HPLC Coupled with Quadrupole Mass Spectrometry and Forensic Analysis of Cocaine.
Putting a Forensic Eye on Mass...
Transcript of Putting a Forensic Eye on Mass...
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Claudio De Nardi Thermo Fisher Scientific - Sales Support Expert LSMS Clinical and Toxicology
Putting a Forensic Eye on Mass Spectrometry
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Clinical and Forensic - A Lot of Overlapping Areas
NewBorn Screening Endocrinology
TDM (Immunosuppressants, Vitamin D, Antiepileptics, etc)
Trace Analysis Explosives
(non biological)
FORENSIC
TOXICOLOGY
CLINICAL
CLINICAL TOXICOLOGY
FORENSIC TOXICOLOGY
TDM (Abuse of Drugs, Alcohol,
Benzodiazepines, etc)
Abuse of Drugs Benzodiazepines, etc
POSTMORTEM
Environmental Toxicology Occupational Health
Antidoping
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Customers Expectations/Requirements
I want to do quantitation
I want to perform targeted screening
with database/library searching
I want to identify unknown
and need retrospective analysis
• I want to be able to screen samples using a database of at least 1000 compounds
• I want to be able to use offline and online spectral libraries
• I want to search for totally unknown compounds in addition to targeted screening
• I need to retrospectively interrogate a sample searching for new/unexpected compounds
• I want ultimate sensitivity
• I want to quantify small concentrations in heavy matrices
• I want to quantify more than 1000 compounds in a run
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Screening Approaches – Some Definitions
Unknown Screening
“Search for the rest”
Targeted Screening
“Search for what you
know”
Untargeted Screening
“Search for what you
suspect”
• Compound search after targeted acquisition using a specific list of compounds
• Detection parameters need to be numerous and selective to avoid false positives
• Compound search following acqusition based on most intense signals NOT on a list of compounds
• Identification based on compound databases and/or spectral libraries
• Search of compounds with an totally untargeted acquisition
• Putative identification typically followed by structural elucidation
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Screening Workflow
• Related to sample matrix (urine, blood, serum, plasma, etc)
• TraceFinder
• Compound
databases
• Spectral libraries
• mzCloud
• Compound Discoverer
• Ion Trap
• Triple Quadrupole
• High Resolution MS
Sample Preparation Data acquisition Data Processing
Filtration Dilute-and-Shoot
Protein Precipitation Solid Phase Extraction TurboFlow
Liquid Liquid Extraction
Supported Liquid Extraction
CLE
AN
EA
SY
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Front Ends and Sample Preparation
Liquid Chromatography
TurboFlow
Paperspray
• Require offline sample preparation
• Suitable for multi-channel solutions
• Require minimal or no sample preparation
• Suitable for multi-channel solutions
• NO sample preparation
• Simple to use
• Fast
Ultimate 3000 Flexibility
Vanquish High-end UHPLC
Transcend II Flexibility
Prelude Robustness
Velox 360 Simplicity
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TurboFlow for Online Sample Extraction
• Online sample extraction that adds less than 2 minutes to the normal LC runtime • Offline sample extraction takes hours!
