Comparison of methods for the determination of xenobiotics in biological samples: Targeted vs. two different General Unknown Screening (GUS) methods

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1 Comparison of methods for the determination of xenobiotics in biological samples: Targeted vs. two different General Unknown Screening (GUS) methods FACTA, Melbourne August Dr Brad Patterson Staff Scientist, Australia and New ZealandF

2 Project Details Samples Provided by Victorian Institute of Forensic Medicine (VIFM) (Dimitri Gerostamoulos, Olaf H. Drummer and Jochen Beyer) VIFM perform all the toxicology analysis for the Department of Justice in Victoria, Australia Hair Screening Post Mortem Blood (overnight toxicology service for coroner) Drugs in Driving (road side drug testing confirmation) Sexual Assault Cases Wanted to assess different approaches to screening samples AB SCIEX

3 HPLC-MS/MS Methods Investigated Triple Quadrupole (QqQ)Targeted Screen (MRM) Hybrid Linear Ion Trap (QqLIT) (MRM-IDA-EPI) Hybrid Linear Ion Trap (QqLIT) (EMS-IDA-EPI) Hi-speed, Hi-resolution QqTOF (TOF MS and MS/MS) VIFM Shanghai Facility AB SCIEX

4 Sample Preparation and Method Details Mass Spec Source Conditions Ion Source TurboIonSpray TurboV Heater Temp (TEM) 750 ºC GS1/GS2 90/90 Declustering Potential (DP) Optmised for Each Compound (50 for EMS) Entrance Potential (EP) 10 Collision Energy (CE) Optimised for each MRM Collision Cell Exit Potential (CXP) Optimised for each MRM IonSpray Voltage (IS) 5500 CAD gas setting (N 2 ) Medium (High for EMS and EPI Methods) Sample Preparation 0.1 ml Blood (plus I.S.) 0.2 ml Trizma Buffer; 1 ml ButCl 5 minutes shaking ButCl into vial, evaporation reconstitute 50 µl B, dil. 450 µl A QTRAP Scan Speeds (where applicable) 10,000 amu/sec 100 µl into LC-MS/MS (QTRAP 5500) HPLC Information Synchronization Mode LC Sync HPLC Flow and Solvents ml/min (A) 0.1% Formic, 50 mm NH 4 CO 2 H in Water (B) 0.1% Formic, 50 mm NH4CO2H in Acetonitrile Column Type Zorbax XBD C18 (4.6x150 mm, 5 µm) Injection Volume 100 µl Mobile Phase B (%) Time (min) AB SCIEX

5 Samples, samples, samples and only 22 more min to talk! 5 x 7 level standard curve (110 compounds +IS) 88 recent forensic cases (post- and ante-mortem blood) 20 post-mortem blood samples 50 post-mortem blood samples spiked at therapeutic concentration of 50 different compounds of interest (i.e. one drug per sample) Blinded Study AB SCIEX

6 MRM Method for Quantitation Current Validated Overnight Toxicology Method at VIFM Routine Screening and Quantitative Method currently in use 226 MRMs with 2 MRMs per compound and 6 deuterated internal standards (Scheduled MRM ) Fully automated reporting AB SCIEX

7 Targeted screening methods (Triggering full scan product ion scans with MRM) Single MRM used to govern collection of EPI (Enhanced Product Ions) requires hybrid instruments to obtain rich fragmentation information Automated library matching provides feedback on identity Dynamic Background Subtraction algorithm ensures EPI triggered at correct moment on chromatographic peak Requires prior knowledge of targets and an optimised MRM AB SCIEX

8 The General Unknown Screening (GUS) Methods Option 1: Full-scan Enhanced Mass Spec (EMS) Scans Collect full scan (EMS) m/z data and use intelligent algorithms (DBS) to trigger the collection of Enhanced Product Ion (EPI) data during the run EMS-IDA-3xEPI (scan times approx 2 sec for good quality qualitative data) Search all LCMS peaks against a library EMS EPI 1 EPI 2 EPI 3 Option 2: High Resolution, High Mass Accuracy Scans Using rapid scanning of a QqTOF instrument to get more data, faster Collect TOF-MS and use information in this scan to trigger collection of up to 50 MSMS every second. Quantitative and qualitative data is collected High mass accuracy, high resolution gives chemical formula of true unknowns AB SCIEX

