Search for What's Missing: Unknown Compound ...€¦ · −Radio-labeled (13. C, 14. C, 3. H) •...
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The world leader in serving science Search for What’s Missing: Unknown Compound Characterization Using LC-MS Kate Comstock Sr. Marketing Specialist, Pharma/Biopharma Marketing
Transcript of Search for What's Missing: Unknown Compound ...€¦ · −Radio-labeled (13. C, 14. C, 3. H) •...
The world leader in serving science
Search for What’s Missing: Unknown Compound Characterization Using LC-MSKate ComstockSr. Marketing Specialist, Pharma/Biopharma Marketing
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Small molecule structure analysis encompasses broad applications:Pharmaceutical, metabolomics, food & environmental, clinical, forensic, industrial chemical, etc.
The following are small molecule structure analyses and are crucial for pharma R&D.• Impurity analysis
− Drug substance (API)− Drug product− Genotoxic− Stability studies - degradants
• Met ID− Drug discovery – pre-clinical development – clinical development − In vitro (hepatocytes, microsomes, whole blood, plasma)− In vivo (whole blood, plasma, urine, bile, fecal homogenates)− Radio-labeled (13C, 14C, 3H)
• E&L analysis− Extractables− Leachables
• Natural products & traditional medicines research− Discovery and Identify new medicine
Small Molecule Structure Analysis
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• Broad range of chemicals with very diverse structures• ChemSpider has 71 M chemical structures.
• Background Interference• Complex biologic matrices, excipients, and solvent background
• Sample Limitations • Example: ADC drug Met ID, the small molecule “warhead” is only small portion of the drug.
• Unknowns• De Novo structural determination is not trivial!
• Many small molecule structure analyses are highly regulated.• Pharma R&D must follow EPA, FDA, EMEA, countries' regulations and guidelines: compliance and GLP.• Toxicity assessments, clinic trails... to ensure drug efficacy and consumer safety
Small Molecule Structure Analyses are Challenging and Complex
All decisions must be based on solid scientific analysis results.High Quality Data is Vital!
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Reference: Mass Spectrometry Identification Categories in USP Chapter <1663>
Data typically available from GC/MS and LC/MS analyses (see A through E below) are used to designate individual extractables identifications in the categories of Confirmed, Confident, or TentativeA. Mass spectrometric fragmentation behaviorB. Confirmation of molecular weightC. Confirmation of elemental compositionD. Mass spectrum matches automated library or literature spectrumE. Mass spectrum and chromatographic retention index match authentic reference compound
Confirmed - A Confirmed identification means that A, B (or C), and D (or E) have been fulfilled. Confident - A Confident identification means that sufficient data to preclude all but the most closely related
structures have been obtained. The combination of D with any of A, B, or C can be used to provide a confident identification
Tentative - A Tentative identification means that data have been obtained that are consistent with a class of molecule only.
Confident Structure ID Requires High Resolution Accurate Mass (HRAM) and MSn Fragments
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Small Molecule Structure Identification
The Instrument of Choice: Orbitrap™ Mass Spectrometer
AccurateMass
The Three Pillars for Small Molecule Structure ID
CONFIDENT STRUCTURE ID
Isotopic pattern fidelity
MS/MS & MSn
Spectra
Compound FingerprintElemental Composition
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SS_calmix_5e4_1M_stdpress #2 RT: 0.07 AV: 1 NL: 2.82E7T: FTMS + p ESI Full ms [150.0000-2000.0000]
524.2 524.4 524.6 524.8 525.0 525.2 525.4 525.6 525.8 526.0 526.2 526.4m/z
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524.2648R=665307
525.2682R=621002
526.2607R=624402
525.8954R=647302524.4520
R=597302524.8989R=624000
526.4311R=477100
The Power of High Resolution MS: Fine Isotope Structure MRFASS_calmix_5e4_1M_stdpress #2 RT: 0.07 AV: 1 NL: 1.25E6T: FTMS + p ESI Full ms [150.0000-2000.0000]
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34S
213C
15N 13C
18O
MRFA
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Ultra High Resolution for Unknown Impurity ID
524.0 524.5 525.0 525.5 526.0 526.5 527.0 527.5m/z
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524.2074R=327537
525.2107R=329117
524.2074R=624278
525.2108R=627302
524.2074R=999105
525.2108R=998597
526.2032R=997988
527.2066R=998143
526.20 526.21 526.22m/z
526.2032
526.2141
526.2075
526.2115
526.2169526.1999
526.2033
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526.2117526.2078
526.2171526.2032
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526.2117526.2078
526.2171526.2015
500-K Res.
