Implementation of HPLC/MS-based methods and solutions for the metabolomic study in new drug discovery

Transcription

1 Implementation of HPLC/MS-based methods and solutions for the metabolomic study in new drug discovery Александр Кирилюк, PhD Москва, ул Краснопролетарская, 7 т

2 What is Metabolite ID? Why is it done? Need to determine what happens to a drug after it is ingested Body acts on drug to break it down, make it more polar and therefore more easily eliminated Goal of Metabolite Identification is to find and identify these metabolites Must determine whether these metabolites are toxic, accumulate in the body, or are eliminated harmlessly Metabolism reactions divided into Phase I And Phase II biotransformations Phase I (functionalization reactions) introduces a functional group or exposes a pre-existing functional group via oxidation, hydroxylation, demethylation Phase II (biosynthetic reactions) addition of hydrophilic endogenous species such as glucuronic acid or sulfate Metabolism occurs primarily in the liver To simulate drug metabolism, researchers use human liver microsomes or hepatocytes for in vitro drug metabolism studies AB SCIEX

3 Metabolism Studies: Pharmacokinetics describes what the body does to the drug Absorption Distribution Metabolism (Identification being a subset) Excretion Pharmacodynamics explores what the drug does to the body a whole lot of Qualitative and Quantitative work to understand effects on drug and body AB SCIEX

4 The average time from synthesis of a potential compound to approval of a new drug application (NDA) has increased significantly to years AB SCIEX

5 Metabolism Excretion Pharmacokinetics / Efficacy (CLint) Drug enzymes Metabolites Interact w/ enzyme Inhibition Induction Drug-Drug Interaction (Ki, Km, dose adjustment) Covalent Binding to Macromolecules Adverse Reaction/ Toxicity (Covalent binding; Oxidative stress; Mito dysfunction) Metabolism is the complete set of chemical reactions that occur in living cells (elimination of drugs) AB SCIEX

6 Metabolite Identification has numerous goals through the pipeline Discovery Identification of Metabolites which are formed when a xenobiotic drug enters the body Identification of sites of metabolism on a compound so it can be redesigned in order to prevent toxic or unwanted metabolism Identification of quick-forming metabolites Screening for toxicity (acyl glucaronides, glutathiones) Typically uses in vitro samples relatively clean matrices Speed is of the essence id 80% of metabolites focusing on most prevalent Development First-in-man definitive metabolite study Must conclusively ID all metabolites Monitoring active metabolites in dirty matrices Lower throughput but need to identify everything that can be detected Often use radioisotope labeled drugs to do mass balance studies ensure can account for all of parent drug AB SCIEX

7 Metabolite ID Workflow Relatively simple experiment Customers run sample and control Control is matrix blank (no parent drug dosed/incubated) Sample contains parent drug and any metabolites that are formed Compare sample and control any peaks in sample that are not in control should be metabolites AB SCIEX

8 Metabolite ID Compile a list of all possible metabolites major minor Sample Preparation (in-vitro, in-vivo) Define strategy to identify confirm characterize AB SCIEX

9 in vitro and in vivo Drug Metabolism Systems Common in vitro samples (Our Standards) Hepatocyte: fresh or cryo-preseved Liver S9 fraction Liver microsome Recombinate P450s and other metabolizing enzyms Common in vivo samples (Our Living Systems) Urine: relative clean; dilute and inject Bile: dirty due bile salt; SPE or dilute Feces: Biliary excretes and parent; SPE Plasma: PPT AB SCIEX

10 Strategy for Metabolite Identification Acquire control & sample of interest Find Metabolites Acquire MS/MS Spectra Confirm Metabolites with MS/MS Spectra Report AB SCIEX

11 Current Technologies Used for Small Molecules TripleQuad Quantitation ID with MRM ratio QTRAP Quantitation ID with MRM ratio ID with MS/MS library searching TripleTOF Quantitation ID with accurate mass ID with MS/MS library searching ID for true unknowns Retrospective data processing Increasing confidence in unknown compound identification AB SCIEX

12 Взаимодополняющие решения для метаболомных задач Hardware, Software, & Kits for Non-Targeted and Targeted Workflows TripleTOF 5600 system solutions QTRAP 5500 system solutions Нецелевой Целевой AB SCIEX

13 Сравнение Нецелевого и Целевого Анализа Non-Targeted Analysis MS MS/MS Targeted Analysis - MRM Advantages: Detects any species Disadvantages: Typically lower dynamic range and sensitivity with a separate ID step required Requires sophisticated informatics Advantages: High sensitivity, throughput and dynamic range High quant quality Simplified informatics Disadvantages: Only detects specific species Upfront method development AB SCIEX

