2. 1 October 14, 2010 GBMSDG Talk Mass Spectrometry as the Premier Analytical Tool in Drug Discovery and Drug Development Walter Korfmacher Exploratory Drug Metabolism Merck Research Laboratories Kenilworth, NJ USA

3. 2 October 14, 2010 GBMSDG Talk Outline New Drug Discovery Challenges New Drug Discovery Challenges Mass Spectrometry Basics Mass Spectrometry Basics Selected In vitro Drug Metabolism Applications Selected In vitro Drug Metabolism Applications Selected In vivo Drug Metabolism Applications Selected In vivo Drug Metabolism Applications Metabolite ID Applications Metabolite ID Applications MS Imaging Applications MS Imaging Applications Conclusions Conclusions

4. 3 Topics Not Covered Proteomics Proteomics Biomarker discovery or assay Biomarker discovery or assay Metabolomics Metabolomics High Throughput Screening High Throughput Screening October 14, 2010 GBMSDG Talk

5. 4 Drug Discovery: From Library to Market Approved Drug Clinical Development Compound Libraries Lead Selection and Optimization Stages of Discovery and Development Number of compounds Early Discovery Lead Optimization Safety Testing Clinical Testing FDA Approval October 14, 2010

6. 5 GBMSDG Talk NEW DRUG DISCOVERY PIPELINE Chemistry Biology--HTS for Receptor Activity In-vitro Stability Screen In-vitro Absorption Screen P450 Enzyme Inhibition Screen CARRS Oral PK Screen Rat IV / PO PK Dog and Monkey IV / PO PK Rising Dose and Multiple Dose Studies and Safety Screens Drugs into Development Metabolite ID DMPK Lead Optimization

7. 6 October 14, 2010 GBMSDG Talk The Challenge How to deal with the multiple compounds at multiple stages in the drug discovery/drug development pipeline. How to deal with the multiple compounds at multiple stages in the drug discovery/drug development pipeline.

8. 7 October 14, 2010 GBMSDG Talk The Solution LC-MS and LC-MS/MS LC-MS and LC-MS/MS

9. 8 October 14, 2010 GBMSDG Talk Why use Mass Spectrometry? Specificity! Non-specific techniques, such as UV and fluorescence, are unable to provide proof of the analyte identity Specificity! Non-specific techniques, such as UV and fluorescence, are unable to provide proof of the analyte identity Ease of Use! Modern mass spectrometry software interfaces are easy to use Ease of Use! Modern mass spectrometry software interfaces are easy to use Versatility! MS is both a qualitative and quantitative technique Versatility! MS is both a qualitative and quantitative technique

10. 9 October 14, 2010 GBMSDG Talk The Challenge Choosing the right tool for the task : Choosing the right tool for the task : MS Toolbox Hammer? OR Wrench?

11. 10 October 14, 2010 GBMSDG Talk Common MS Tools

12. 11 October 14, 2010 GBMSDG Talk What is LC-MS? Liquid Chromatography Coupled to a Mass Spectrometer (In this case the Mass Spectrometer is a Single Quadrupole instrument) HPLCColumnIon sourceMass analyzerDetector APCI or ESI

13. 12 October 14, 2010 GBMSDG Talk The Challenge—Compound Synthesis At a big Pharma site, one might find hundreds of medicinal chemists who might produce 200- 1000 new compounds each week. These have to be assayed. At a big Pharma site, one might find hundreds of medicinal chemists who might produce 200- 1000 new compounds each week. These have to be assayed.

14. 13 October 14, 2010 GBMSDG Talk The Solution—LC-MS The LC-MS system based on a single quadrupole MS is a very useful tool for medicinal chemists who want to know if their synthesis is working correctly—did they make the right compound? Often this is set up as an open access tool. The chemists set up the run and get results within 24 hours.

15. 14 October 14, 2010 GBMSDG Talk The Challenge—Lead Optimization In Vitro Screening At a big Pharma site, one might get 100-200 new compounds each week that have to be screened in various in vitro assays. These have to be assayed separately for each screen. At a big Pharma site, one might get 100-200 new compounds each week that have to be screened in various in vitro assays. These have to be assayed separately for each screen.

