Mass Spectrometry in Forensic Science. Erin Shonsey January 29, Overview

Transcription

1 Mass Spectrometry in Forensic Science Erin Shonsey January 29, 2010 Overview Introduction to forensic sciences Uses of mass spectrometry in forensic sciences Typical instrumentation in forensic sciences Applications of new instrumentation 1

2 Introduction to Forensic Sciences Forensic Sciences is defined as: the application of a broad spectrum of sciences to answer questions of interest to the legal system. Introduction to Forensic Sciences Typical analytical sections within a forensic science laboratory: Drug Chemistry Analysis of pills, powders, liquids, plant materials, and other suspicious items for illegal drug content Toxicology Analysis of biological samples for alcohol, prescription medication, drugs of abuse, and other chemicals that are not naturally occurring in the body DNA Extraction and amplification of DNA from biological fluids for identification Firearms Bullet pattern recognition and analysis of gun powder Fire Debris -- Identification of ignitable liquids used in arsons 2

3 Standards for Accepting the Scientific Validity of a Procedure, Technique, and Principle Alabama Frye standard: the court must decide if the questioned procedure, technique, and principles are generally accepted by a relevant community Federal Rule 702: a witness qualified as an expert may testify in the form of an opinion Federal Daubert: Has it been tested? Has it been published and peer reviewed? Potential rate of error Existence and maintenance of standards controlling the techniques operation Accepted in the relevant scientific community Mass Spectrometry in Forensic Science A gas chromatograph with a mass spec detector is the final tool used in the analysis of drug chemistry and toxicology samples for identification and confirmation. 3

4 Typical forms of Mass Spectrometry in Every Forensic Science Lab Gas Chromatography-Mass Spectrometry (GC-MS) Typical forms of Mass Spectrometry in Every Forensic Science Lab Gas Chromatography-Mass Spectrometry (GC-MS) 4

5 Typical forms of Mass Spectrometry in Gas Chromatography-Mass Every Forensic Science Spectrometry Lab(GC-MS) Spectrum Typical forms of Mass Spectrometry in Every Forensic Science Lab Gas Chromatography-Mass Spectrometry (GC-MS) Spectrum Spectra are searched against a library of known compounds in an effort to identify every peak in the TIC A standard is analyzed on the instrument to generate a known retention time and spectrum of the compound for that instrument 5

6 Problems Encountered with the GC/MS Lose the parent ion of the compound upon ionization in the instrument Example: Methadone Problems Encountered with the GC/MS Derivatize the compound for analysis with GC/MS which decreases detection of low level compounds Example: THC 6

7 Problems Encountered with the GC/MS Heat labile compound will be identified as a related compound, but not the actual compound Example: Clorazepate to Nordiazepam New Technology Four new instruments have been brought into the department in October 2008 AccuTOF-DART mass spectrometer 3200 QTRAP mass spectrometer with LC 3200 QTRAP mass spectrometer with DART HS-GC-MSD 7

8 Different forms of Mass Spectrometry Liquid Chromatography Electrospray Ionization Mass Spectrometry (LC-ESI-MS) Sample solution Gas -5 KV Atmospheric pressure N 2 Collision gas Q1 Q2 Q3 Detector Vacuum Different forms of Mass Spectrometry Direct Analysis in Real Time with Time of Flight Mass Spectrometry N 2 TOF detector Electrostatic reflector 8

9 HS-GC-MSD This instrument provides opportunity for qualitative and quantitative identification of volatile compounds TIC of Volatiles Mix 9

10 Spectrum of Peak at 1.44 min Isopropanol Spectrum of Peak at 1.61 min Acetone 10

11 Spectrum of Peak at 1.70 min 1-propanol (IS) TIC of Ethanol Standard 11

12 Spectrum of Peak at 1.29 min Ethanol Spectrum of Peak at 1.70 min 1-propanol (IS) 12

13 Summary Method development is underway with the HS-GC-MSD Good separation and spectra from the volatiles mix and ethanol standard Ready to start validation Developing method for commonly abused inhalants Developing a screening for other volatile compounds Example: GHB AccuTOF-DART MS The DART is the first open air, ambient ion source for a mass spectrometer Coupled to a time of flight instrument exact mass measurements can be used in the putative identification of compounds 13

