Time of Flight The Power of Accurate Mass · 2016-09-04 · Mass Resolving Power Why resolution...
Transcript of Time of Flight The Power of Accurate Mass · 2016-09-04 · Mass Resolving Power Why resolution...
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Time of Flight The Power of Accurate Mass
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What is "Accurate Mass"?
• Mass measurements accurate to several decimal places (rather than "nominal" or "unit" mass measurements of ± 0.1 Da)
• Made possible by higher resolution mass analyzers
• Formerly limited to magnetic sector instruments
• Now available with Time of Flight (TOF) and Fourier Transform (FT) instruments (FT-MS and "orbitrap")
• Most Common Uses:
– Allows confirmation of molecular formula for proposed compounds
– Allows calculation of possible molecular formulae for unknowns
– Allows rapid selective and specific screening for target compounds
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Figures of Merit for Accurate-Mass Instrument Performance
Resolution = (m/z)/w1/2
e.g., 922.0092/0.087 = 10,597
Mass accuracy (mass error)
(1000.001-1000.000)/1000)x106 = 1 ppm
Therefore: 0.001 Da (1 mDa) error
@ MW 300 Da is approximately 3 ppm
921.5 922.0 922.5 923.0 923.5 924.0 924.5 m/z, amu
922.0092
923.0121
W1/2 = 0.087
610theory
theory- measured accuracy Mass
+TOF MS: 5.474 to 5.547 min from steroid2TOFap42.wiff Agilent, subtracted (4.696 to ... Max. 1.2e5 counts.
120 140 160 180 200 220 240 260 280 300 320 340 360 380 400m/z, amu
0.00
1.00e4
2.00e4
3.00e4
4.00e4
5.00e4
6.00e4
7.00e4
8.00e4
9.00e4
1.00e5
1.10e5
1.16e5
In
te
ns
ity
, c
ou
nts
289.2159
271.2051
269.1893
287.1997
Epitestosterone
Resolution 6890
theor 289.2162
meas 289.2159
mass error
-0.0003 Da
-1.06 ppm
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Mass Resolving Power Why resolution contributes to accurate mass:
narrow mass peak widths allow accurate centroiding
R=13,500 R=9,300
Arb.
Units
m/z m/z m/z 118 119 120 622 623 624 2722 2724
R=4,700
(Masses from
standard Agilent
tune mix
solution)
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How Much Accuracy in Needed for Identification?
(new concept for LC/MS: mass accuracy in "ppm")
Reserpine (C33H40N2O9) has a protonated ion at m/z 609.28066
Single quadrupole MS reports mass to +/- 0.1 = 165 ppm
Number of possible formulas using only C, H, O & N, at various
mass errors:
• 165 ppm 209 (single quadrupole resolution)
• 10 ppm 13 (older TOF instruments)
• 5 ppm 7
• 3 ppm 4
• 2 ppm 2
Accurate mass limits the number of possible formulae for a
given m/z measurement.
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Agilent Time-of-Flight (TOF) LC/MS
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Agilent’s orthogonal TOF design
Octopole
Rough Pump
Ion Pulser
Turbo 2
DC Quad
Turbo 1 Turbo 1 Turbo 1
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A Time-of-Flight "Scan"
Pulser Detector
1. Pulse a packet of ions up flight tube(a transient)
2. Measure ion arrivals at detector each nanosecond
3. Large number of data points in each transient
4. Sum 2000 – 10000 transients into one scan
5. Produces spectra with excellent ion statistics
(both center of mass and abundance)
Flight time is converted to m/z against calibration: e.g., 20.010 µsec →m/z 121.0508
See an animation at: http://www.chem.agilent.com/cag/other/lc-msd-tof.wmv
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Benefits of Atmospheric Pressure Ionization (API)
Ionizing difficult compounds (API), no derivatization
Accurate mass measurement (four decimal places, < 2ppm)
High resolution for distinguishing near-isobaric masses: - 5000 at low mass, > 10,000 at higher masses
Fast scanning >10 spectra/sec, many spectra across even a narrow UPLC peak
Excellent sensitivity for full-scan data (some instruments)
Wide mass range when needed
All-scan, all-the-time data allows retrospective search for compounds not originally sought.