• Allows for the direct injection of complex matrices
• A cleaner sample reaches the mass spec • less background noise → better sensitivity • longer LC column lifetime • cleaner mass spec → less routine cleaning
• You can inject more (up to 100µL) • better sensitivity
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Paperspray – No Sample Preparation
http://www.prosolia.com/resources/videos/velox-360
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Paperspray on TSQ Quantiva – DoA Quantification in Urine
• LOQs in the 0.5 to 50 ng/mL • LODs in the 0.5-1 ng/mL • No sample preparation • No chromatography
amphetamineY = 0.060314+0.00694195*X R^2 = 0.9098 W: 1/X
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cocaethyleneY = 0.000134274+0.00128168*X R^2 = 0.9958 W: 1/X
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cocaineY = 0.00086511+0.00260058*X R^2 = 0.9923 W: 1/X
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methamphetamineY = 0.00628846+0.010073*X R^2 = 0.9967 W: 1/X
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PCPY = 0.00805257+0.00922576*X R^2 = 0.9954 W: 1/X
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amitriptyline Y = 0.146009+0.0161053*X R^2 = 0.9870 W: 1/X
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temazepamY = 0.0262972+0.00528053*X R^2 = 0.9802 W: 1/X
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Amitriptyline Amphetamine Cocaethylene
Cocaine Codeine
Methamphetamine PCP
Temazepam
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Mass Spectrometers – A Leading Portfolio
HR/AM
MS, MSn
Appl
ied
Mar
kets
Research
Markets
Non-targeted Analysis
Targeted Analysis
Quantitative Qualitative
• Biomarker Discovery • Proteomics • Metabolism
• Metabolomics • Proteomics • Bioanalysis
• Food Safety • Environmental • Clinical/Toxicology
• Metabolomics • PTM Analysis • Lipidomics
Transform Your Science
Ion Traps Triple Quads
Tribrid Orbitrap MS
Exactive Series MS
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Triple Quadrupoles for Targeted Screening and Quantitation
TSQ Endura Extreme Quantitative Value
• Best-in-class performance
• Unprecedented usability
• Exceptional robustness
TSQ Quantiva Extreme Quantitative Performance
• Attogram sensitivity
• Unprecedented usability
• Exceptional robustness
Chromatogram for
Quantitation
Chromatogram for
Confirmation
Internal Standard
Ion Overlay
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Triple Quadrupoles for Quantitation
• Quantitation of 72 Drugs of Abuse on Urine on TSQ Quantiva • Simply dilute-and-shoot • Polarity switching • One SRM for quantitation and one or two for confirmation • Calibration range 5 to 5000 ng/mL
QC %RSD QCA 1.4
QCB 2.6
QUANTIFIER QUALIFIER
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Why Q Exactive and MS for Forensic Labs?
Affordable Focus version • Price comparable to high end Triple Quad
Versatile lab of the future • Targeted screening • Untargeted screening • Unknown screening • Quantitative confirmation
Ultimate performance • High sensitivity • High selectivity • High Resolution & Mass Accuracy • Polarity switching
Ease of use • Simple calibration, holds for days • No need for optimization • No compound-specific setup • Robust platform, low maintenance
Your High Resolution Accurate Mass lab of the future
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Resolution – Q Exactive vs. Q-Tof
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Res
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m/z
Q Exactive
Q-Tof Vendor S
Q-Tof Vendor A
Q-Tof Vendor B
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0
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200000
300000
400000
500000
1955 1965 1975 1985 1995 2005 2015
Orbitrap Tof / QTof
Race for Resolution – Q Exactive vs. Q-Tof
Year
Res
olut
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Bendix Tof
LTQ Orbitrap
Exactive Series
Orbitrap Elite
Orbitrap Fusion
First Q-Tof
QExactive Plus
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Benefits of Using Q Exactive for Screening
Resolving power Separate target compounds from interference
Mass stability and mass accuracy
Calibration maintained for days to weeks Unrivaled scan to scan mass accuracy and precision
Fast polarity switching
Quick positive-negative switch maintaining accurate mass/charge determination
No trade-off between resolution and sensitivity Both resolution and sensitivity are retained
Minimum scan to scan variance Excellent signal/noise, no need for averaging
Experiment flexibility Easy to use, a plug and play device
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Screening Approach Using a Q Exactive
Fragmentation positive
R = 17.500
FullMS negative
R = 70000
Fragmentation negative
R = 17.500
FullMS positive
R = 70000
Fragmentation types: 1. “Confirmational MS2” for targeted screening