16. 15 October 14, 2010 GBMSDG Talk The Solution—LC-MS/MS The LC-MS/MS system based on a triple quadrupole MS system is the tool of choice for most quantitative discovery bioanalytical applications. The application of this tool varies with the screen.

17. 16 October 14, 2010 GBMSDG Talk What is LC-MS/MS aka Triple Quadrupole Technology? Liquid Chromatography Coupled to a Tandem Mass Spectrometer (In this case the Mass Spectrometer is a Triple Quadrupole instrument) Q1Q2Q3 HPLCColumnIon sourceMass analyzerDetector

18. 17 October 14, 2010 GBMSDG Talk Quantitation Gold Standard: MS/MS – Selected Reaction Monitoring (SRM)* SelectFragmentSelect precursor ion in Q1precursor ion in Q2product ion in Q3 e.g. m/z 216(Collision Cell)e.g. m/z 174 * Often referred to as Multiple Reaction Monitoring (MRM)

19. 18 October 14, 2010 GBMSDG Talk Effects of Stages of Analysis on Signal, Noise, and Signal-to-Noise 1234 S/N Signal Noise Stages of Analysis LC LC-MS LC-MS/MS ? Important Concept!!

20. 19 October 14, 2010 GBMSDG Talk LC-TIC LC-MS

21. 20October 14, 2010GBMSDG Talk 10 ng/ml LC-MS/MS

22. 21 October 14, 2010 GBMSDG Talk Discovery In Vitro Screening P450 Assay--Enzyme Inhibition Screen P450 Assay--Enzyme Inhibition Screen Caco-2 cells—Absorption Screen Caco-2 cells—Absorption Screen Liver Microsomes/Hepatocytes--Metabolic Stability Screen Liver Microsomes/Hepatocytes--Metabolic Stability Screen Plasma Protein Binding Plasma Protein Binding Each In Vitro Assay Uses LC-MS/MS for the analytical step (typically a triple quadrupole MS system).

23. 22 October 14, 2010 GBMSDG Talk High Throughput CYP Inhibition Assay Example Generic LC-MS/MS method Generic LC-MS/MS method 1 minute gradient 1 minute gradient Monitors 3 substrates in a single LC-MS/MS run Monitors 3 substrates in a single LC-MS/MS run Template is used for creating sample list Template is used for creating sample list Automatic results calculation and import into Activity Base Automatic results calculation and import into Activity Base

24. 23 October 14, 2010 GBMSDG Talk Evaluate direct and mechanism-based inhibitors for P450 enzymes (3A4, 2D6, 2C9) to assess potential for drug-drug interactions. Purpose: In Vitro Evaluation of CYP Inhibition incubation cocktail ( 3A4, 2D6, 2C9) detection LC-MS- P450 source human liver substrates testosteroneDextromethorphanTolbutamine products 6  -hydroxytestosteronedextrophan4-hydroxytobutamide 3A4 2D6 2C9 Method: incubation cocktail detection LC-MS- P450 source human liver substrates testosteroneDextromethorphanTolbutamine products 6  -hydroxytestosteronedextrophan4-hydroxytobutamide 3A4 2D6 2C9 incubation detection LC-MS- P450 source human liver microsomes substrates testosteroneDextromethorphanTolbutamine products 6  -hydroxytestosteronedextrophan4-hydroxytobutamide 3A4 2D6 2C9 Method:

25. 24 October 14, 2010 GBMSDG Talk In Vitro Evaluation of CYP Inhibition Stock solution from CDC Serial dilution incubation/pre-incubation 1. Coincunation 2. Coincubation 3. Preincubation 4. Preincubation three concentrations/cpd: 20  M, 2  M and 0.2  M duplicates/each conc. 30 compounds/set

