Advantages and Disadvantages of

High-end Mass Spectrometry in a

Forensic Toxicology Lab

Raymond Van Orden

Forensic Scientist Supervisor
Controlled Substances and Toxicology

For Forensic Use.

 Objectives
1) Discuss advantages/disadvantages of each
mass spectrometry system
GC/MS
LC/MS
Tandem 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) Blood
2) Urine
3) Post mortem or Anti mortem
4) Other matrices?

For Forensic Use.

 Goals of Mass Spec
• Quantitative or Qualitative
` Quantitative
Ń Calibration curve
Ń Confirmation
x Retention time
x Ion Ratio
x Quant/Qual ion coelution

` Qualitative
Ń Retention Time
Ń Spectral Match (ion trap or scan)
Ń Product ion ratios
For Forensic Use.
 Factors affecting Mass Spec
Performance
• 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

For Forensic Use.

 Single Quad GC/MS
• 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
For Forensic Use.
 Single Quad LC/MS
• 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.
For Forensic Use.
 Collision Induced Dissociation
• 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

` Factors that influence CID spectra:

Ń Collision energy
Ń Collision gas choice and pressure
Ń CID time

For Forensic Use.

 Collision Induced Dissociation
• Most Common Proton acceptors
– R – NH2 Better +Ion
formation
– R – NH – R’
– R2 – PH, R2PH3
– R – OH, R - SH
– R – OR, R – C = O
– Hydrocarbons/Aromatics
Worse +Ion
formation

For Forensic Use.

 Collision Induced Dissociation
• 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
` Water (H2O) 18 Daltons, is a common neutral loss
Ń Try to avoid using losses of 18
` Uncommon to lose a mass of 4-14 Daltons and 21-25
Daltons

For Forensic Use.

 Tandem MS
• 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

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.

 Why Tandem MS

• 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)

For Forensic Use.

 Getting started
• Maximize separation (LC/GC first)
– Can use existing LC or GC methods
– Good chromatography assists with
higher sensitivity and confidence

For Forensic Use.

 Getting started
• 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

Make sure buffers are filtered!

For Forensic Use.

 For Forensic Use.
12

Quetiapine
11

PCP Diphenhyd
CE
10
Cocaine D3-Coc
TFMPP
Methylphenidate 9
Pheniramine Propylhexadrine
Ketamine
Norketamine

8
MDEA BE D8-BE
DMS
Dehydro

7
MDMA D5-
Phentermine
MDMA

6
MDA
x105 Chromatograph of Analyzed

Methamp
D11-Meth

5
Amphetamine
Methcathinone D10-

4
Pseudoephedrine
Amp
Ephedrine BZP
Cathinone

3
Compounds

Cathine
PPA

2
EME D3-EME

1
1.6

1.4

1.2

1.0

.8

.6

.4

.2
 LC can provide:
• Baseline separation of stereoisomer's
– PPA (Norephedrine) and Cathine (Norpsuedoephedrine)
– Ephedrine and Pseudoephedrine
• Which makes identification and quantification simple

Ephedrine

Pseudoephedrine
Cathine
PPA

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.

 Getting started
• 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
For Forensic Use.
 Third Quad Optimization
• Group MRM

For Forensic Use.

 Third Quad Optimization
• Dynamic MRM

8.03

1.02
3.36 8.74
6.05

For Forensic Use.

 Third Quad Optimization
• Dynamic MRM

For Forensic Use.

 Noted Problems
• 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

For Forensic Use.

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

For Forensic Use.

 Benefits of LC/GC tandem MS
• (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

For Forensic Use.

 Summary
1) Each system has advantages and
disadvantages, but combining them makes the
laboratory more efficient and productive

For Forensic Use.

Questions?

For Forensic Use.