Triggers all compounds of a specific list 2. “Discovery MS2” for untargeted screening
Triggers compounds having the highest intensity 3. “AIF” or “vDIA” MS2 for unknown screening
Fragments everything
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TraceFinder Software for Data Acquisition and Processing
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Routine Quantitation Targeted and Untargeted
Screening Unknown Screening
• Rugged and robust quantitation for multiple markets, across LC and GCMS systems
• Provides multiple forms of confirmation for targeted screening applications (spectral library, MS/MS2 database, isotopic pattern confirmation)
• Allows for screening for unknowns in conjunction with quan or targeted screening
Providing a complete quantitation and screening workflow for routine applications
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TraceFInder WorkFlow for Targeted / Untargeted Screening
Further confirms based on mass of fragments reported in the database 5
Compares theoretical and experimental mass spectrum of the parent using a specific spectral library 4
Identifies chromatographic peaks based on parent mass from a specific database 1
Confirms identification based on retention time in the database 2
Compares theoretical and experimental isotopic pattern for the parent 3
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TraceFinder and Targeted / Untargeted Screening
• multiple forms of confirmation: spectral library, MS/MS2 database, isotopic pattern confirmation
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TraceFinder and Targeted / Untargeted Screening
• multiple forms of confirmation: spectral library, MS/MS2 database, isotopic pattern confirmation
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TraceFinder and Targeted / Untargeted Screening
• multiple forms of confirmation: spectral library, MS/MS2 database, isotopic pattern confirmation
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TraceFinder and Unknown Screening
Confirm obtained ChemSpider hit by comparing theoretical fragmentation to experimental fragmentation 5
Search proposed molecular formulas with ChemSpider databases 4
Detect all chromatographic peaks above user specified threshold 1
Identify detected peaks using local database 2
For peaks not identified in step #2 propose molecular formula based on accurate mass and isotopic pattern 3
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Example of a hit from Unknown Screening
Unknown peak m/z 166.1224
C10H16ON C4H17N5P
ChemSpider Search Pholedrine Ephedrine Pseudoephedrine 3-(Diethyloamino) phenol 4-(Butyloamino) phenol
TraceFinder Identification
MassFrontier Confirmation
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100148.1119
91.0546
115.0543
117.0699133.0885
56.0502
132.080865.0392
70.0658 104.062278.0469 95.049653.0392 166.1225 175.454562.0037
NH•
N
N•
NH
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100148.1119
91.0546
115.0543
117.0699133.0885
56.0502
132.080865.0392
70.0658 104.062278.0469 95.0496166.122553.0028 175.454562.0037
N 2H •
NH
NH H
N•
H
Isomers
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100148.1119
91.0546
115.0543
117.0699133.0885
56.0502
132.080865.0392
70.0658 104.062278.0469 95.0496166.1225 175.4545
N
N•
N
•N
3-(Diethylamino) phenol 4 fragments
Theoretical vs. experimental fragmentation
NH
OH
NH
OHN
OH
Ephedrine 7 fragments
Pholedrine 7 fragments
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Database and Spectral Library of compounds of interest to the clinical research and forensic toxicology communities: • Prescription drugs • Natural and industrial toxins • Drugs of abuse • Drugs of interest for monitoring research • Performance enhancing drugs
An updated version will be available later this year with 4000 compounds and metabolites
The Importance of Databases and Spectral Libraries
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MzCloud
4,427 compounds - 7,309 trees - 1,066,072 spectra
https://www.mzcloud.org/
• A novel mass database / library of MS/MS and MSn spectra (R = 140.000 @ m/z 200)
• Structural info for compounds even if they are not represented in the library through identification of substructures
• Multi-energy, multi-fragment level, Multi-fragment technique
• Open consortium to establish a large public domain library which our software will link to
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Compound Discoverer 2.0
TraceFinder
Targeted Profiling
m/z Cloud
Compound Identification
Compound Discoverer
Untargeted Discovery
Complete small molecule research and structure identification in a Next Generation
platform
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Compound Discoverer 2.0
• Unknown compound detection
• Search ChemSpider
• Automatic background filtering • Composition prediction
• Mass list search • Pattern Scoring • Search mzCloud