14 3200 QTRAP-DART MS Coupled to a hybrid triple quadrupole/trap instrument molecular ions can be individually fragmented for identification of sample components DART Ionization Penning ionization: energy is transferred from metastable ions (M*) Positive ions: He* ionizes water which transfers a proton to the sample Negative ions: Penning electrons are rapidly thermalized and captured by oxygen which ionizes the sample onmechanisms/tabid/450/default.aspx 14

15 DART Ionization He He He He He* He He He* He*He He He He* He*He* He*He* He* H 2 O [(H 2 O+ ) MH + H 2 O n H]+ He* H 2 O MH + MH + He* H H 2 O [(H 2 O + ) n H] + H 2 O 2 O He* MH + MH + H [(H 2 O + ) n H] + 2 O H 2 O MH + Time of Flight Detector Accelerating pulse TOF detector t = (d/ (2U))(( m/z)) t = time d = flight tube distance U = accelerating voltage m = mass z = charge 15

16 AccuTOF Mass Spectrometer N 2 Orthogonal acceleration time of flight Repelling plate TOF detector The reflector doubles the length of the flight tube Electrostatic reflector Different forms of Mass Spectrometry DART Ionization Tandem Mass Spectrometry N 2 Collision gas Q1 Q2 Q3 Detector Vacuum 16

17 Quadrupoles have variable ion transmission modes m2 m4 m1 m3 m4 m3 m2 m1 mass scanning mode m2 m1 m4 m3 m2 m2 m2 m2 single mass transmission mode Molecular Ion Scanning N 2 Detector Q1 Q2 Q3 Vacuum 17

18 Product Ion Scanning N 2 N 2 Collision gas Detector Q1 Q2 Q3 Vacuum Multiple Reaction Monitoring (MRM) N 2 N 2 Collision gas Q1 Q2 Q3 Detector Vacuum 18

19 Sample Introduction with the AccuTOF-DART MS Liquid samples are introduced with a glass capillary tube closed at one end Solid samples are introduced into the stream with tweezers Sample Introduction with the AccuTOF-DART MS x10 3 Intensity ( Every ) sampling device is analyzed for contamination prior to use Time[min] 19

20 Types of Samples Analyzed with the AccuTOF-DART MS White Powder Analyzed with the AccuTOF-DART MS 0 3 Intensity ( ) m/z Name Neutral comp. Meas. Calc. Diff(u) Abund. Methamphetamine C10H15N Phentermine C10H15N Amantadine C10H17N Phendimetrazine C12H17NO

21 2.8e6 2.6e6 Molecular Ion Mass Spectrum Possible Methamphetami ne 2.4e6 2.2e6 2.0e6 Q1 of White Powder Intensity, cps 1.8e6 1.6e6 1.4e6 1.2e6 1.0e e e e e m/z, Da Intensity, cps 2.1e5 2.0e5 1.9e5 1.8e5 1.7e5 1.6e5 1.5e5 1.4e5 1.3e5 1.2e5 1.1e5 1.0e5 9.0e4 8.0e4 7.0e4 6.0e4 5.0e4 4.0e4 3.0e4 2.0e4 1.0e Product Ion Mass Spectrum Name Fit RevFit Purity CE METHAMPHETAMINE BZP PHENTERMINE METH TPC METH TPC TFMPP Metanephrine m/z, Da

22 Total Analysis Time = 5 min Intensity, cps 3500 METHAMPHETAMINE 1 METHAMPHETAMINE 2 RATIO % OF STANDARD STANDARD CASE % Time, min Plant Material Analyzed with the AccuTOF-DART MS x10 3 Intensity (110649) m/z Name Neutral comp. Meas. Calc. Diff(u) Abund. Cannabidiol C21H30O Tetrahydrocannibinols C21H30O

23 Intensity, cps 4.8e e5 4.4e5 4.2e5 4.0e5 Name Fit RevFit Purity CE THC e MDMA e5 BZP e5 MDMA e5 MDMA e5 TFMPP e5 Phenobarbitol PSEUDOEPHEDRINE e METHAMPHETAMINE e5 Epinephrine e5 93.1Normetanephrine e5 Metanephrine e5 Epinephrine e Metanephrine e5 1.2e e e4 6.0e e e m/z, Da 6.3e4 6.0e4 5.5e Total Analysis Time = 5 min 5.0e4 4.5e e4 Intensity, cps 3.5e4 3.0e4 2.5e4 THC 1 THC 2 RATIO % OF STANDARD STANDARD 2.49E E CASE 1.11E E % 2.0e4 1.5e4 1.0e Time, min 23