9 Resolution and Mass Accuracy Accelerator TOF Analyzer High transmission ion optics at entrance to TOF region Two-stage mirror Better correction for spread in ion energies and position in the pulser 15 kv acceleration Reduces influence of energy spread among ions High transmission grids (93%) Reduced scattering/deflection in mirror Stable software controlled power supplies and low-jitter pulser and timing module AB SCIEX

10 Information Dependant Acquisition Cycle for a TOF MS/MSMS Screening Method TOFMS IDA 10xTOFMSMS TOF MS Full Scan msec 10 x 40 msec Total Cycle Time of 600 msec Information Dependant Acquisition Benefiting from the speed and sensitivity for MS/MS coverage High resolution & great mass accuracy at low mass AB SCIEX

11 Quantitation from Screening Method TOFMS Screening Method Haloperidol ( ng/ml) AB SCIEX

12 Information Dependant Acquisition Cycle for a TOF MS/MSMS Screening Method TOFMS IDA 10xTOFMSMS TOF MS Full Scan AB SCIEX

13 Instrument Calibration Batch Auto Calibration Calibrant Delivery System (CDS) connected to union of Turbo V Source Calibration performed every 5 injections (~ 2 hrs) during run No calibrant flow during methods AB SCIEX

14 Analysing the Screening Data TOFMS Screening Method 1. Target Lists 2. Statistical workflow to find peaks not in target list 3. Complete unknown searches (not covered here but uses advanced LC-MS Peak finding algorithms to search thousands of possible peaks) AB SCIEX

15 1. Analysing Target Lists (XIC Manager) TIC (10xTOFMSMS) (Dependant Scans) TIC (TOFMS) Name and Formula Adduct Required Retention Time (if known) AB SCIEX

16 Case Alprazolam Even with 10 MSMS and 1 TOF MS every 600 msec, excellent quant data is available AB SCIEX

17 Case Alprenolol AB SCIEX

18 Results how do the screens perform? TOFMS-IDA-TOFMS/MS (Target list of 1200 compounds applied) /50 compounds identified AB SCIEX

19 2. Statistical Methods of Data Analysis Given a group of blank samples, how does my unknown differ? vs AB SCIEX

20 Example of t-test vs. m/z (scenario 1 vs. 20 blank samples) t-value for Scenario 1 to Selectivity /8.9 (102) C Isotope Cl Isotope 6000 t-value /8.9 (114) 13 C Isotope t-value /6.2 (31) 265.1/8.9 (108) m/z AB SCIEX

21 Extracting Peaks of Interest XIC Of Selected Peak TOF MS Isotope Pattern TOF MSMS + Library Hit AB SCIEX

22 Are they all that easy? well, no (scenario 2 vs. 20 blank samples) t-value for Scenario 2 to Selectivity /4.2 (27) 350 t-value /9.4 (228) 313.1/11.8 (289) 300.1/9.4 (232) 221.1/9.5 (32) /9.5 (34) /9.5 (357) 178.1/1.3 (12) 416.2/7.6 (715) 527.4/4.4 (1171) 221.1/6.2 (31) 576.4/16.4 (1342) 358.2/5.7 (466) 459.3/9.1 (915) 641.4/8.0 (1514) 696.4/15.9 (1592) 742.5/11.5 (1670) m/z AB SCIEX

23 Confirming Compounds not in Library TOF MS Isotope Pattern TOF MSMS TOF MSMS Fragments AB SCIEX

24 Results how do the screens perform? TOFMS-IDA-TOFMS/MS (MarkerView Software Statistical Analysis) /50 compounds identified AB SCIEX

25 Results how do the screens perform? Unblinded List of possible compounds examined Search revealed 222 entries...with same formula (C 16 H 17 ClN 2 S) /40 39/40 compounds identified Desmethylchlorpromazine AB SCIEX

26 Value of Collecting high quality MSMS AB SCIEX

27 Closing Comments We will have MRM-based methods for a while yet It s fast, very sensitive and widely accepted Hybrid instruments are able to collect MSMS for a large number of targets Current statistical workflows can learn so we can exclude lipids and many other known endogenous materials Have moved into an era where quantitative and qualitative work can be done in the same run and with real samples Authentic standards are ALWAYS important AB SCIEX

28 Acknowledgments Dr Jochen Beyer (Victorian Institute of Forensic Medicine) Feng Qin (AB SCIEX, Shanghai) AB SCIEX

29 Trademarks/Licensing For Research Use Only. Not for use in diagnostic procedures. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX is being used under license AB SCIEX AB SCIEX

30 Questions AB SCIEX