1-M Res.
1-M Simulation 18O
34S
2H13C
213C
15N13C
33S13C
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Orbitrap MS for Small Molecule Structure Analysis
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Thermo Scientific LTQ Orbitrap MS – First Generation Hybrid MS
Since 2005 ASMS introduction, Orbitrap MS has become the gold standard for small molecular structure analysis.
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Thermo Scientific Q Exactive MS Family – Quan/Qual Workhorse
Thermo Scientific™ Q Exactive™ Plus MS
Thermo Scientific™ Q Exactive ™ MS
Thermo Scientific™ Q Exactive™ Focus MS
Resolving Power: 140K @ m/z 200Scan Range: 50-6,000Scan Rate: 12 Hz at 17.5KOptional: 280K
Resolving Power: 70K @ m/z 200Scan Range: m/z 50-2,000Scan Rate: 12 Hz at 17.5K
Resolving Power: 140K @ m/z 200Scan Range: 50-6,000Scan Rate: 12 Hz at 17.5K
First bench-top Orbitrap MS High performanceEasy to use, robust Polarity switching <1 secHCD MS2
Thermo Scientific™ Q Exactive™ HF MS
Resolving Power: 240K @ m/z 200Scan Range: 50-6,000Scan rate: 18Hz at 15K
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Transforming Small Molecule Identification and Characterization
Thermo Scientific™ Orbitrap ID-X™ Tribrid™ MS – Optimized and dedicated to small molecule structure analysis• High performance
• Very high resolution (500K at m/z 200) and high scan rate (30 Hz OT/40 Hz IT) • MSn, CID/HCD multiple dissociation techniques in a single run, OT/IT parallel detection …
• Ultimate flexibility and capability for data acquisition • Comprehensive, feature-specific filters enable triggering MSn of low abundant components• Predefined method templates for quick start
• Novel data acquisition – AcquireX• Automatic background exclusion, greatly improves efficiency, quality and accuracy of analysis
• Advanced data processing SW suite and database • Thermo Scientific™ Compound Discoverer™ 3.0 software, Thermo Scientific™ Mass Frontier™ 8.0 software, and mzCloud™.
mzCloud is a trademark of HighChem LLC, Slovakia
• Mass Range m/z 50 – 2000 • Mass Accuracy <1ppm internal, <3ppm external• Max. Mass Resolution >500,000• Scan rate 30 Hz OT MS2 /40 Hz IT MS2
• Dissociation HCD, CID• MS/MS and MSn
• Polarity switching on the fly
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Schematic for Thermo Scientific Orbitrap ID-X MS – Improved Instrumentation
Ultra High Field Orbitrap Mass AnalyzerAccurate mass measurement and resolves isobaric species
Dual Pressure Linear Ion TrapSensitive MSn mass analysis of HCD and CID fragments
Optional Easy-IC Ion SourceGenerates internal calibrant ion for real-time mass calibration
Ion Routing MultipoleAllows parallel analysisHCD at any fragmentation level
Mass Selection QuadrupoleEfficient and selective precursor ion isolationActive Beam Guide
Prevents neutrals and high velocity clusters from entering mass selecting quadrupole S Lens
Efficiently focuses ions into a tight beam minimizing in-source dissociation of fragile compounds http://planetorbitrap.com
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General Workflow for Small Molecule Structure Analysis
Orbitrap ID-X MS
CompoundDiscoverer 3.0
Confident Result
CompoundIdentified. _________. _________. _________. _________
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Time (min)
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10.1 12.113.211.010.5 13.8 15.88.4
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Urine-8-12hr-1 #4384 RT: 8.74 AV: 1 NL: 8.12E6F: FTMS + p ESI Full ms [120.0000-1200.0000]
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311.2049267.1746219.0477 597.3562198.1280
High Quality HRAM MS & MSn
Mass Frontier 8.0
Novel Software & Database
AcquireX Data Acquisition
Metabolite ID
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Case Study: Metabolite Identification of 5 Drugs in Human Liver Microsomal Incubation
O
ONHNS
OH
OO
O
N2H
AmprenavirC25H35N3O6S(M+H)+ 506.23193Cas# 161814-49-9
O
ONHOH
NHN
NH
O
O
