1. Implmentation of TMS at BHH

2. Mass Spectrometry ‘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’

3. General Principles

4. LC Shimadzou Prominence System 2 x Binary Pumps 1 x Autosampler 1 x Column Oven

5. General Principles

6. Inlet (Source) TurboIonSpray (ESI and APCI capable) flow rates from 2 to 1000 uL/min

7. Interface

8. General Principles

9. Ionisation If a quantity of energy is supplied to a molecule equivalent to the ionisation energy of the molecule, a molecule ion is formed M+ There are several ways of doing this Electron impact (EI) Chemical Ionisation (CI) Atmospheric pressure Ionisation (API) Electrospray (ESI)

10. ESI Stream of solution sprayed out of capillary at high voltage (ca. 3 – 5 kV) Charged droplets formed by spray (“Taylor cone”) Solvent evaporated by stream of warm N 2 As droplet shrinks, charge density increases until analyte ions ejected Pseudomolecular [M+H] + ions formed Solvent pumped away and ions admitted to mass spectrometer

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

14. APCI A sample solution flows through a heated tube where it is volatilized and sprayed into a corona discharge with the aid of nitrogen nebulization. Ions are produced in the discharge and extracted into the mass spectrometer. APCI i s best suited to relatively polar, semi-volatile samples. An APCI mass spectrum usually contains the quasi-molecular ion, [M+H]+.

16. General Principles

17. Mass Separation Quadrupole mass filters Four rods arranged precisely with DC and RF alternating voltages applied to pairs Ions enter quadrupole region Because of RF voltage and DC offset the polarity of each pair of rods continually changes

18. Mass Separation Ion in quadrupole is alternately repelled and attracted to given rod Ion follows helical path through quadrupole For given RF and DC voltage settings only certain m/z ions have stable trajectory to detector – the rest collide with rods By changing values of the voltages different m/z ions can be focussed onto the detector Quadrupole mass filter transmits one m/z ratio at once

19. Mass Separation

20. General Principles

21. Detection Photographic plate Faraday cage Electron multiplier Photomultiplier Charge collectors

22. Electron Multiplier

23. Amplified current measured and related to ion count Sensitive Allows for rapid scanning

24. TMS

28. Data Acquisition Modes Simple MS Scanning Using one quadrapole Product ion scan (daughter) MS1 static MS2 scanning Precursor ion scan (parent) MS1 scanning MS2 static

29. Data Acquisition Modes Neutral loss scan MS1 and MS2 scanning But synchronised Multiple reaction monitoring (MRM) MS1 and MS2 static Enhanced sensitivity

30. MRM Highly specific Only data on analytes of interest is collected All other compounds are ignored More sensitive than full scan mode Quadrapoles can spend longer scanning as fewer transitions to monitor Faster flow rates into ion source Possible quicker analyses

31. MRM

32. TMS

33. Compound Dependent Parameters 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 Entrance Potential (EP)- Focuses the ions through the high pressure Q0 region Collision cell entrance potential (CEP)- Focuses ions into the collision cell

34. Compound Dependent Parameters Collision Energy (CE) - The amount of energy precursor ions receive as they are accelerated into the collision cell. Collision Gas (CAD) Collision Cell Exit Potential (CXP)

38. Source Optimization Parameters Gas 1 - Helps Generate small Droplets of sample flow Gas 2 - Turbo gas, helps evaporate the spray droplets and prevents solvent entering the system Temperature - Temperature of the Turbo Gas

39. Source Optimization Parameters Curtain Gas - Prevents solvent droplets from entering and contaminating the ion optics Ion Spray voltage - The voltage applied to the needle that ionises the sample at the ion source Nebulizer or needle current - The current applied to the corona discharge needle in APCI Interface Heater - prevents contamination of ion optics