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Forensic Tox Compound Analysis
The Value of the Agilent Solution
Robust hardware
Sample Preparation Products
HPLC and UHPLC Columns
Automated software workflows
Comprehensive Accurate
Mass Libraries
LC/MS Application Kits
First-class service and
application support
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Page 11
Agilent TOF Screening Solution
Agilent 62** TOF
- 20K resolution
- 5 decades in-scan
- Low Pg Sensitivity
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Qualitative Screen Report – Compound 9 (Cocaine)
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Results For Compound 9 – PCD Search Confirmed as Cocaine
EIC of Cocaine
Mass 303.1471
(50 ng/ml)
Isotope Scoring 0.99931 linear correlation
Using TOF for Quantitation
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Agilent Personal Compound Database - Forensics/Toxicology
Database containing >800 Forensic/Doping Analytes
•Automated Reporting
•Controlled Substances
•Explosives
•WADA Exclusion List
•Accurate Mass Confirmation
•Retention time Confirmation
•Spectral Match Scoring
(based on Isotopic Pattern &
Accurate Mass spacing)
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Isobars – The Need for Adequate Chromatographic Separation.
Isobaric Mass Ions – “Defined as different ions that have identical mass”
Morphine
(C17H20NO3)
[M+H]+ = 286 m/z
Hydromorphone
(C17H20NO3)
[M+H]+ = 286 m/z
Codeine
(C18H22NO3)
[M+H]+ = 300 m/z
Hydrocodone
(C18H22NO3)
[M+H]+ = 300 m/z
methamphetamine
(C10H16N)
[M+H]+ = 150 m/z
Phentermine
(C10H16N)
[M+H]+ = 150 m/z
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Isobaric Pairs
Morphine
Codeine
Hydromorphone
Hydrocodone
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Target Compound Screening Results: RT and mass matches
Narrow-mass EIC for compound m/z
Spectrum with overlaid structure,
theoretical isotope abundance
pattern
Result
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TOF screen results: opiates (isobars are separated)
morphine
codeine
hydrocodone
oxymorphone
hydromorphone
6-AM
oxycodone
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ALL IONS ADDING FRAGMENT CONFIRMATION
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All Ions MS/MS-What is it?
Step 1: Fragmentation without Isolation on TOF or Q-TOF
a) First Scan: Low fragmentation energy to analyze precursors
b) Second Scan: High fragmentation energy to analyze fragment ions
Low energy spectrum
High energy spectrum
Shows
Precursor
Ions
Shows
Fragment
Ions
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All Ions MS/MS-What is it?
Step 2: Software Extracts, Correlates, and Confirms
a) Find by Formula uses library to extract precursors from low energy MS
channel
b) All Ions MS/MS uses library to extract fragments from high energy
channel, gives coelution score
Software automatically
matches precursors with
fragment ions:
Coelution Plot with
Coelution Score
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Evaluation of All Ions MS/MS Automated Workflow Untargeted Drug Screening in Postmortem Blood
Step 1: Find by Formula Extracts Precursors using PCDL database
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Evaluation of All Ions MS/MS Automated Workflow Untargeted Drug Screening in Postmortem Blood
Step 2: Extract Corresponding Fragment Ions
Fragments from PCDL
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Evaluation of All Ions MS/MS Automated Workflow Untargeted Drug Screening in Postmortem Blood
Step 3: Precursor and Fragment Correlation/Coelution
Overlaid Precursor and
Fragment Ion
Chromatograms
Coelution Plot
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Differentiation and Identification of Isomers Untargeted Drug Screening in Postmortem Blood
Coeluting with ~50x
Concentration Difference
Alprazolam
Methadone
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Differentiation and Identification of Isomers Untargeted Drug Screening in Postmortem Blood
Similar precursor
m/z values and
overlapping isotopic
clusters
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Compound Confirmation-Methadone Untargeted Drug Screening in Postmortem Blood
Red outline overlays
theoretical isotopic pattern
and abundance with
acquired precursor data
for methadone
Methadone confirmed
with seven qualified
fragments
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Compound Confirmation-Alprazolam Untargeted Drug Screening in Postmortem Blood
Red outline overlays
theoretical isotopic pattern
and abundance with
acquired precursor data
for alprazolam
Alprazolam confirmed
with two qualified
fragments, despite
low concentration
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High Productivity: Compound Details View
Co-elution Plot
Overlaid
Chromatograms
Compound List Compound ID Results
Low Energy (MS) Spectrum
High Energy Fragment Spectrum
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All Ions on Random Dollar Bill for Cocaine
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What do you think?