26. 25 October 14, 2010 GBMSDG Talk LC-MS/MS for p450 Inhibition Screen- Run time is less than 1 minute Substrate for CYP3A4, 6  -hydroxytestosterone Substrate for CYP2D6, dextrophan Internal Standard Substrate for CYP2C9, 4-hydroxytolbutamide m/z 305  269 m/z 258  157 m/z 287  171 m/z 347  121

27. 26 October 14, 2010 GBMSDG Talk CYP Inhibition Screen Throughput  Throughput: ~ 150 compounds /week, ~ 7000 samples/week  Analytical cycle-time: 48 hr from delivery to results NOTE: The advantage for this assay is that it is the same regardless of the test compound—no compound method development needed.

28. 27 October 14, 2010 GBMSDG Talk CYP Inhibition Screen Review

29. 28 October 14, 2010 GBMSDG Talk The Challenge— Metabolic Stability Screening At a big Pharma site, one might get 100-200 new compounds each week that have to be screened for metabolic stability. At a big Pharma site, one might get 100-200 new compounds each week that have to be screened for metabolic stability. The challenge is that LC-MS/MS methods have to be developed for each compound. The challenge is that LC-MS/MS methods have to be developed for each compound.

30. 29 October 14, 2010 GBMSDG Talk The Solution—LC-MS/MS + Software + Hardware Use a generic HPLC method. This works for 80-90% of the compounds. Use a vendor-supplied software tool for automated MS/MS method development, e.g.: QuickQuan™ (Thermo-Fisher)QuickQuan™ (Thermo-Fisher) QuanOptimise™ (Waters-Micromass)QuanOptimise™ (Waters-Micromass) DiscoveryQuant™ (AB-Sciex)DiscoveryQuant™ (AB-Sciex) Optimizer TM (Agilent)Optimizer TM (Agilent) Use robots for automated sample handlingUse robots for automated sample handling

31. 30 October 14, 2010 GBMSDG Talk Metabolic Stability Assay Example

32. 31 October 14, 2010 GBMSDG Talk Metabolic Stability Assay Layout shows how a robotic liquid handler can be used to perform the incubation and sample preparation steps in a metabolic stability assay.

33. 32 October 14, 2010 GBMSDG Talk Metabolic Stability Assay Scheme shows how a well organized system is needed to provide high throughput metabolic stability data.

34. 33 October 14, 2010 GBMSDG Talk In Vivo Assays Various types of in vivo assays are performed as part of new drug discovery and development Various types of in vivo assays are performed as part of new drug discovery and development The goal may be to understand absorption (A), distribution (D), metabolism (M), or excretion (E) properties of a compound The goal may be to understand absorption (A), distribution (D), metabolism (M), or excretion (E) properties of a compound The goal may be to get pharmacokinetic (PK) information on a compound The goal may be to get pharmacokinetic (PK) information on a compound

35. 34ASMS 2010 ADME-PK Studies Brain-- D Drug Levels— LC-MS/MS MS Image— MALDI-MS/MS Liver— D Drug Levels— LC-MS/MS Plasma— A Drug Levels— LC-MS/MS PK Parameters Dose NCE (Drug) PO/IV Ref: “Using Mass Spectrometry for Drug Metabolism Studies” W. Korfmacher, ed., CRC Press, 2005.

36. 35 October 14, 2010 GBMSDG Talk The Challenge— In Vivo PK Screening At a big Pharma site, one might get 50 - 100 new compounds each week that have to be screened for in vivo PK. At a big Pharma site, one might get 50 - 100 new compounds each week that have to be screened for in vivo PK. The challenge is that LC-MS/MS methods have to be developed for each compound. The challenge is that LC-MS/MS methods have to be developed for each compound.

37. 36 October 14, 2010 GBMSDG Talk The Solution—LC-MS/MS + Software + Planning Use a generic HPLC method. This works for 80- 90% of the compounds. Use a vendor-supplied software tool for automated MS/MS method development. Use robots for automated sample handling.Use robots for automated sample handling. Develop a standard PK screening assay.Develop a standard PK screening assay.

