LC- MSMS and GC- MS

Advancement in Toxicology

Amir Naimi
Toxicologist
Dorevitch Pathology
 GC-MS and LC-MSMS
GC/MS LC/MSMS

• Long been the technique • Has been the natural

of choice for progression of
identification and chromatographic/mass
quantification. spectral techniques.

• In some cases extreme • Many of the

efforts are employed in compounds that are
sample preparation to difficult to analyse by
make difficult compounds GC/MS such
viable for GC/MS as amines, glucuronides
analysis. and semi-volatile
compounds are ideal for
•Sample preparation often LC/MSMS.
includes liquid-liquid
extraction (LLE), solid Many advantages to
phase extraction (SPE), switching assays from
and/or derivatization. GC/MS to LC/MSMS.
 The Techniques
 Gas chromatography / Mass spectrometry
 Liquid chromatography / Tandem Mass spectrometry
 Confirmation techniques of choice
 LC-MS is currently competing with GC-MS for the status of the reference
analytic technique in toxicology
 Chromatography separates analytes
 A mass spectrometer detects, identifies and quantitates - highly specific
 Historically a slow and labour-intensive manual process.
- requires extensive sample preparation
- instrument run times can be quite long depending on profile
 GC-MS
Gas Chromatography Mass Spectrometry
 Analysis and quantitation of volatile and semi volatile compounds that are thermally
stable
 Prepared volatile compound injected as liquid into GC inlet

> High inlet temperatures vaporise the

volatile liquid

> Mixture in gas form is then carried by

carrier gas into and through the
capillary column where elution rate is
controlled by a temperature controlled
compartment (GC).
> The mixture of interest is separated by
their relative interaction with the coating of the column (stationary phase) and the
carrier gas (mobile phase)
> The latter part of the column ends at the entrance to ion source
where compounds eluting from the column are converted to ions.
> Fragments are then produced in mass spectrometer with characteristic
relative abundances that provide a 'fingerprint' for that molecular structure.
 Response Chromatographic separation

Retention time

Fragmentation
Pattern -
“Fingerprint”
 LC-MSMS
Liquid Chromatography Tandem Mass Spectrometry

 Chromatographic separation based on mass and polarity

 Elution from HPLC column at known times and enter
mass spectrometer
 Protonation / deprotonation in ion source
 Q1 monitors precursor ions
> Collusion induced dissociation (CID) in collusion cell (Q2)
 Q3 monitors fragments of precursor ions
 Mass detector quantifies
 LC-MSMS
Configuration

Q0 Q1 Q2 Q3

Scanning Collision Cell Scanning

 Q1 and Q3 are standard mass filter quadrupoles

 Q2 is the “collision cell” where mass fragmentation occurs

– Q2 does not filter ions
– Accepts all ions sent to it by Q1 and passes all ions formed by collision to
Q3 to be sorted
 LC-MSMS
Configuration

Detector/
Liquid
Ionization Mass Analyzer Data
Chromatography
Collection

-Solvents - ESI - Triple Quadripoles

-Columns - APCI - Ion Traps
- APPI - Hybrids
 LC-MSMS
Ionisation

 Electro spray Ionisation (ESI) :

Ionization process which uses electrical fields to generate charged droplets and
subsequent analyte ions by ion evaporation for MS analysis

- Increasing polarity and molecular weight and thermal instability favours electrospray
Majority of drugs of abuse are highly polar and are easily analysed using electrospray
High molecular weight proteins also require electrospray

 A Gas Phase Chemical Ionization (APCI):

A gas phase chemical ionization (CI) process where the solvent acts as
the CI reagent gas to ionize the sample

 Atmospheric Pressure Photoionization (APPI):

Krypton lamp producing ultraviolet light ionizes gas phase analytes or dropants
with subsequent gas-phase reactions