LopinavirC37H48N4O5(M+H)+ 629.36975Cas# 192725-17-0
S
N
ONH
NHNH N
SOOH O
ON
RitonavirC37H48N6O5S2(M+H)+ 721.32004Cas# 155213-67-5
N
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NH
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SO O
O
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BosentanC27H29N5O6S(M+H)+ 552.19113Cas# 147536-97-8
N SNH
O
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OO
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TipranavirC31H33F3N2O5S(M+H)+ 603.21350Cas# 174484-41-4
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Traditional Data Dependent Acquisition of MS/MS Spectra for Drug Metabolites
Pähler and Brink, Drug Discov Today Technol., 2013, 10, e207-213
Targeted Approach Non-targeted Approach
DDA Selection Criteria:• m/z inclusion list based on predicted
metabolites• Isotope pattern• Mass defect
DDA Selection Criteria:• Intensity based (“top N” most intense)• All ion fragmentation
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AcquireX – A Novel Data Acquisition Workflow for Drug Impurity & Metabolites ID
AcquireX with Orbitrap ID-X MS – Tackles the Identification Bottleneck Automatically Automatically generates an exclusion list from a control sample.
• Excludes the background ions from triggering MSn
• Intelligent data-dependent acquisition - only triggers the ions of interest that are not present in the control.
Automatically generate an inclusion list of samples for deep scan when needed. High quality MSn data in one run and no repeat injections, no need for user to build
exclusion and inclusion list offline Acquire useful data - better than data-independent acquisition (DIA)
AcquireX greatly improves analysis efficiency, quality, and accuracy!
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AcquireX Acquisition Workflow: High Performance and Easy to Use
Predefined Method Templates for Met ID
AcquireX Acquisition Workflow
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AcquireX Data Acquisition Workflow and Sequence Setup
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Conventional and AcquireX Workflow Comparison: Amprenavir as an Example
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8.8348.8348.8348.8348.8348.8348.8348.8348.8348.8348.8348.8348.8348.8348.8348.8348.8348.8348.8348.8348.8348.8348.8348.8348.8348.8348.8347.6217.6217.6217.6217.6217.6217.6217.6217.6217.6217.6217.6217.6217.6217.6217.6217.6217.6217.6217.6217.6217.6217.6217.6217.6217.6217.621
5.1415.1415.1415.1415.1415.1415.1415.1415.1415.1415.1415.1415.1415.1415.1415.1415.1415.1415.1415.1415.1415.1415.1415.1415.1415.1415.141
6.4466.4466.4466.4466.4466.4466.4466.4466.4466.4466.4466.4466.4466.4466.4466.4466.4466.4466.4466.4466.4466.4466.4466.4466.4466.4466.446
9.3039.3039.3039.3039.3039.3039.3039.3039.3039.3039.3039.3039.3039.3039.3039.3039.3039.3039.3039.3039.3039.3039.3039.3039.3039.3039.303
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8.2778.2778.2778.2778.2778.2778.2778.2778.2778.2778.2778.2778.2778.2778.2778.2778.2778.2778.2778.2778.2778.2778.2778.2778.2778.2778.277
5.2535.2535.2535.2535.2535.2535.2535.2535.2535.2535.2535.2535.2535.2535.2535.2535.2535.2535.2535.2535.2535.2535.2535.2535.2535.2535.253
9.0179.0179.0179.0179.0179.0179.0179.0179.0179.0179.0179.0179.0179.0179.0179.0179.0179.0179.0179.0179.0179.0179.0179.0179.0179.0179.017
Parent
DDA triggered 8 metabolites
AcquireX triggered 21 metabolites
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Data Processing Using Thermo Scientific Compound Discoverer 3.0 (CD 3.0) SoftwareUtilizing unique features of CD 3.0, metabolites both expected and unknown were identified
using pre-made “Expected and Unknown” processing workflow
Find expected through dealkylation & dearylation as well as phase I & phase II biotransformation
Flag & provide score for compounds which has matching MS2 fragments with parent compound
Provide score for detected expected compounds; annotates on MSn spectra by fragment ion search, including transformation mass shifted
