Instrumentation and application of LC-MS/MS in bioanalysis

Instrumentation and application of LC-MS/MS in bioanalysis

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MR. AMIT PATEL
Associate Research Scientist
Pharmaceutical Technology Center
Cadila Healthcare Limited
INSTRUMENTATION AND APPLICATION
OF LC-MS/MS IN BIOANALYSIS

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Principle of MS
Mass spectrometry (MS) is an analytical technique that
measures the mass-to-charge ratio (m/z) of charged particles.
Mass spectrometry is based upon the motion of a charged particle,
called an ion, in an electric or magnetic field.
 
The mass to charge ratio (m/z) of the ion effects this motion.
 

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Why need of MS in Bioanalysis ?
•Sensitivity (Detection at very low concentration level)
•Selectivity (Selectively detect the analyte of interest)
•Qualitative analysis (Identify the presence of analyte & metabolite)
•Quantitative analysis (Quantitate the analyte & metabolite of interest)
•High Throughput (Analyze the large no. of samples)
Apart from the above further reasons are,
•Structure Elucidation (In Drug Discovery)
•Unknown Mass Identification (Identification of Degradation)
•Impurity Profiling (Identification of mass of impurities)

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Brief Introduction of LC-MS/MS
•Sensitive analytical system.
•LC separation + MS/MS identification.
•Suitable for wild range of compound-matrix combinations
analysis.
•Easy-to-use.
•High sensitivity.
Liquid chromatography tandem mass spectrometry (LC–MS/MS),
has led to major breakthroughs in the field of quantitative
bioanalysis since 1990s due to its inherent specificity,
sensitivity, and speed. It is now generally accepted as the
preferred technique for quantitation of small molecule drugs,
metabolites in biological matrices (plasma, blood, serum, urine,
and tissue).

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Applications of MS
•Drug Development
Determination of drugs and metabolites in plasma or 
other biological fluids.
•Food Safety
Melamine  dosing,  Pesticides  residue,  myotoxins, 
additives.
•Life Science
Proteomics, metabolomics, polysaccharides
•Clinical Science
Neonatal  Screening,  Therapeutic  Drug  Monitoring, 
Occupational Bio-monitoring
•Forensic Science
Drug Abuse

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Components of Mass Spectrometer
•Mass  Spectrometer  operates  at  very  high  vacuum 
condition……….. To avoid the collision between ions inside the 
ion path.

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General setup of a Triple Quad System
Ion Transport
Ion Detection
Ion Fragmentation
Ion Filtering
Ion Filtering
Ion Production

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Tandem Mass Spectrometry

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Tandem Mass Spectrometry
•Tandem mass spectrometry, also known as MS/MS.
•Parent / Precursor ions are formed in the ion source and separated by mass-
to-charge ratio in the first stage of mass spectrometry (MS1). 
•Ions  of  a  particular  mass-to-charge  ratio  (Parent  /  precursor  ions)  are 
selected and fragment ions (product / daughter ions) are created by collision. 
•The resulting ions (product / daughter ions) are then separated and detected 
in a second stage of mass spectrometry (MS2)

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LC-MS/MS System

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Electrospray Ionization (ESI)
Gas
Nebulized
Spray
LC
Heater
Sample
Cone
MS
CapillaryIonization
Chamber
760 torr
10
-6
torr
-
+
Gas1- Nebulizer Gas
Gas2- Heater Gas
Ionizer: Charged Capillary

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Ion Source: Overview
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++++
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Curtain Plate
CUR
GS1 IS
TEM &GS2
ihe
Orifice Plate
Nebulizer Gas (GS1)
Heater Gas (GS2)
Ion Spray Capillary Voltage (IS)
Temperature (TEM)
Curtain Gas (CUR)
Interface Heater (ihe)

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Electro-Spray Ionization (ESI)

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PROS CONS
Soft ionization technique Lower flow rates
Suitable for Thermo Labile
Compounds
Ion Suppression
Suitable for wide range of analytes
Highly efficient ion production
Electro-Spray Ionization (ESI)

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ESI: Ion Supression
~90%reduction

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Atmospheric Pressure Chemical Ionization (APCI)
Gas
Nebulized
Spray
LC
Heater
MS
CapillaryIonization
Chamber
760 torr
10
-6
torr
-
Sample
Cone
Corona
discharge
needle
Gas1- Nebulizer Gas
Gas2- Heater Gas
Ionizer: Charged Corona Needle

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Atmospheric Pressure Chemical Ionization (APCI)

