Tandem Mass Spectroscopy Basics

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 Inlet IonizeMass

analyzeDetect

LCShimadzou Prominence

System2 x Binary Pumps1 x Autosampler1 x Column Oven

General Principles

LC Inlet IonizeMass

analyzeDetect

Inlet (Source)

TurboIonSpray(ESI and APCI capable)

flow rates from 2 to 1000 uL/min

Interface

General Principles

LC Inlet IonizeMass

analyzeDetect

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 N2

• 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 fragmentationLarge 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

LC Inlet IonizeMass

analyzeDetect

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

LC Inlet IonizeMass

analyzeDetect

Detection

•Photographic plate

•Faraday cage

•Electron multiplier

•Photomultiplier

•Charge collectors

Electron Multiplier

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

MRMMS1 Collision Cell

Static CID

Set to knownprecursor m/z

Set to knownproduct m/z

344

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-MSCluster/Adduct

Source of Source of ClusterCluster OccurenceOccurence Mass of Cluster Mass of Cluster

IonIon

[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 phase 4mM 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

Drug Q1 Q3

Amphetamine 136 91

  136 119.2

Amphetamine-D6 142.2 125.1

  142.2 93.1

  142.2 66.9

Metamphetamine 150.1 91

  150.1 119

7-aminonitrazepam 252.1 121.2

  252.1 224.2

Nordiazepam 271.1 165.2

  271.1 208.3

EDDP 278.2 234

  278.2 249.1

Nitrazepam 282.2 236.2

  282.2 180.2

Diazepam 285.1 154.1

  285.1 192.9

Morphine 286.1 152.1

  286.1 165.2

Diazepam-D5 290.1 154.1

  290.1 198.2

Benzoylcgonine 290.1 168.1

  290.1 150.1

Morphine-D6 292.2 152.1

  292.2 181.2

Benzoylcgonine-D3 293.1 171.3

  293.1 153

Codeine 300.2 152.1

Urine top std

Accuracyaverage accuracy Stdev

benzoylcgonine Low std 104.45 4.16High std 97.77 3.39

QC 106.65 5.776-MAM Low std 96.9 21.53

High std 101.55 14.71QC 121.36 26.70

Nitrazepam Low std 102.59 8.39High std 98.69 6.45

QC 85.87 2.32EDDP Low std 104.12 3.48

High std 98.01 2.64QC 121 3.67

Methadone Low std 104 2.58High std 98.09 2.42

QC 103.1 1.85Codeine Low std 95.55 12.17

High std 102.22 13.46QC 102.28 14.83

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)