38. 37 October 14, 2010 GBMSDG Talk In Vivo PK Screening Source: Drug Discovery Today Volume 13, Numbers 7/8 April 2008 Authors: Bo Liu, Jonathan Chang, William P. Gordon, John Isbell, Yingyao Zhou and Tove Tuntland, Department of Pharmacology, Genomics Institute of the Novartis Research Foundation (GNF), San Diego, USA

39. 38 October 14, 2010 GBMSDG Talk PK Screening Example: CARRS PK Screening Example: CARRS Provide basic pharmacokinetic information for all rapid rat compounds. AUC (0-6hr) Concentration vs Time Profile (0-6 hr) Throughput: Up to 96 compounds per week

40. 39 October 14, 2010 GBMSDG Talk CARRS ASSAY Protein Precipitation Sample Preparation Generic UPLC conditions (1-2 min run time) Triple Quadrupole MS for assay (Two-point standard curve) Automated MS method development (QuanOptimize)

41. 40 October 14, 2010 GBMSDG Talk Preclinical PK Studies Typical study is one compound dosed oral (PO) and IV (Intravenous) in a laboratory animal. The goal is to get PK parameters in various preclinical species. Typical study is one compound dosed oral (PO) and IV (Intravenous) in a laboratory animal. The goal is to get PK parameters in various preclinical species. Typically this produces 50-60 plasma samples. Typically this produces 50-60 plasma samples. Sample preparation is protein precipitation. Sample preparation is protein precipitation. A multipoint standard curve is prepared for the assay A multipoint standard curve is prepared for the assay Analysis is by LC-MS/MS on a triple quadrupole MS/MS system. Usually a generic internal standard is used for the assay. Analysis is by LC-MS/MS on a triple quadrupole MS/MS system. Usually a generic internal standard is used for the assay.

42. 41 October 14, 2010 GBMSDG Talk Discovery PK Analysis Flowchart The MS/MS instrument is normally a triple quadrupole system

43. Rapid MS Method Development ion a Discovery Environment Xu et al. Anal. Chem.--2005 GBMSDG Talk October 14, 2010

44. 43 October 14, 2010 GBMSDG Talk Typical Discovery PK Assay 1 – 10,000 ng/mL Response ratio

45. 44 October 14, 2010 GBMSDG Talk Discovery Metabolite ID Generally this has two components: Generally this has two components: Lead Optimization Phase--would use unlabelled compounds and in vitro samples to look for major routes of metabolism. Lead Optimization Phase--would use unlabelled compounds and in vitro samples to look for major routes of metabolism. Pre-Recommendation Phase—Look for problem metabolites plus in vitro comparison of human to animal metabolism. Pre-Recommendation Phase—Look for problem metabolites plus in vitro comparison of human to animal metabolism.

46. 45 October 14, 2010 GBMSDG Talk Discovery Metabolite ID Mass Spectrometry serves two purposes: Mass Spectrometry serves two purposes: Finding metabolites-MS systems can be used in various ways to find metabolites in multilple biological matrices (e.g., plasma, bile, urine). Finding metabolites-MS systems can be used in various ways to find metabolites in multilple biological matrices (e.g., plasma, bile, urine). Structure elucidation—MS systems can be used to to obtain partial or complete structural identification for the metabolites Structure elucidation—MS systems can be used to to obtain partial or complete structural identification for the metabolites

47. 46October 14, 2010GBMSDG Talk Triple Quad Scan Functions: to find metabolites Neutral Loss Scan Neutral Loss Scan no prior knowledge of the parent is required—this is used to look for certain classes of metabolites (e.g., glucuronide, sulfate or glutathione conjugates) no prior knowledge of the parent is required—this is used to look for certain classes of metabolites (e.g., glucuronide, sulfate or glutathione conjugates) Precursor Ion Scan Precursor Ion Scan only fragmentation pattern of parent is required—may find unexpected metabolites only fragmentation pattern of parent is required—may find unexpected metabolites SRM/MRM SRM/MRM the fragmentation pattern of parent is used to predict the fragment ions for likely metabolites—some vendors have software tools that make it easy to build a scan set the fragmentation pattern of parent is used to predict the fragment ions for likely metabolites—some vendors have software tools that make it easy to build a scan set

