High Resolution MS in Forensic Toxicology Screening Osama Abu-Nimreh - PowerPoint PPT Presentation

High Resolution MS in Forensic Toxicology Screening Osama Abu-Nimreh CMD Sales Support Specialist MECEC , Dubai The world leader in serving science

Screening Approaches in LC/MSMS • Screening applications are commonly used in forensic and clinical toxicology laboratories. • Targeted screening : compound is identified and confirmed using databases and/or libraries. • Unknown screening: no databases and libraries available. Compound is identified using MS 2 and or MS n data. • Screening applications utilize different types of mass spectrometers • Ion Traps : MS and MS n experiments. Pos/Neg switching • Triple quadrupole : 2 SRMs/analyte. Confirmation using the Ion Ratio. • HRAM instruments (OrbiTrap) : Full Scan followed by AIF for the Exactive Plus. Full Scan followed by MS 2 experiments for the Thermo Scientific ™ Q- Exactive ™ Plus. Full Scan followed by 4 vDIA events for the Thermo Scientific™ Q-Exactive Focus. For Research use Only. Not for Use in Diagnostic Procedures 2

Screening – General Workflows Step 1: Sample Step 2 : Data Step 3 : Processing Preparation acquisition Data Depending on sample Different approaches: TraceFinder type ( urine, plasma, -Ion trap -ToxID serum, whole blood ): -Triple quadrupole -HRAM screening -Dilution -Orbitrap (HRAM MS) -LLE -SPE -Online TurboFlow extraction -Protein precipitation 3

Mass Resolution FWHM m • Resolution  R  m (FWHM)  m • Orbitrap (HRAM) MS • Quadrupole MS 400  400   R 1000  R 100000 0 . 4 0 . 004 4

How Accurate Is Your Mass?  Mass accuracy  m m    6 meas true / 10 m z m true  6  500 . 1 500 . 0    • Quadrupole MS m / z 10 200 ppm 500  6  500 . 10314 500 . 10214    • Orbitrap MS m / z 10 2 ppm 500 . 10314 TOF MS 5

Selectivity Increases With Higher Mass Accuracy RT: 8.45 NL: 1.20E5 100 m/z= 484.25313- 90 484.25507 F: FTMS {1,0} + p APCI Full 80 ms [300.00-850.00] MS pivo_5_UH 70 60 2 ppm Abundance 50 Relative 40 30 20 10 8.55 7.99 7.26 7.41 7.49 7.76 7.91 10.31 10.50 10.61 7.67 9.46 10.16 0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8 10.0 10.2 10.4 10.6 Time (min) RT: 8.45 NL: 1.20E5 100 m/z= 484.24441- 90 484.26379 F: FTMS {1,0} + p APCI Full 80 ms [300.00-850.00] MS pivo_5_UH 70 60 Abundance 50 20 ppm Relative 40 30 8.21 7.62 20 7.85 7.59 7.40 8.25 7.37 7.81 7.92 7.99 10 8.61 7.30 7.26 10.61 8.67 8.88 9.11 9.46 10.09 10.16 10.31 10.50 0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8 10.0 10.2 10.4 10.6 Time (min) NL: 1.25E5 RT: 8.45 100 m/z= 484.20567- 90 484.30253 F: FTMS {1,0} + p APCI Full 80 ms [300.00-850.00] MS pivo_5_UH 70 60 Abundance 50 Relative 9.29 8.72 40 8.67 100 ppm 8.99 30 8.78 9.11 8.21 7.85 8.83 7.62 8.13 20 7.59 7.81 8.10 7.38 8.35 7.33 10 7.27 9.46 9.50 10.50 10.61 9.64 10.09 10.16 10.34 0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8 10.0 10.2 10.4 10.6 Time (min) *Zachariasova M et al, Anal. Chim. Acta 6

Specificity = Resolution + Mass Accuracy Resolution: 10k, 30k, 50k, 100k 54 ppm Butyl-Phthalate, 279.15909 apart (ubiquitous background ion) 100 90 80 70 Relative Abundance 60 Ethinyl-Estradiol, 279.17434 50 40 30 20 10 0 279.12 279.14 279.16 279.18 279.20 m/z 7

The Industry’s Leading Thermo Scientific ™ MS Portfolio Non-targeted Exactive Exactive Exactive Tribrid Orbitrap Tribrid Orbitrap Tribrid Orbitrap Analysis Series MS Series MS Series MS MS MS MS HR/AM • Metabolomics • Biomarker Discovery • Proteomics • Proteomics Transform Research Applied • Bioanalysis Markets Markets • Metabolism Quantitative Qualitative Your Science • Food Safety • Metabolomics • Environmental • PTM Analysis • Clinical/Toxicology • Lipidomics Triple Quads Triple Quads Triple Quads Ion Traps Ion Traps Ion Traps MS, MS n Targeted Analysis 8