40. Common Problems Interfering compounds (Iso Baric Compounds) Ion Suppression Ionisation Problems

44. Ion Suppression Salts can interfer with ionisation and can cluster to complicate spectrum Strong Bases or quaternary amines can interfer with positive mode analytes e.g. Triethylamine (TEA) Acids - Sulfuric and TFA interfer in negative mode experiments Phosphate Buffer and non-volatile ion-paring agents (e.g. SDS) can cause severe suppression and complex spectra Non - covalent Dimers in Ion Spray Dimer signal = (MW*2)+1 can cause linearity at high concentrations

45. Adducts and Clusters Formed in LC-MS Cluster/Adduct Source of Cluster Occurence Mass of Cluster Ion [M + CH3COO]-Acetic AcidAPCI & IS -ve modeM + 59 [M + Cl]-Chlorinated SolventAPCI & IS -ve modeM + 35 [M + NH4]+AmmoniaAPCI & IS +ve modeM + 18 [M + Na]+Sodium SaltsAPCI & IS +ve modeM + 23 [M+K]+Potassium SaltsAPCI & IS +ve modeM + 39 [M + CH3CNH]+AcetonitrileAPCI & IS +ve modeM + 42 [M+CH3OHH]+MethanolAPCI & IS +ve modeM + 33 [M+H3O]+WaterAPCI & IS +ve modeM + 19

46. Plans Vitamin D Drugs of Abuse Testing Steroid Analysis R&D

51. Simultaneous Detection of following Drugs by LC-MS/MS Benzoylecgonine (Cocaine metabolite) Opiates EDDP (Methadone metabolite) Methadone Morphine 6-MAM (Heroin metabolite) Codeine Dihydrocodeine Amphetamine Methamphetamine Ecstasy metabolites MDA MDMA MDEA Cannabis D9-tetrahydrocannabinol (THC) 11-nor-D9-carboxy THC (THC- COOH) Benzodiazepines Tamazepam Diazepam Nitrazepam 7-amino Nitrazepam Nordiazepam Buprenorphine Gamma Hydroxybutyrate (GHB)

52. DOA screening by LC-MS/MS Due to specificity of the tandem MS no need for a confirmation method Advantages - Multiple transitions should decrease interference by metabolites 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

53. Proposed Service for Heartlands Extraction of Urine 5ml urine + 2ml extraction solvent (dichloromethane, dichloroethane, heptane, propan-2-ol) + 3g extraction buffer (NaCl, NaHCO3, Na2CO3) + 50ul IS Mix for 10mins Centrifuge Decant upper aqueous layer and evaporate to dryness Reconstitute in mobile phase

54. LC-MS/MS Method Inject 5uL extracted sample LC separation on C8 column (50mm x 4um (Vydec)) Mobile phase4mM ammonium acetate in 5% MeOH Gradient elution 95% Methanol Electrospray ionisation Transitions monitored(at least 2 for each drug) Approximate run time 6.5 mins Approximate cost of DOAS screen by LC-MS/MS £8 Complex interpretation of results

55. DrugQ1Q3 Amphetamine13691 136119.2 Amphetamine-D6142.2125.1 142.293.1 142.266.9 Metamphetamine150.191 150.1119 7-aminonitrazepam252.1121.2 252.1224.2 Nordiazepam271.1165.2 271.1208.3 EDDP278.2234 278.2249.1 Nitrazepam282.2236.2 282.2180.2 Diazepam285.1154.1 285.1192.9 Morphine286.1152.1 286.1165.2 Diazepam-D5290.1154.1 290.1198.2 Benzoylcgonine290.1168.1 290.1150.1 Morphine-D6292.2152.1 292.2181.2 Benzoylcgonine-D3293.1171.3 293.1153 Codeine300.2152.1

56. Urine top std

57. Accuracy

59. Steroids on the LC-MS/MS PCOS screen Simultaneous detection of: Testosterone 4-Androstenedione DHEAS 17-OHP Urinary free cortisol Prednisolone for compliance monitoring

60. Proposed method for Steroids APCI Extraction ? Zinc sulphate and methanol IS Testosterone D3 40ul sample required to inject onto LC Gradient method (methanol elution) 8 min/sample

61. Testosterone by LC- MS/MS (ESI)