48. 47 October 14, 2010 GBMSDG Talk MS Tools Triple Quadrupole MS systems are the premier analytical tool for LC-MS quantitative assays. They are also useful for metabolite ID applications Q-TOF MS systems are best used for metabolite ID applications and for Imaging MS applications QTrap MS systems are excellent tools for quantitative analyses as well as for metabolite ID applications

49. 48 October 14, 2010 GBMSDG Talk QTrap MS Publication In Vivo PK Samples Simultaneously quantifying parent drugs and screening for metabolites in plasma pharmacokinetic samples using selected reaction monitoring information-dependent acquisition on a QTrap instrument: Simultaneously quantifying parent drugs and screening for metabolites in plasma pharmacokinetic samples using selected reaction monitoring information-dependent acquisition on a QTrap instrument: Li et al. (Covance), RCMS, 1943, 2005.

50. Typical Metabolite Profiling Experiments and Instrumentation HPLC Radiometric Flow detector Mass Spectrometer Injector Sample from in vivo or in vitro studies 051015202530354045 Time (min) 0 20 40 60 80 100 LC/MS Chromatogram 051015202530354045 Time (min) 0 20 40 60 80 100 Radiocarbon Chromatogram 10-50% 50-90% Radioactivity helps to locate the metabolites in the samples GBMSDG Talk October 14, 2010

51. 50 October 14, 2010 GBMSDG Talk Product Ion Scan Select Precursor Ion Scan Products Fragmentation m1+m1+ m2+m2+ m2+m2+ m2+m2+ Product ion spectrum of a particular compound A key technique for obtaining structural information.

52. 51 October 14, 2010 GBMSDG Talk Discovery Metabolite ID (A) HPLC Radiochromatogram of 14C-Gemfibrozil at 25 mm Incubated in Human Liver Microsomes Fortified with NADPH and UDPGA; (B) Reconstructed Ion Chromatogram; (C) Full Scan Mass Spectrum of M1 [M-H] -. Xia, Y.Q. et al., Use of a quadrupole linear ion trap mass spectrometer in metabolite identification and bioanalysis, Rapid Commun. Mass Spectrom., 17(11), 1137, 2003. MS/MS spectrum of M1

53. 52 October 14, 2010 GBMSDG Talk What is MIST? M ass Spectrometry M ass Spectrometry I nvestigators I nvestigators S ecurity S ecurity T rust T rust MIST will ensure job security for MS metabolite ID experts MIST will ensure job security for MS metabolite ID experts

54. 53 October 14, 2010 GBMSDG Talk What is MIST? Metabolites Metabolites I n I n S afety S afety T esting T esting MIST will ensure job security for MS metabolite ID experts MIST will ensure job security for MS metabolite ID experts

55. 54 October 14, 2010 GBMSDG Talk Key MIST Points 1. Human metabolites that can raise a safety concern are those formed at greater than 10 percent greater than 10 percent of parent drug’s systemic exposure of parent drug’s systemic exposure at steady state. at steady state. 2. Metabolites identified only in human plasma or Metabolites present at disproportionately higher levels in humans than in any of the animal test species should be considered for safety assessment. Bottom line: Find human metabolites and then be sure they are “covered” in the tox species. Bottom line: Find human metabolites and then be sure they are “covered” in the tox species.

