Implmentation of TMS at BHH

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’

General Principles

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

General Principles

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

Interface

General Principles

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)

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

ESI Soft ionisation technique; little fragmentation Large molecules may be protonated more than once Peaks seen for same compound with different m/z ratios

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]+.

General Principles

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

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

Mass Separation

General Principles

Detection Photographic plate Faraday cage Electron multiplier Photomultiplier Charge collectors

Electron Multiplier

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

TMS

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

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

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

MRM

TMS

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

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)

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

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

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

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

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

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

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)

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

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

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

DrugQ1Q3 Amphetamine Amphetamine-D Metamphetamine aminonitrazepam Nordiazepam EDDP Nitrazepam Diazepam Morphine Diazepam-D Benzoylcgonine Morphine-D Benzoylcgonine-D Codeine

Urine top std

Accuracy

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

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

Testosterone by LC- MS/MS (ESI)