Co NH
NHO
O
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Unknown Structure Elucidation Based on CD 2.1 “Predicted Compositions”
226.18013222.14873224.18520 225.19609
223.00858223.15370
222.12064226.11403225.18463
225.14859223.16905
225.12189
226.12236222.96649 225.11076224.11839
223.11507[M+H]+1
223 224 225 226m/z
0
2
4
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8
10
Inte
nsity
[cou
nts]
(10^
6) IPA_H2O_Extract_2 (F1) #2428, RT=10.245 min, MS1, FTMS (+) C13 H18 O S as [M+H]+1
225.12189
225.11076
225.09 225.10 225.11 225.12 225.13 225.14m/z
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Inte
nsity [
co
un
ts]
(10
^3
)
IPA_H2O_Extract_2 (F1) #2428, RT=10.245 min, MS1, FTMS (+) C13 H18 O S as [M+H]+1
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Unknown Structure Elucidation Using Compound Annotation Editor and FISh Scoring
Propose structure based on “Predicted Composition” and MS/MS fragments
Apply “FISh Scoring” to search the embedded “HighChem Fragment Library”
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FISh Scoring Result: Fragment Ion Matching and Fragment Structure Annotation
224.11726
206.10915139.05728
105.07021107.0858664.97821
161.04135C10 H9 S [M-e]+1175.0573
73.06555C4 H9 O [M-e]+1205.10481C13 H17 S [M-e]+1
151.05766C9 H11 S [M-e]+1
162.04982C10 H10 S [M-e]+1
223.11511C13 H19 O S [
177.07326C11 H13 S [M-e]+1
163.05760C10 H11 S [M-e]+1
50 100 150 200m/z
0
100
200
300
400
500
600
700
800
Inte
nsity
[cou
nts]
(10^
3)
IPA_H2O_Extract_2 (F1) #2429, RT=10.250 min, MS2, FTMS (+), (HCD, DDF, 223.1151@(30;45;60), +1) C13 H18 O S FISh Coverage: 9 D
S
O
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Specialized Traces
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Specialized Traces Provide Additional Information
DAD Signal Overlay
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CD 2.1 Differential Analysis Feature: Volcano Plot
Using the interactive volcano plot to find compounds that are significantly different between two sample groups
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Thermo Scientific Compound Discoverer Software Home Page http://mycompounddiscoverer.com
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• Plasticizers • Flame Retardants • Impact Modifiers • Antioxidants • Antimicrobials • UV Stabilizers • Colorants • Lubricants & processing aids
Some of the Common Additives