Tandem Mass Spectroscopy Basics


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A talk outlining the basics of method development using a tandem mass spectrometer

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Tandem Mass Spectroscopy Basics

  1. 1. Implmentation of TMS at BHH
  2. 2. Mass Spectrometry <ul><li>‘ Mass spectrometry is the study of systems causing the formation of gaseous ions, with or without fragmentation, which are then characterised by their mass to charge ratios (m/z) and relative abundances’ </li></ul>
  3. 3. General Principles
  4. 4. LC <ul><li>Shimadzou Prominence System </li></ul><ul><li>2 x Binary Pumps </li></ul><ul><li>1 x Autosampler </li></ul><ul><li>1 x Column Oven </li></ul>
  5. 5. General Principles
  6. 6. Inlet (Source) <ul><li>TurboIonSpray </li></ul><ul><li>(ESI and APCI capable) </li></ul><ul><li>flow rates from 2 to 1000 uL/min </li></ul>
  7. 7. Interface
  8. 8. General Principles
  9. 9. Ionisation <ul><li>If a quantity of energy is supplied to a molecule equivalent to the ionisation energy of the molecule, a molecule ion is formed M+ </li></ul><ul><li>There are several ways of doing this </li></ul><ul><ul><li>Electron impact (EI) </li></ul></ul><ul><ul><li>Chemical Ionisation (CI) </li></ul></ul><ul><ul><li>Atmospheric pressure Ionisation (API) </li></ul></ul><ul><ul><li>Electrospray (ESI) </li></ul></ul>
  10. 10. ESI <ul><li>Stream of solution sprayed out of capillary at high voltage (ca. 3 – 5 kV) </li></ul><ul><li>Charged droplets formed by spray (“Taylor cone”) </li></ul><ul><li>Solvent evaporated by stream of warm N 2 </li></ul><ul><li>As droplet shrinks, charge density increases until analyte ions ejected </li></ul><ul><li>Pseudomolecular [M+H] + ions formed </li></ul><ul><li>Solvent pumped away and ions admitted to mass spectrometer </li></ul>
  11. 11. ESI Soft ionisation technique; little fragmentation Large molecules may be protonated more than once Peaks seen for same compound with different m/z ratios
  12. 14. APCI <ul><li>A sample solution flows through a heated tube where it is volatilized and sprayed into a corona discharge with the aid of nitrogen nebulization. </li></ul><ul><li>Ions are produced in the discharge and extracted into the mass spectrometer. </li></ul><ul><li>APCI i s best suited to relatively polar, semi-volatile samples. An APCI mass spectrum usually contains the quasi-molecular ion, [M+H]+. </li></ul>
  13. 16. General Principles
  14. 17. Mass Separation <ul><li>Quadrupole mass filters </li></ul><ul><li>Four rods arranged precisely with DC and RF alternating voltages applied to pairs </li></ul><ul><li>Ions enter quadrupole region </li></ul><ul><li>Because of RF voltage and DC offset the polarity of each pair of rods continually changes </li></ul>
  15. 18. Mass Separation <ul><li>Ion in quadrupole is alternately repelled and attracted to given rod </li></ul><ul><li>Ion follows helical path through quadrupole </li></ul><ul><li>For given RF and DC voltage settings only certain m/z ions have stable trajectory to detector – the rest collide with rods </li></ul><ul><li>By changing values of the voltages different m/z ions can be focussed onto the detector </li></ul><ul><li>Quadrupole mass filter transmits one m/z ratio at once </li></ul>
  16. 19. Mass Separation
  17. 20. General Principles
  18. 21. Detection <ul><li>Photographic plate </li></ul><ul><li>Faraday cage </li></ul><ul><li>Electron multiplier </li></ul><ul><li>Photomultiplier </li></ul><ul><li>Charge collectors </li></ul>
  19. 22. Electron Multiplier
  20. 23. Electron Multiplier <ul><li>Amplified current measured and related to ion count </li></ul><ul><li>Sensitive </li></ul><ul><li>Allows for rapid scanning </li></ul>
  21. 24. TMS
