Advantages and Disadvantages of High-end Mass Spectrometry in a

Forensic Toxicology Lab

Raymond Van OrdenForensic Scientist Supervisor

Controlled Substances and Toxicology

For Forensic Use.

Objectives

1) Discuss advantages/disadvantages of each mass spectrometry system

GC/MSLC/MSTandem MS

1) Best approach to implementing tandem MS systems

For Forensic Use.

What kind of samples are you testing ?

Makes a difference to what kind of system you want to use

1) Blood2) Urine3) Post mortem or Anti mortem4) Other matrices?

For Forensic Use.

• Quantitative or Qualitative

Goals of Mass Spec

QuantitativeCalibration curveConfirmation

Retention timeIon Ratio Quant/Qual ion coelution

QualitativeRetention TimeSpectral Match (ion trap or scan)Product ion ratios

For Forensic Use.

• Sample concentration• Matrix• Analyte type• Buffers and purity• Purity of Organic solvents• Purity of curtain gas and collision cell gas• Run time• Number of samples run

Factors affecting Mass Spec Performance

For Forensic Use.

• Higher concentration samples• Usually involves derivitization depending on

analyte• SIM• Electron impact ionization (EI) or chemical

ionization (CI)• Most common in labs• Get “fingerprint” identification, good for library

matches of unknowns when in scan mode

Single Quad GC/MS

For Forensic Use.

• Tends to have better sensitivity than GC• No derivitization needed• Have to over come the fear of LC• Uses collision induced dissociation –

“softer technique” not as many ions• Have to be aware of adducts• Not good for library matches, gives you the

molecular ion in most cases.

Single Quad LC/MS

For Forensic Use.

• CID spectra affected most by:– Type and pressure of collision gas– Initial kinetic energy of the ions– Structure of the ions– Instrument configuration– Charge site

Collision Induced Dissociation

Factors that influence CID spectra:Collision energyCollision gas choice and pressureCID time

For Forensic Use.

• Most Common Proton acceptors– R – NH2– R – NH – R’– R2 – PH, R2PH3– R – OH, R - SH– R – OR, R – C = O– Hydrocarbons/Aromatics

Collision Induced Dissociation

Better +Ionformation

Worse +Ionformation

For Forensic Use.

• Atmospheric Pressure ionization processes most often produce even electron ions

• The most favorable losses will be neutral fragments (stable molecules) and production of another even electron ion

Collision Induced Dissociation

Water (H2O) 18 Daltons, is a common neutral lossTry to avoid using losses of 18

Uncommon to lose a mass of 4-14 Daltons and 21-25 Daltons

For Forensic Use.

• Used with either GC or LC• Removes matrix interferences• Provides reliable confirmation• Allows for the selective quantitation of target

compounds in high background samples• Better S/N in complex matrices than can be

achieved by single quad scan and SIM approaches

Tandem MS

For Forensic Use.

Scan

MRM

For Forensic Use.

Who should use QQQ (MS/MS)?

User doing Selected Ion Monitoring(SIM) for target compound analysis in laboratories, needing additional sensitivity and selectivity with less sample prep, to meet more demanding analytical requirements.

For Forensic Use.

• LC or GC-MS/MS: Routine Targeted trace analysis in complex matrix MRM Sensitivity unsurpassed (up to a few hundred compounds)

• Complex matrix with less clean-up (within reason)

Why Tandem MS

For Forensic Use.

• Maximize separation (LC/GC first)– Can use existing LC or GC methods– Good chromatography assists with

higher sensitivity and confidence

Getting started

For Forensic Use.

• For LC beginners– Choose a good buffer in appropriate

pH range of your compounds– Select an appropriate column– Usually use a gradient technique

• Mixture of MEOH and buffer

Getting started

Make sure buffers are filtered!

For Forensic Use.

EME

D3-EM

E

PPAC

athineC

athinoneB

ZPEphedrine

PseudoephedrineM

ethcathinone Am

phetamine

D11-M

ethM

ethamp M

DA

Phentermine

MD

MA

Dehydro

DM

S D8-B

EM

DEA

BE

Norketam

ineK

etamine

Pheniramine

PropylhexadrineM

ethylphenidateTFM

PPC

ocaineD

3-Coc

CE

Quetiapine

Diphenhyd

PCP

.2

.4

.6

.8

1.0

1.2

1.4

x105

1.6

Chromatograph of Analyzed Compounds

1 2 3 4 5 6 7 8 9 10 11 12

D10-

Am

p

D5-

MD

MA

For Forensic Use.

• Baseline separation of stereoisomer's– PPA (Norephedrine) and Cathine (Norpsuedoephedrine)– Ephedrine and Pseudoephedrine

• Which makes identification and quantification simple

PPA

Cathine

Ephedrine

Pseudoephedrine

LC can provide:

For Forensic Use.

Specific Hints

• Filter, Filter, Filter– All buffers

• Good Sample prep– SPE, filter samples

• Guard Column or Frits (“crud catchers”)• “Garbage in garbage out!”• Takes time, but optimize each analyte• Monitor pressures

– Column, nebulizer, collision cell• Monitor Vacuum• Monitor Log book

For Forensic Use.

• Optimize Mass spec– Tune (manual or auto?)– Spray chamber

• Drying gas temp. and flow• Nebulizer• Capillary voltage

– Quad 1• Capillary voltage• Fragmentor voltage

– Collision Cell• Collision energy

– Quad 3 (grouped or dynamic)• Maximize daughter ions• EMV

Getting started

For Forensic Use.

• Group MRMThird Quad Optimization

For Forensic Use.

• Dynamic MRM

Third Quad Optimization

1.023.36

6.05

8.03

8.74

For Forensic Use.

• Dynamic MRMThird Quad Optimization

For Forensic Use.

• Changing of fragmentor from 80 (Meth) to 100 (Phentermine) half way through created a split in the peak

• Fragmentor values changed for Phentermine, MDA, MDMA, and D5-MDMA from 100 to 80 and changed the Meth and Phentermine RT window from 1 min to 2 min

Noted Problems

For Forensic Use.

• Improper setting of retention time for methamphetamine and phentermine, daughter ion caused a jump in the 91 ion half way through the peak

Noted Problems

For Forensic Use.

• (LC) Eliminates the need for derivatization• Can separate and ID similar compounds• Superior sensitivity and selectivity of analytes• When operating in MRM mode

– Quantification of low levels of compounds in biological matrices

– Wide dynamic range

Benefits of LC/GC tandem MS

For Forensic Use.

Summary1) Each system has advantages and

disadvantages, but combining them makes the laboratory more efficient and productive

For Forensic Use.

Questions?

For Forensic Use.