24 Clandestine Tablet Analyzed with the AccuTOF-DART MS x10 3 Intensity ( ) m/z Name Neutral comp. Meas. Calc. Diff(u) Abund Benzylpiperazine C11H16N Caffeine C8H10N4O TFMPP C11H13F3N Intensity, cps 2.0e5 1.9e5 1.8e5 1.7e5 1.6e5 1.5e5 1.4e5 1.3e5 1.2e5 1.1e5 1.0e5 9.0e4 8.0e4 7.0e4 6.0e4 5.0e4 4.0e4 3.0e4 2.0e4 1.0e Name Fit RevFit Purity CE BZP METHAMPHETAMINE PHENTERMINE METH TPC m/z, Da

25 7.8e4 7.5e4 7.0e4 6.5e4 6.0e4 Total Analysis Time for 2 Items = 8 min Analyst Time for 2 Items = 3 hrs 5.5e4 5.0e4 Intensity, cps 4.5e4 4.0e4 3.5e4 3.0e4 BZP 1 BZP 2 RATIO % OF STANDARD STANDARD 1.51E E CASE Item #1 4.66E E % CASE Item #2 6.21E E % 2.5e4 2.0e4 1.5e4 1.0e Time, min Clandestine Tablet Analyzed with the AccuTOF-DART MS x10 3 Intensity ( ) m/z Name Neutral comp. Meas. Calc. Diff(u) Abund. Phentermine C10H15N Methamphetamine C10H15N Benzylpiperazine C11H16N Butamben C11H15NO MDMA C11H15NO TFMPP C11H13F3N

26 5.2e e5 5.0e4 5.0e4 1.05e5 4.8e4 4.8e4 1.00e5 4.6e4 4.6e4 4.4e4 Name Fit RevFit Purity CE 9.50e4 4.4e4 4.2e4 MDMA e4 4.2e4 4.0e4 METHAMPHETAMINE e4 8.50e4 3.8e4 BZP e4 8.00e4 3.6e4 PHENTERMINE e4 Warfarin e4 3.4e4 Name Fit RevFit Purity CE 3.4e4 PSEUDOEPHEDRINE e4 3.2e4 METHAMPHETAMINE e4 Epinephrine Name Fit RevFit Purity 25 CE 3.0e4 BZP e4 3.0e4 BZP THC PHENTERMINE e4 6.00e4 2.8e4 METHAMPHETAMINE Normetanephrine METH TPC e4 METH TPC e4 2.6e4 PHENTERMINE METH TPC e4 2.4e4 METH TPC Lorazepam TFMPP e e4 2.2e4 Metanephrine Milrinone e4 2.0e4 2.0e4 Epinephrine e4 1.8e4 1.8e4 3.50e4 1.6e e4 1.4e4 2.50e4 1.2e4 1.0e e e e m/z, Da Intensity, Intensity, cps cps Analysis Time = 10 min Intensity, cps METHAMPHETAMINE 1 METHAMPHETAMINE 2 RATIO % OF STANDARD STANDARD CASE % MDMA 1 MDMA 2 RATIO % OF STANDARD STANDARD CASE % BZP 1 BZP 2 RATIO % OF STANDARD STANDARD CASE % Time, min 26

27 Clandestine Laboratory Samples Analyzed with the AccuTOF- DART MS x10 3 Intensity ( Methamphetamine Pen tube (Pseudo)ephedrine m/z Intensity ( Methamphetamine (Pseudo)ephedrine Aluminum Foil m/z x10 3 Intensity (50970) (Pseudo)ephedrine 40 Blender Jar x m/z Intensity (182939) (Pseudo)ephedrine White Powder m/z x Intensity (72800) Methamphetamine Aluminum Foil m/z 27