  22. 28. Data Acquisition Modes <ul><li>Simple MS Scanning </li></ul><ul><ul><li>Using one quadrapole </li></ul></ul><ul><li>Product ion scan (daughter) </li></ul><ul><ul><li>MS1 static MS2 scanning </li></ul></ul><ul><li>Precursor ion scan (parent) </li></ul><ul><ul><li>MS1 scanning MS2 static </li></ul></ul>
  23. 29. Data Acquisition Modes <ul><li>Neutral loss scan </li></ul><ul><ul><li>MS1 and MS2 scanning </li></ul></ul><ul><ul><li>But synchronised </li></ul></ul><ul><li>Multiple reaction monitoring (MRM) </li></ul><ul><ul><li>MS1 and MS2 static </li></ul></ul><ul><ul><li>Enhanced sensitivity </li></ul></ul>
  24. 30. MRM <ul><li>Highly specific </li></ul><ul><li>Only data on analytes of interest is collected </li></ul><ul><li>All other compounds are ignored </li></ul><ul><li>More sensitive than full scan mode </li></ul><ul><li>Quadrapoles can spend longer scanning as fewer transitions to monitor </li></ul><ul><li>Faster flow rates into ion source </li></ul><ul><li>Possible quicker analyses </li></ul>
  25. 31. MRM
  26. 32. TMS
  27. 33. Compound Dependent Parameters <ul><li>De-clustering potential ( DP ) - the potential difference between the ground (usually the skimmer) and the orifice plate. Used to minimize solvent cluster ions, which may attach to the sample. The higher the voltage the greater the amount of fragmentation </li></ul><ul><li>Entrance Potential ( EP )- Focuses the ions through the high pressure Q0 region </li></ul><ul><li>Collision cell entrance potential ( CEP )- Focuses ions into the collision cell </li></ul>
  28. 34. Compound Dependent Parameters <ul><li>Collision Energy ( CE ) - The amount of energy precursor ions receive as they are accelerated into the collision cell. </li></ul><ul><li>Collision Gas ( CAD ) </li></ul><ul><li>Collision Cell Exit Potential ( CXP ) </li></ul>
  29. 38. Source Optimization Parameters <ul><li>Gas 1 - Helps Generate small Droplets of sample flow </li></ul><ul><li>Gas 2 - Turbo gas, helps evaporate the spray droplets and prevents solvent entering the system </li></ul><ul><li>Temperature - Temperature of the Turbo Gas </li></ul>
  30. 39. Source Optimization Parameters <ul><li>Curtain Gas - Prevents solvent droplets from entering and contaminating the ion optics </li></ul><ul><li>Ion Spray voltage - The voltage applied to the needle that ionises the sample at the ion source </li></ul><ul><li>Nebulizer or needle current - The current applied to the corona discharge needle in APCI </li></ul><ul><li>Interface Heater - prevents contamination of ion optics </li></ul>
  31. 40. Common Problems <ul><li>Interfering compounds (Iso Baric Compounds) </li></ul><ul><li>Ion Suppression </li></ul><ul><li>Ionisation Problems </li></ul>
  32. 44. Ion Suppression <ul><li>Salts can interfer with ionisation and can cluster to complicate spectrum </li></ul><ul><li>Strong Bases or quaternary amines can interfer with positive mode analytes e.g. Triethylamine (TEA) </li></ul><ul><li>Acids - Sulfuric and TFA interfer in negative mode experiments </li></ul><ul><li>Phosphate Buffer and non-volatile ion-paring agents (e.g. SDS) can cause severe suppression and complex spectra </li></ul><ul><li>Non - covalent Dimers in Ion Spray </li></ul><ul><ul><li>Dimer signal = (MW*2)+1 </li></ul></ul><ul><ul><ul><li>can cause linearity at high concentrations </li></ul></ul></ul>
  33. 45. Adducts and Clusters Formed in LC-MS Cluster/Adduct Source of Cluster Occurence Mass of Cluster Ion [M + CH3COO]- Acetic Acid APCI & IS -ve mode M + 59 [M + Cl]- Chlorinated Solvent APCI & IS -ve mode M + 35 [M + NH4]+ Ammonia APCI & IS +ve mode M + 18 [M + Na]+ Sodium Salts APCI & IS +ve mode M + 23 [M+K]+ Potassium Salts APCI & IS +ve mode M + 39 [M + CH3CNH]+ Acetonitrile APCI & IS +ve mode M + 42 [M+CH3OHH]+ Methanol APCI & IS +ve mode M + 33 [M+H3O]+ Water APCI & IS +ve mode M + 19
  34. 46. Plans <ul><li>Vitamin D </li></ul><ul><li>Drugs of Abuse Testing </li></ul><ul><li>Steroid Analysis </li></ul><ul><li>R&D </li></ul>