56. 55 October 14, 2010 GBMSDG Talk New MS Tool for Finding Metabolites: HRMS High Resolution Mass Spectrometry (HRMS) has become the tool of choice for finding metabolites in complex biological matrices. High Resolution Mass Spectrometry (HRMS) has become the tool of choice for finding metabolites in complex biological matrices. The improved mass resolution can be used to differentiate metabolites from endogenous background The improved mass resolution can be used to differentiate metabolites from endogenous background Software tools can use HRMS to find metabolites Software tools can use HRMS to find metabolites The accurate mass of a detected metabolite can help to confirm its identity by leading to its empirical formula The accurate mass of a detected metabolite can help to confirm its identity by leading to its empirical formula

57. 56 October 14, 2010 GBMSDG Talk Two HRMS Systems LTQ-Orbitrap The TOF MS provides mass resolution of 10,000- 50,000 The Orbitrap MS provides mass resolution of 10,000-100,000

58. 57 October 14, 2010 GBMSDG Talk Why High Mass Resolution? TOF-MS of Sidenafil Example Scan: 200 - 800 MS window: 1 Da MS window: 0.1 Da MS window: 0.01Da MS window: 0.001 Da

59. 58 October 14, 2010 GBMSDG Talk “Fish-out” drug-derived peaks from endogenous peaks in a complex biological matrix Key utility in non-radio-labeled drug-administration An alternative to triple quadrupole tools: neutral loss scan (NLS) and precursor ion scan (PIS) Use of Mass Defect Filter for Post- Acquisition Processing of Accurate Mass (High Resolution) LC-MS Data. M. Zhu et al., Drug Metab. Dispos. 2006, 34, 1722-1733

60. 59 October 14, 2010 GBMSDG Talk Mass Defect Filter Reference J. Mass Spectrom., 2009, 44, 999-1016.

61. 60 October 14, 2010 GBMSDG Talk Exact Mass and Isotopic Abundance of Common Elements

62. 61 October 14, 2010 GBMSDG Talk (B) (C) Source: JMS 2003, 38, 1110-1112 Mass Defect Filter Example TIC 14 C Processed MS

63. 62 October 14, 2010 GBMSDG Talk Mass spectra of metabolite x at retention time 35.5 min (A) Full scan spectrum of metabolite x form the unprocessed total ion chromatogram. (B) Detail of (A) in mass range 450-550 Da. (C) Full scan spectrum of metabolite x (the molecular ion was at m/z 503.0737 from the MDF processed total ion chromatogram. (B) (C) Source: JMS 2003, 38, 1110-1112 Mass Defect Filter Example

64. 63 October 14, 2010 GBMSDG Talk Metabolite ID Software Tool: BgS-NoRA Published in RCMS, 23, 1563 (2009).

65. 64 October 14, 2010 GBMSDG Talk Metabolite ID Software Tool: BgS-NoRA Mouse urine spiked with diclofenac microsomal incubation sample TIC TIC after Background Subtraction TIC after BgS-NoRA P = parent M1, M2, M3 = metabolites

66. 65October 14, 2010GBMSDG Talk Metabolite ID Software Tool: BgS-NoRA Mouse urine spiked with diclofenac microsomal incubation sample—peak at 7.8 min Unprocessed data Mass spectrum after data processed with BgS-NoRA

67. 66 October 14, 2010 GBMSDG Talk Which tool is Best for Metabolite ID? Published in RCMS, 24, 939 (2010).

68. 67 October 14, 2010 GBMSDG Talk Additional Development Stages that use MS Analysis

69. 68 October 14, 2010 GBMSDG Talk TK Study Support Typical study is one compound dosed oral (PO) in a laboratory animal. The goal is to get TK (toxicokinetic) parameters. Typical study is one compound dosed oral (PO) in a laboratory animal. The goal is to get TK (toxicokinetic) parameters. This is a GLP (Good Laboratory Practices) study. This is a GLP (Good Laboratory Practices) study. Sample preparation is typically SPE. Sample preparation is typically SPE. A multipoint standard curve is prepared for the assay. A multipoint standard curve is prepared for the assay. Analysis is by LC-MS/MS on a triple quadrupole MS/MS system. Usually a SIL (stable isotope label) internal standard is used for the assay. Analysis is by LC-MS/MS on a triple quadrupole MS/MS system. Usually a SIL (stable isotope label) internal standard is used for the assay.