XIC traces for analytescontaining user-defined isotopic patterns
Provide score for analytes containing user-defined isotopic patterns
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“Generate Expected Compounds” Prediction in CD 3.0
-C5H6O3
parent 506.2320 @9.52min 392.2002 @6.50min
Hydrolysis
Dealkylation and Dearylation followed Phase I, Phase II transformation better prediction for expected compounds
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Compound Class for Unexpected Metabolite Identification
m/z 506.2319 m/z 351.2278 m/z 237.1961
Compound Class (parent compound fragments)Facilitate unexpected compounds identification
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Fragment Ion Search (FISh) Annotations
101.36971171.11336
176.14333190.01756
201.15109246.14876
149.07094
237.19618
202.15903
245.16487
163.1231
141.1024
188.1432116.1068
218.1538158.0965 217.1698190.0863144.0807 172.0756
147.0804
234.11259C13 H16 N O3 [M-e]+1
200.14343C14 H18 N [M-e]+1
117.06980C9 H9 [M-e]+1164.1069
189.10223C11 H13 N2 O [M-e]+1 207.11284C11 H15 N2 O2 [M-e]+1131.08548C10 H11 [M-e]+1164.07056C9 H10 N O2 [M-e]+1
146.06000C9 H8 N O [M-e]+1
134.09637C9 H12 N [M-e]+1257.0955
146.09636C10 H12 N [M-e]+1
208.09682C11 H14 N O3 [M-e]+1
120.08070C8 H10 N [M-e]+1
129.06979C10 H9 [M-e]+1
4429C6 H6 N O [M-e]+1
124.03922C6 H6 N O2 [M-e]+1(+Oxidation)
172.00624C6 H6 N O3 S [M-e]+1(+Oxidation)
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6) Amp_1Hr_ID (F5) #2717, RT=9.004 min, MS2, FTMS (+), (HCD, DDF, 522.2267@(20;40;65), +1) Amprenavir + (Oxidation) C25 H35 N3 O7 S, MW: 521.21957, Area: 3910188 FISh Coverage: 54 Matched, 34 Unmatched, 26 Skipped
Amprenavir[ ] +O
[ ] +O
Color coded transformation shifted fragment ion annotations help localize the site of biotransformation.
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More Metabolites were Triggered for MSn Using Acquire X than Conventional DDA
RT [min] Molecular Weight Formula Transformations DDA AcquireX
2.29 236.1889 C14H24N2O Sulfonamide hydrolysis + amide hydrolysis Y4.84 407.18788 C20H29N3O4S Amide hydrolysis + oxidation Y5.02 350.22056 C19H30N2O4 Sulfonamide hydrolysis Y Y5.14 364.23621 C20H32N2O4 Sulfonamide hydrolysis + methylation Y5.25 407.18788 C20H29N3O4S Amide hydrolysis + oxidation Y Y6.09 407.18788 C20H29N3O4S Amide hydrolysis + oxidation Y Y6.41 697.24513 C30H43N5O10S2 Oxidation + Cys-Gly-conjugation Y6.50 391.19296 C20H29N3O3S Amide hydrolysis Y Y6.61 519.20392 C25H33N3O7S Oxidation (+O-2H) Y Y6.88 826.28773 C35H50N6O13S2 Oxidation + GSH Conjugation Y7.19 624.22876 C28H40N4O8S2 Cysteine Conjugation Y7.32 521.21957 C25H35N3O7S Oxidation Y Y7.62 405.20861 C21H31N3O3S Amide hydrolysis + methylation Y7.81 681.25674 C31H43N3O12S Glucuronidation y Y7.84 521.21957 C25H35N3O7S Oxidation Y Y8.01 537.21449 C25H35N3O8S Di-oxidation Y8.28 503.20901 C25H33N3O6S Dehydration Y8.37 503.20901 C25H33N3O6S Dehydration Y9.02 521.21957 C25H35N3O7S Oxidation Y9.11 503.20901 C25H33N3O6S Dehydration Y9.52 505.2246 C25H35N3O6S Amprenavir Parent Y Y
10.61 519.24031 C26H37N3O6S Methylation Y
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Metabolites Identified from Amprenavir HLM Incubations
H2N
SN
OH
O
O HN O
OO
Amprenavirm/z 506
HNOH H
N O
OO
m/z 351
HNOH
NH2
m/z 237
H2N
SN
OH
O
O NH2
m/z 408 (rt 4.8, 5.3 min)
H2N
SN
OH
O
O NH2
m/z 392
OH
m/z 408 (rt 6.1 min)
H2N
SN
OH
O
O NH2
OH
HNOH H
N O
OO
H3C
m/z 365
H2N
SN
OH
O
O HN O
OO
Gluc
m/z 682
H2N
SN
OH
O
O HN O
OO
H2N
SN
OH
O
O HN O
OO
OH
HO
m/z 522 (rt 7.3, 7.8 min)
m/z 522 (rt 9.0 min)
H2N
SN
OH
O
O HN O
OO
m/z 520
O
H2N
SN
OH
O
O HN O
OO
-2H
m/z 504 (rt 8.3, 8.4 min)
m/z 698
H2N
SN
OH
O
O HN O
OO
+O+GSH
H2N
SN
OH
O
O HN O
OOCys
m/z 827
m/z 625
NH
SN
OH
O
O HN O
OO
m/z 520 H2N
SN
OH
O
O HN O
OO+O+Cys-Gly
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Isotope Triggered Trace Level Metabolite MSn
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Use of Isotopically Enhanced Labels for Plant Metabolite Identification