- Lower polarity and molecular weight favours APCI or APPI

 Ion Suppression
 A form of matrix effect that LC-MS techniques suffer.
 Negatively affects response, precision and accuracy.
 Results from the presence of less volatile compounds changing
the efficiency of droplet formation or droplet evaporation,
which in turn affects the amount of charged ions in the gas
phase that ultimately reaches the detector.
 Ion Suppression

 Occurs at early stages of ionisation at MS interface

 When a particular component is eluted from HPLC to MS
and influences the ionisation of a co-eluting compound.
 Reduces response
 Even if the interfering compounds are not recorded there
presence will still reduce (supress) response.
 Effects MS-MS as well as single MS
 LC–MS-MS suppression problems are much more evident
due to the specificity of the method
 Causes
 Many possible sources;
Endogenous compounds, sample matrices, exogenous
substances, contamination > Sample prep
 Influential Factors
 High concentration
 Mass
 Basicity
 Elution time

> If the interfering molecule

shares the same retention
window as the analyte of interest.
 The Solution
 Chromatographically
> Change elution time of compound of interest into region where
suppression is not observed. Generally involves increasing run
times.
 “Correcting” the degree of suppression.
> Ensuring compound of interest and internal standard co-elute.
Ion suppression for both compounds should become equal if the
peaks chromatographically coincide therefore “correcting” for the
degree of suppression.

• Sample prep
> Using an effective sample preparation procedure which usually
involves either liquid/liquid (LLE) or Solid phase (SPE) can
remove ion suppressing species from sample matrix.
 Internal Standard
 Known concentration of a substance added to every sample that
is analysed.
 Compound that is very similar but not identical to substance of
interest in sample. Often isotopically labelled.
> Sample preparation should have the same effects for the IS as
the compound of interest.
> Anything adversely effecting the
analyte of interest will also effect
the drug of interest.
 Parent Fragmentation
Qualifier

Internal standard
 LC-MSMS

 Ideal method for Toxicology would be one method confirming all drug groups
though there are limitations in doing this;

Obstacles

- Not all pure standards are currently available in conjugated form

- May require separate sample preparations depending on compound
 GC-MS LC-MSMS
GC-Strengths
No mobile phase preparation
- pH meters, balances, stability etc.
Very low gas consumption
Precise retention times
Reagent impurity not an issue
- No reagent impurity peaks
GC-Weaknesses
Derivatization
- May be required to improve injection or chromatography, sample needs to be
volatile in order for mobile phase to carry
- Added time and cost
Injection process
- Can be troublesome with high throughput laboratories
- Less precise
Extra sample preparation
- Hydrolysis of glucuronides
 GC-MS LC-MSMS
LC-Strengths
Precise accurate injections
- No loss from vaporization
Ideal for thermally unstable compounds
Ideal for non-volatile compounds
- No need for derivatization
- High molecular weight possible
Less sample preparation
- Derivatization
- Enzyme hydrolysis
Ideal for polar metabolites (glucuronides)
Tandem MS/MS selectivity is greatly enhanced

LC-Weaknesses
Sensitivity dependent onmobile phase conditions
May require monitoring of both (+) and (-) ions
- Separate analyses
- Sensitivity compromised if run simultaneously
WINNER!!
 LC-MSMS
Advancement in Toxicology

 Methods to detect and quantitate drug glucuronide metabolites.

 Opiate Glucuronide, THC drug standards currently available
 Simple dilute and shoot method (when matrix effects have been
eliminated)
- No hydrolysis
 Simplified sample preparation means possibility for more drugs
on one analytical run
 Example of Improvement in
Toxicology
Opiates
 Previously on GC/MS involved slow enzyme hydrolysis (2hrs)
and derivatization step (15 minutes).
 LC/ MS does not require enzyme hydrolysis as it can be used
for glucuronide detection. Derivatization step not required as
mobile phase is liquid.
Saved 2.5hrs total in manual preparation time.
Benzodiazepines
 Unfortunately no Benzo glucuronide drug standards available
so hydrolysis is still necessary.
 Derivatization is no longer needed as mobile phase is liquid.
The Ideal Method
THANK YOU