28 x10 3 Intensity (57470) Methamphetamine Rubber Tubing m/z x Intensity (98301) Methamphetamine (Pseudo)ephedrine White Powder m/z 6.8e4 6.5e e4 Intensity, cps 5.5e4 5.0e4 4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e4 Name Fit RevFit Purity CE PSEUDOEPHEDRINE METHAMPHETAMINE BZP Metanephrine Epinephrine Normetanephrine Epinephrine PHENTERMINE Normetanephrine MDMA TFMPP Milrinone Phenobarbitol Warfarin Warfarin METH TPC Metanephrine m/z, Da 28

29 Clandestine Laboratory Samples Analyzed with the DART- QTRAP METHAMPHETAMINE 1 METHAMPHETAMINE 2 RATIO % OF STANDARD STANDARD 2.78E E White Powder 1.51E E % Pen Tube 6.85E E % Aluminum Foil 9.25E E % Aluminum Foil 3.02E E % Rubber Tubing 7.89E E % PSEUDO 1 PSEUDO 2 RATIO STANDARD 5.75E E White Powder 5.17E E % White Powder 4.08E E % Pen Tube 5.75E E % Blender 5.94E E % Total Analysis Time for 7 Items = 1 hr Analyst Time for 7 Items = 2 days Real Time Sample Analysis with the AccuTOF- DART MS Efficient screening instrument Soft ionization keeps the molecular ion intact Mass accuracy allows matches within 5 mmu of the theoretical mass of a compound No extraction is required for sample analysis Raw samples the preferred sample High-throughput Typical analysis time for a sample is 1-2 min 29

30 Real Time Sample Analysis with the DART- QTRAP MS Compound fragmentation is possible without extraction CID fragmentation allows retention of molecular ion in fragmentation spectrum These can be searched against an in house library for identification MRM analysis gives ion ratios for a second level of compound identification in comparison to a standard Complex mixtures do not present a problem for analysis The instrument has the ability to isolate a single compound for fragmentation LC-MRM-MS assay for Drug Detection and Quantitation Sample solution Gas -5 KV Atmospheric pressure N 2 Collision gas Q1 Q2 Q3 Detector Vacuum 30

31 LC-ESI-MS/MS Spectrum of Methadone m/z, amu LC-MRM-MS analysis for Drug Quantitation Collision gas Q1 Q2 Q3 31

32 LC-ESI-MRM-MS 310 Collision gas 265 Q1 Q2 Q3 Dwell Time (msec) Collision Energy (CE) Compound Molecular Weight Parent ion Product ion Declustering Potential (DP) Retention Time pka Alprazolam Amitriptyline Cocaethylene Cocaine Fentanyl Imipramine Mepivacaine Methadone Methamphetamine Oxycodone THC Trazodone TIC of Check Mix Retention Order Oxycodone Methamphetamine Mepivacaine Cocaine Cocaethylene Trazodone Fentanyl Imipramine Methadone Amitriptyline Alprazolam THC Time, min 32

33 Information Dependent Acquisition (IDA) Survey Scan (1) Survey Scan (2) Enhanced Resolution Survey scan be EMS, EMC, Neutral Loss, Precursor Scan, MRM or EPI (combinations of 2 surveys) Improve Resolution/Accuracy IDA Criteria Level 1 Two levels of criteria Dependent Dependent Scan Scan (s) (s) Dependent Scan (s) Dependent Scan (s) IDA Criteria Level 2 Add to Exclusion List Multiple dependant scans (EPI, Product Ion and MS3) Inclusion/Exclusion List Second Second Dependent Dependent Scan Scan (s) Second Dependent Scan (s) (s) Second Level Dependant Scan (MS 3 ) Courtesy of Ricky Ciner IDA Analysis of Check Mix TIC XIC MRM EPI 33

34 Library Search of Fragment Spectrum Summary LC-ESI-MS can be used in the qualitative and quantitative analysis of drugs in toxicological specimens The instrumentation is advantageous in that chemicals do not have to be derivatized The soft ionization aids detection of the parent ion of the compound 34

35 Overall Summary Mass spectrometry is a powerful tool in a forensic science lab New instrumentation is expanding the sample analysis possibilities beyond current limitations No one technique is robust enough for everything, therefore a combination of techniques is ideal for screening and confirmation of drug and toxicology samples Acknowledgements UAB Dr. Stephen Barnes Marion Kirk Ray Moore Dr. Matthew Renfrow Landon Wilson ADFS Dr. Dale Carpenter Andrea Headrick Dr. Jack Kalin Gary Wallace 35