C13
C13
C13 HC13 H
C13
C13
F
O
Cl
Cl
N 2H
ON 2H
O
F
O
Cl
Cl
N 2H
ON 2H
O
C14H11Cl2FN2O3Mass 344.01308
(M+H)+ 345.02035Cas# 943831-98-9
C813C6H11Cl2FN2O3
Mass 350.03320(M+H)+ 351.04048
Blend of A and B yields significant +2, +6, +8 isotopic pattern
A
B A+B
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347.0174
349.0145348.0208
351.0405
353.0375
355.0346
6.0201
1.997150
100
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353.0375
355.0346
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Feature-Specific Filters for Trace Level Labeled Metabolite ID
The feature-specific filters in ID-X method editor allow user to build sophisticated instrument method to capture trace level metabolites present in complex matrices, triggering MSn for confident identification and structure elucidation.
Isotope triggering By delta mass: Sulfur, Chlorine By isotope peak relative intensity By customer-defined mass difference
Comprehensive, feature-specific filters to trigger low abundant components MSn
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Feature Specific Filters Capture Ions of Interest for MSn
Targeted Isotopic Ratio
Targeted Mass Difference
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Isotopic Filtering of Mass Spectra
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Xenobiotics: Labeled Metabolite Identification in Complex Matrices
Structure Elucidation Workflow
• Xenobiotic material is applied as mixture of natural and a stable isotope-labeled material to create an ‘un-natural’ isotope cluster in resulting metabolites
• ‘Mass-Difference’ filtered data dependent method acquires OT-MS/MS, (monoisotopic), Ultra High Resolution OT-SIM (Selected Ion Monitoring), IT-MS3 and IT-MS2 (full isotope window)
• Unique workflow provides comprehensive spectral data that enables full structural elucidation of unknown metabolites
Ultra High Resolution, High Specificity Experimental Design
isotope34S
HR monoisotopic MS2
UHR 500K SIM
IT ‘full isotope’ MS2IT-MS3
Data courtesy of Dr. Jeff Gilbert, Dow AgroSciences
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MS3 Analysis of Trace Level Components
Customized Workflow
• ‘Targeted Mass Difference’ filter allows specific triggering of MS/MS scans on precursors that have peaks with a specified mass difference
• High specificity in experimental design allows the detection of trace components with 500K resolution for elemental composition determination
• High sensitivity MS3 analysis provides fragmentation spectra allowing structure validation
• This highly selective workflow increases throughput by 2X vs. a traditional targeted workflowTime (min)
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Glucose Conjugate of Demethylated Parent
200 300 400 500 600 700 800 900 1000m/z
635.1054
631 632 633 634 635 636 637 638m/z
635.1054
632.1081
636.1086633.1112634.2493
637.1114
2.997
High Specificity MS3 Fragmentation of Metabolites
Isotope Mass Trigger
Data courtesy of Dr. Jeff Gilbert, Dow AgroSciences
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Mass Difference Triggered MSn of Plant Metabolites in Complex Biomatrix
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Orbitrap ID-X MS AcquireX Deep Scan Workflow for MetIDand Metabolomics Research
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AcquireX Deep Scan Workflow
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AcquireX | A New Acquisition Paradigm
Collect more meaningful data, not just more data to maximize productivity
Compared with Traditional DDA, AcquireX Deep Scan WorkflowSignificantly Increases ID by Triggering More MS/MS and MSn
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Compound Discoverer 3.0 for Data Processing
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Data Analysis Software, Database and Spectral Library Suite
Compound Discoverer 3.0 software
Thermo Scientific™TraceFinderTM softwaretargeted screening and Quan.