Q Exactive MS - a 3D view Hyperbolic Quadrupole Mass filter RF-Lens C-Trap HCD Cell Orbitrap Mass Analyzer 9

Q Exactive: Hardware 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 10

Orbitrap - Principle of Operation • A short ion packet of one m/z from c- trap enters the field tangentially • C-trap is only used as an ion storage device • Ions are squeezed towards the central electrode by increasing voltage on the central electrode • In the axial direction, ions are forced to move away from the narrow gap towards the wider gap near the equator. This initiates axial oscillations • After the voltage increase stops, ion trajectories become a stable spiral 11

Orbitrap - Principle of Operation  Ion m/z separation depends on • Frequency of harmonic oscillations and is proportional to sq root of m/z  Three frequencies create oscillations • Frequency of rotation • Frequency of radial oscillations • Frequency of axial oscillations  Resolving power is • Inversely proportional to the square root of m/z • Proportional to acquisition time  Sensitivity is independent of acquisition speed.  Red rings smallest m/z ; Blue ring larger m/z ; Green ring largest m/z Makarov A. Anal. Chem. 2000, 72 , 1156-1162. 12

Principle of Orbitrap MS Operation r z φ Hyper-logarithmic potential distribution: “ideal Kingdon trap”   k      2 2 2 U ( r , z ) z r / 2 R ln( r / R ) m m 2  Characteristic frequencies: Frequency of rotation ω φ • Frequency of radial oscillations ω r • Frequency of axial oscillations ω z • k  2 2       R m R m             z 1 2 z r z     m / q R R 2 Makarov A. Anal. Chem. 2000, 72 , 1156-1162. 13

Triple Quadrupole is great tool! but ?  It is only targeted!  Selectivity provided by tandem MS/MS (SRM transition needed)  False positives are reality!  Need to setup instrument (SRM) before analysis  Realistic breakpoint is 200-300 compounds in a run  Time consuming data processing HRAM is a solution!  Can perform the same level of quantitation as MS/MS  Selectivity obtained by accurate mass measurement (only m/z needed)  No false positives!  No need to setup instrument (SRM) before analysis  Unlimited number of compounds in a run – perfect for screening  Automated data processing 14

Orbitrap Mass Analyzer Features • Fundamental difference to other HRAM instruments • Parameter measured is frequency , not time/voltage/current • Resolution allows more accurate m/z determination • Less prone to ambient conditions changes • Usually stable within <2 ppm during several days • No need for lock mass in “routine work” • Small footprint • Easy to setup 15

High Resolution MS Technology Race 500000 Orbitrap Orbitrap Fusion 450000 Tof / QTof Mass resolution (FWHM) 400000 350000 ORBITRAP’s spectacular climb in performance in 10-year span! 300000 QE Plus* 250000 Orbitrap Elite 200000 150000 Exactive Series 100000 LTQ Orbitrap 50000 First Q-Tof Bendix Tof 0 1955 1965 1975 1985 1995 2005 2015 Time progression (year) 16

Orbitrap technology – Workflow examples Step 1: Sample Step 2 : Data Step 3 : Processing Preparation acquisition Data 17

Q Exactive Focus - Acquisition apporaches • There are 3 approaches possible for screening: • DDE : Data Dependent Experiment. Here the system selects the more intense ions reported in the Full scan MS spectra to fragment those on MS² mode. If the ion has a low intensity it is probable that it won’t be selected for MS² and therefore not confirmed by the processing software. • AIF : All Ion Fragmentation.Here the system fragments all the ions present in the MS spectra in the collision cell. Lack of specificity. • DIA : Data Independent Analysis. Here fragmentation is performed in different mass ranges. It is more specific than AIF but less specific than DDE. Today we use the two approaches DDE and vDIA for screening purposes, we strongly suggest the vDIA approach for a better fragmentation. Drug identification based on : Accurate mass of the parent ion Accurate mass of the fragment ions Isotopic pattern Library match Chromatographic retention time window For Research use Only. Not for Use in Diagnostic Procedures 18

3 ways of Quantitation/Screening for Routine Work Full MS or targeted SIM/ddMS2 • Post-acquisition - extracted ion chromatograms of parent ions of interest • Relies on high resolution for selectivity • Useful for less complex background • No method development/preparation needed Full MS/ All Ion Fragmentation – vDIA* • Post-acquisition - extracted ion chromatograms of parent ions of interest • Scheduled target (inclusion) list (Rt, m/z ) • Minimum method development (e.g., predefine parent ions, tr) • Also for screening purposes PRM (Parallel Reaction Monitoring) • Post-acquisition – extracted ion chromatograms of parent -> fragment transitions acquired • Scheduled target list (Rt, m/z , collision energy) • Most sensitive and selective even in highly complex matrices *vDIA method not available in the United States of America 19