  35. 51. Simultaneous Detection of following Drugs by LC-MS/MS <ul><li>Benzoylecgonine (Cocaine metabolite) </li></ul><ul><li>Opiates </li></ul><ul><ul><li>EDDP (Methadone metabolite) </li></ul></ul><ul><ul><li>Methadone </li></ul></ul><ul><ul><li>Morphine </li></ul></ul><ul><ul><li>6-MAM (Heroin metabolite) </li></ul></ul><ul><ul><li>Codeine </li></ul></ul><ul><ul><li>Dihydrocodeine </li></ul></ul><ul><li>Amphetamine </li></ul><ul><li>Methamphetamine </li></ul><ul><li>Ecstasy metabolites </li></ul><ul><ul><li>MDA </li></ul></ul><ul><ul><li>MDMA </li></ul></ul><ul><ul><li>MDEA </li></ul></ul><ul><li>Cannabis </li></ul><ul><ul><li>D9-tetrahydrocannabinol (THC) </li></ul></ul><ul><ul><li>11-nor-D9-carboxy THC (THC-COOH) </li></ul></ul><ul><li>Benzodiazepines </li></ul><ul><ul><li>Tamazepam </li></ul></ul><ul><ul><li>Diazepam </li></ul></ul><ul><ul><li>Nitrazepam </li></ul></ul><ul><ul><li>7-amino Nitrazepam </li></ul></ul><ul><ul><li>Nordiazepam </li></ul></ul><ul><li>Buprenorphine </li></ul><ul><li>Gamma Hydroxybutyrate (GHB) </li></ul>
  36. 52. DOA screening by LC-MS/MS <ul><li>Due to specificity of the tandem MS no need for a confirmation method </li></ul><ul><li>Advantages - Multiple transitions should decrease interference by metabolites </li></ul><ul><li>Problems - Free drug only detected by tandem MS method. Guidelines for limits use total drug concentration. Any drugs that are conjugated will require a hydrolysis step to remove the modification and then total drug conc can be determined </li></ul>
  37. 53. Proposed Service for Heartlands <ul><li>Extraction of Urine </li></ul><ul><ul><li>5ml urine </li></ul></ul><ul><ul><li>+ 2ml extraction solvent (dichloromethane, dichloroethane, heptane, propan-2-ol) </li></ul></ul><ul><ul><li>+ 3g extraction buffer (NaCl, NaHCO3, Na2CO3) </li></ul></ul><ul><ul><li>+ 50ul IS </li></ul></ul><ul><ul><li>Mix for 10mins </li></ul></ul><ul><ul><li>Centrifuge </li></ul></ul><ul><ul><li>Decant upper aqueous layer and evaporate to dryness </li></ul></ul><ul><ul><li>Reconstitute in mobile phase </li></ul></ul>
  38. 54. LC-MS/MS Method <ul><li>Inject 5uL extracted sample </li></ul><ul><li>LC separation on C8 column (50mm x 4um (Vydec)) </li></ul><ul><li>Mobile phase 4mM ammonium acetate in 5% MeOH </li></ul><ul><li>Gradient elution 95% Methanol </li></ul><ul><li>Electrospray ionisation </li></ul><ul><li>Transitions monitored (at least 2 for each drug) </li></ul><ul><li>Approximate run time 6.5 mins </li></ul><ul><li>Approximate cost of DOAS screen by LC-MS/MS £8 </li></ul><ul><li>Complex interpretation of results </li></ul>
  39. 55. 152.1 300.2 Codeine 153 293.1   171.3 293.1 Benzoylcgonine-D3 181.2 292.2   152.1 292.2 Morphine-D6 150.1 290.1   168.1 290.1 Benzoylcgonine 198.2 290.1   154.1 290.1 Diazepam-D5 165.2 286.1   152.1 286.1 Morphine 192.9 285.1   154.1 285.1 Diazepam 180.2 282.2   236.2 282.2 Nitrazepam 249.1 278.2   234 278.2 EDDP 208.3 271.1   165.2 271.1 Nordiazepam 224.2 252.1   121.2 252.1 7-aminonitrazepam 119 150.1   91 150.1 Metamphetamine 66.9 142.2   93.1 142.2   125.1 142.2 Amphetamine-D6 119.2 136   91 136 Amphetamine Q3 Q1 Drug
  40. 56. Urine top std
  41. 57. Accuracy
  42. 59. Steroids on the LC-MS/MS <ul><li>PCOS screen </li></ul><ul><ul><li>Simultaneous detection of: </li></ul></ul><ul><ul><li>Testosterone </li></ul></ul><ul><ul><li>4-Androstenedione </li></ul></ul><ul><ul><li>DHEAS </li></ul></ul><ul><ul><li>17-OHP </li></ul></ul><ul><li>Urinary free cortisol </li></ul><ul><li>Prednisolone for compliance monitoring </li></ul>
  43. 60. Proposed method for Steroids <ul><li>APCI </li></ul><ul><li>Extraction ? Zinc sulphate and methanol </li></ul><ul><li>IS Testosterone D3 </li></ul><ul><li>40ul sample required to inject onto LC </li></ul><ul><li>Gradient method (methanol elution) </li></ul><ul><li>8 min/sample </li></ul>
  44. 61. Testosterone by LC-MS/MS (ESI)