mzVault 2.0 Library Search and Manager
TM
HighChem LLC
Thermo E&L Compound Database
Mass Frontier 8.0spectral interpretation software
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Thermo Scientific Compound Discoverer Software (CD 3.0)
Complete Small Molecule Structure Analysis Platform
Component extraction based on HRAM and isotope pattern
Elemental composition prediction
Customizable feature set for advanced data processing
Searching online/offline spectral libraries and multiple databases for ID
Statistics and differential analysis
https://mycompounddiscoverer.com/
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Customizable Feature Set for Data Processing
Expected biotransformation list
Define adducts based on your experiments
Define ions based on your experiments
Add searchable spectral libraries tailored to your application, e.g., mzVault
Add searchable mass list tailored to your application
Ions belong to the same compound class e.g., parent fragments
• Default lists are available. • All lists can be customizable
to tailor fit your application. e.g., mzVault spectral library,
E&L mass list
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Fragment Ion Search - FISh Algorithm
• Fragment Ion Search – A means of detecting related components in complex samples• Data is scanned using fragmentation knowledge from a known structure• Components that share common fragments are detected• Fragments can be modified (metabolically or otherwise) and still be detected
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The Wizard Allows You to Build Processing Workflow
Creating a study and analysis use the guided “New Study and Analysis wizard” and build-in workflow templates
Descriptionfor each step
Compound Discoverer 3.0 Software
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Node-based Processing Workflow
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Result View – Data Interpretation
sub-tables
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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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Compound Class Scoring: Tool to Find Structurally Related Compounds
• Create compound class using common fragments from parent and known metabolites
• Use Mass FrontierTM to predict fragments from parent mol file. Copy and paste the fragment structures to construct compound class.
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“Structure Proposal & Apply FISh” for Unknown ID
FISh stands for “Fragment Ion Search”
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mzCloud - Advanced High Resolution Mass Spectral Database
https://www.mzcloud.org
Free, Available to everyone, everywhere
mzCloud is a trademark of HighChem LLC, Slovakia
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mzCloud Spectral Library
Spectral treeReference library
Search by different terms
Metadata available forselected library entry
Recalibrated spectra
Permanent citable links for entry, tree, and spectra
Breakdown curves
mzCloud is a trademark of HighChem LLC, Slovakia
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Thermo Scientific mzVault 2.0
Exact Mass-Enabled Library Search and Management- Searching library – local mzCloud- Creating customer library
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Summary
High resolution mass spectrometry is the necessary tool for drug metabolite identification. Only Orbitrap MS delivers high quality HRAM data with high mass accuracy, isotopic pattern fidelity, and MSn for confident small molecule structure characterization.
Orbitrap ID-X MS AcquireX data acquisition workflows generate background exclusion lists and inclusion lists automatically, which minimizes matrix interferences and increase triggering MSn of drug-related ions. AcquireX improves overall metabolite ID and small molecule structure analysis efficiency, quality, and accuracy!
Compound Discoverer 3.0, Mass Frontier 8.0, and mzCloud suite facilitate confident small molecule structure analyses.
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Thermo Scientific Orbitrap ID-X MS for Natural Product Research and Traditional Medicine
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