70. 69 October 14, 2010 GBMSDG Talk Clinical PK Study Support Typical study is one compound dosed oral (PO) in humans. The goal is to get PK parameters. Typical study is one compound dosed oral (PO) in humans. The goal is to get PK parameters. This is a treated as a GLP (Good Laboratory Practices) study. This is a treated as a GLP (Good Laboratory Practices) study. Sample preparation is typically SPE. Sample preparation is typically SPE. A multipoint standard curve is prepared for the assay. A multipoint standard curve is prepared for the assay. Analysis is by LC-MS/MS on a triple quadrupole MS/MS system. Usually a SIL (stable isotope label) internal standard is used for the assay. Analysis is by LC-MS/MS on a triple quadrupole MS/MS system. Usually a SIL (stable isotope label) internal standard is used for the assay.

71. 70 October 14, 2010 GBMSDG Talk Impurities and Degradants These studies are performed to support safety studies or clinical studies. These studies are performed to support safety studies or clinical studies. The goal is to measure any significant impurities or degradants that are in the pharmaceutical test compound batch used for these studies. The goal is to measure any significant impurities or degradants that are in the pharmaceutical test compound batch used for these studies. Generally, one would use a combination of triple quadrupoles as well as QTOF MS systems as well as the Orbitrap MS system to characterize these compounds. Generally, one would use a combination of triple quadrupoles as well as QTOF MS systems as well as the Orbitrap MS system to characterize these compounds.

72. 71 October 14, 2010 GBMSDG Talk MS Imaging—a Specialty use of MS

73. 72 October 14, 2010 GBMSDG Talk MS Imaging using the MALDI QqTOF Quadrupole Mass Filter Collision Cell Time-of- Flight Analyzer Detector h +

74. 73 October 14, 2010 GBMSDG Talk Optical Image Radioautographic ImageMALDI-MS/MS Image 1000 µm MALDI-MS/MS Image is in good agreement with the radioautographic image Rat Brain Tissue Slice --Rat dosed with clozapine Hsieh Y, et al., Rapid Commun Mass Spectrom. 2006;20(6):965-72.

75. 74 October 14, 2010 GBMSDG Talk Mass Spectral Data Confirms the Presence of Clozapine

76. 75 October 14, 2010 GBMSDG Talk Next Step: Whole Mouse Slice MS Imaging

77. 76 October 14, 2010 GBMSDG Talk liver stomach GI tract Ion image of fexofenadine (metabolite). Ion image of terfenadine (parent). Optical image of whole-body mouse slice Mouse Whole Body Image—Mouse Dosed with Terfenadine Result: These MS images allow us to “visualize” first-pass metabolism. Source: Chen et al., Drug Metab. Lett., 2, 1-4 (2008) 100 mpk p.o. 100 mpk p.o. and sacrificed and sacrificed 4 h post dosing 4 h post dosing

78. 77 Description of Technology Automated liquid extraction-based surface sampling technique utilizing the robotic Advion Nanomate chip- based nanoelectrospray platform. Method invented at Oakridge National Laboratory (ORNL). Developed and commercialized at Advion. Liquid microjunction created between the robotically controlled pipette tip dispensing solvent and surface (e.g. tissue section, blood spots on paper). The microfluidics chip contains an array of nanoelectrospray nozzles etched in a silicon wafer eliminating carryover as one tip and one nozzle is used per sample. Can be used for a variety of samples including tissue sections, dried blood spots on paper, samples on MALDI plates for complimentary information by electrospray ionization (ESI), TLC plates, and other planar separation media. Advion Nanomate LESA (Liquid Extraction Surface Analysis) System Advion NanomateESI Chip Liquid microjunction between pipette tip and tissue surface Schematic of chip based nano-ESI infusion http://www.advion.com/biosystems/triversa-nanomate/LESA/index.php GBMSDG TalkOctober 14, 2010