14 Общий Подход в Метаболомике Metabolic Profiling Full scan MS MS/MS Targeted Metabolomics QQQ MS/MS Differential Pattern Information ID of Relevant Metabolites ID / Quantitation by MS/MS Metabolite Concentration Shifts Functional Annotation AB SCIEX

15 Strategies for Metabolite ID pmrm approach Most sensitive technique Comprehensive list of potential metabolites Can screen hundreds of MRM s in a single analysis MS/MS for confirmation collected in automated single injection analysis Multiple Precursor Ion / Neutral Loss Structure specific filtering Ideal for reactive metabolite detection (GSH conjugation) AB SCIEX

16 QTRAP System with LightSight Software Key Benefits for Metabolite ID Advanced workflows High throughput GSH reactive metabolite screening enabled by: Fast polarity switching High LIT sensitivity and speed 20,000 Da/s Fast precursor ion and neutral loss scanning Automated method building LightSight Software Non-targeted metabolite identification Very short dwell times 1-2 ms High Q3 scanning speed and sensitivity AB SCIEX

17 Detection of GSH Conjugates of Reactive Metabolites O OH NH NH H 2 N O O SH O OH OH O O HO O HN H 2 N O NH HS (+) Neutral Loss of 129 (-) Precursors of m/z 272 Applicable to a wide variety of GSH adducts Sensitive and selective Optimum collision energy can vary significantly based on substrate Glutamic acid loss is common Highly selective since it is done in negative ion mode Collision energy is largely independent of substrate structure High sensitivity Requires polarity switch back to positive ion to obtain full product ion spectrum AB SCIEX

18 Comprehensive GSH Conjugate Detection Intensity 2,000 Da/s (+)NL 129 and ( ) Prec of m/z 272 Dual Scans With Fast Polarity Switching (+)NL 129 (-)Prec 272 IDA Enhanced Resolution (+)ER (+)EPI Spectrum from GSH.wiff (sample 13) - troglitazone GSH t60...roglitazone GSH-IDA2, Experiment 4, +EPI (80-800) from min Precursor: Da CH 3 CH H O 2.0e7 3 C O O HO 1.5e7 CH 3 S NH 1.0e7 5.0e GS 440 O ,000 Da/s EPI Scan (+)EPI 0.0e Mass/Charge, Da AB SCIEX

19 Why is the QTRAP System Ideal for this Analysis? Detection: Fast scanning and high sensitivity in precursor ion and neutral loss modes. Fast polarity switching Confirmation Fast scanning (20,000 Da/s) and high sensitivity in Enhanced Product Ion mode for structure elucidation. Rich fragmentation in product ion spectra Usability Automatic method building of complex methods and user friendly data analysis Combined Neutral Loss and Precursor Ion scans provide complementary detection, maximum coverage, and full product ion spectra in the same run AB SCIEX

20 Advanced Transformation List Created in LightSight 2.1 Software AB SCIEX

21 Survey Scan (1) Survey Scan (2) IDA: Powerful & Flexible Survey scan be EMS, EMC, Neutral Loss, Precursor Scan, MRM or EPI (combinations of 2 surveys) Enhanced Resolution 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 ) AB SCIEX

22 Phase II Metabolites Neutral Loss of 80 Neutral Loss of 176 Enhanced Resolution Get Mass of Glucuronide and Sulfate Conjugate Get Isotope Profile Acquire Isotope Ratio Add to Exclusion List Acquire MSMS Spectrum MS 3 MS 3 Get Product Ion Spectrum Fragmentation of Aglicon AB SCIEX

23 Comprehensive Data Processing and Method Building Environment in LightSight Software Potential Metabolites List MS Data Automatic XIC of Selected Metabolite MS/MS Data Now Windows 7 Compatible! AB SCIEX

24 Automatic QTRAP System Method Building Acquisition methods for the QTRAP System combining precursor ion and neutral loss in the same method. Create acquisition methods with 2 survey scans AB SCIEX

25 Theoretical MRM List Generated by LightSight 2.1 Software Q1 Q3 Dwell DP CE CXP Q1 Q3 Dwell DP CE CXP LightSight 2.1 Software automatically lowers declustering potential for glucoronides AB SCIEX

26 QTRAP 5500 (Nefazadone) pmrm AB SCIEX

27 Спасибо AB SCIEX