79. 78 Whole Body Distribution of a Drug and its Metabolites by QWBA vs LESA T 7.5 mg/kg IV ‘Cold’ Propranolol MS Tissue Imaging/Sampling: Sections transferred to glass slides with UV-activated adhesive or collected on adhesive tape and sent to ORNL for MS tissue imaging/profiling 7.5 mg/kg IV [ 3 H] Propranolol QWBA (Quantitative Whole Body Autoradiography): QWBA study with metabolite ID by radioprofiling in conjunction with nanospray MS or accurate mass MS at Merck Male CD-1 Mice T T

80. 79 QWBA Brain Lung Liver Stomach Kidney 20 µM-eq 40 µM-eq 21 µM-eq 31µM-eq 45 µM-eq Autoradioluminograph [ 3 H]Propranolol Drug Related Material 40 µm Mouse Whole Body Sagittal Section: 60 min post [ 3 H]Propranolol IV Dose QWBA Results Confirmed High Levels of [ 3 H] Propranolol Related Material in Brain, Lung, Liver, and Kidney 0 100 GBMSDG TalkOctober 14, 2010

81. 80 Identification of [ 3 H] Drug Related Material (DRM) in Tissues Unchanged parent detected in lung and brain. Major metabolites in liver and kidney identified as hydroxypropranolol glucuronide metabolites by LC- MS/MS-rad. GBMSDG TalkOctober 14, 2010

82. 81 Normal Operation using the Advion Nanomate System Mass spectrometer Sample Sampling tip Nozzle Aspirate sample Transfer sample Apply HV, spray voltage GBMSDG TalkOctober 14, 2010

83. 82 Operation using the Nanomate System for Surface Sampling – ORNL Invention (Advion LESA) Mass spectrometer Sample On Surface Solvent Sampling tip Aspirate solvent Dispense solvent on sample Aspirate sample solution Transfer sample Spray sample Nozzle Vilmos Kertesz, Gary J. Van Berkel „Fully Automated Liquid Extraction-Based Surface Sampling and Ionization Using a Chip-Based Robotic Nanoelectrospray Platform” J. Mass Spectrom., 2010 Mar;45(3):252-60. ~ 1 mm spot size

84. LESA: MS for Detection of Propranolol (7.5 mg/kg IV) and a Major Metabolite in Tissues Dosed tissue Control tissue stomach liver lung kidney muscle brain kidney muscle brain lung liver stomach Propranolol Hydroxypropranolol glucuronide Lung Liver Stomach Kidney Brain Muscle Lung Liver Kidney Muscle t,min Rapid and automated technique to sample tissues including ones on tape used for QWBA. Successful detection of propranolol and its major glucuronide metabolite not seen by DESI-MS. Stomach Brain

85. 84 October 14, 2010 GBMSDG Talk Conclusions Mass spectrometry is used at multiple stages of new drug discovery and development. Various types of mass spectrometers are utilized including single quadrupoles, triple quadrupoles, Q-Traps, Q-TOFs and Orbitrap MS systems. The need for the multiple types of MS system is due to the variety of assays that are mandated at the different stages in the new drug discovery process. Mass spectrometry is used at multiple stages of new drug discovery and development. Various types of mass spectrometers are utilized including single quadrupoles, triple quadrupoles, Q-Traps, Q-TOFs and Orbitrap MS systems. The need for the multiple types of MS system is due to the variety of assays that are mandated at the different stages in the new drug discovery process.

86. 2008 2009 MS Reference Books Available at Amazon.com 2009 2005

87. 86 October 14, 2010 GBMSDG Talk Acknowledgments (Thanks to the following for one or more slides) Waters-MicroMass Waters-MicroMass Thermo-Fisher Thermo-Fisher AB-Sciex AB-Sciex Agilent Agilent Marissa Vavrek Marissa Vavrek Rick King Rick King Swapan Chowdhury Joanna Zgoda-Pols Michelle Reyzer Yunsheng Hsieh Fangbiao Li

88. 87 October 14, 2010 GBMSDG Talk Acknowledgements

89. 88 October 14, 2010 GBMSDG Talk Thank you for your attention! ?