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PROS CONS
Hard ionization technique in
comparison with ESI
Not suitable for Thermo Labile
Compounds
Reduced effect of Ion Suppression High temperature is required
Compatible with high mobile phase
flow rate
Fragmentation takes place for
weak bond molecules
Atmospheric Pressure Chemical Ionization (APCI)

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Atmospheric Pressure Photo Ionization (APPI)

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Gas1- Nebulizer Gas
Gas2- Heater Gas
Atmospheric Pressure Photoionization (APPI)
Gas
Nebulized
Spray
LC
Heater
MS
CapillaryIonization
Chamber
760 torr
10
-6
torr
-
Sample
Cone
UV
lamp
Ionizer: UV Lamp

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Matrix Assisted Laser Desorption Ionization (MALDI)
Use of Matrix
Intermediate for charge transfer
Protect fragile analyte molecule getting broken

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Matrix-Assisted Laser Desorption/Ionization (MALDI)
Laser beam
+
+
-
MS
Vacuum
Applied for
– High MW, ionization not
possible using ESI, APCI,
APPI
– High MW separates these
compounds from the
background signal of the
matrix

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Matrix Assisted Laser Desorption Ionization (MALDI)
PROS CONS
Fast and Highly Accurate Low Resolution in Mass
Can be used for large mass range
Not suitable for Photosensitive
analytes
Soft ionization with High SensitivityFrequent cleaning is necessary

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LC-MS Ionization Application

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LC-MS/MS System

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Ion filtration based on their m/z ratio using DC/RF voltage ratio applied on quadrupole.
Triple Quadrupole

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A quadrupole is
formed by four (2 set)
paralel round rods
rods
support
++ +
-
-
One set is electrically
connected with a positive
DC voltage and the other
one with a negative DC
voltage.
An additional RF voltage at
a fixed frequency which
has an amplitude that
oscillates between positive
and negative is also
applied to all four surfaces.
Basic of Quadrupole

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Single/Selected Ion Monitoring (SIM)
Q0 Allows all ions to pass through
Q1 Selects an [M+H]
+
or [M-H]
-
ion
Q2 passes the selected ion without fragmentation.
Q3 passes the selected ion
Q0 Q1 Q2 Q3
Argon or N
2

CAD Gas
Precursor ion
selection
Ion accumulation
No Fragmentation
Exit lens
Passing of
Precursor ion

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Multiple Reaction Monitoring (MRM)
Q0 Allows all ions to pass through
Q1 Selects an [M+H]
+
or [M-H]
-
ion
Q2 fragments the selected ion.
Q3 monitors only one daughter ion
Q0 Q1 Q2 Q3
Argon or N
2

CAD Gas
Precursor ion
selection
Ion accumulation
Fragmentation
Exit lens
Daughter ion
selection

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Time of Flight (TOF)
Ion filtration can be done by time taken for flight from
one point (start) to another point (end).
Higher m/z ions requires more time to flight and low
m/z ions requires lesser time comparatively.

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Schematic Diagram of Q-TOF

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PROS CONS
Higher Resolution and Accuracy
Lesser Sensitivity as compared to
Quadrupole
Specially used in Protein & Peptide
analysis
Not suitable for Quantitative
Analysis
Best for Qualitative Analysis
Time of Flight (TOF)

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Ring ElectrodeConventional Ion Trap
•Depending on different voltage settings, ions at a specific m/z is
trapped, ejected and detected.
•Iontrap provides possibilities to fragment trapped ions further.
•Iontrap combined with quadrupole provide sensitivity.
Ion Trap

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Quadrupole- Ion Trap
Q1 selects a parent ion.
Q2 fragments the selected ion
Q3 traps then scans out all fragment ions.
Fragmentation of fragment is possible which provide more
information for structure
identification.
Selection of exit ion can be customized as per requirement.
Q0 Q1 Q2 Q3
N
2
CAD Gas
linear ion trap 3x10
-5
Torr
Precursor ion
selection
Ion accumulation
Fragmentation
Exit lens

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LC-MS/MS System

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Fragmentation
(Collision Associated Dissociation)

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CAD gas (N
2
/Ar)
Q0 Q1 Q2 Q3
CXP
CEPCE
Deflector
CEM or PEM
Mechanism:
1.CEP guides parent ion into Q2 (Collision cell).
2.Ions enters into Q2 cell (Collision cell).
3.Collide with inert collision gas (N
2
/Ar) with high collision energy (CE).
4.Parent ion’s breakdown to fragments due to collision with inert gas.
5.Daughter ion (fragment) loses kinetic energy due to collision and
require potential (CXP) to thrown out from collision cell.
6.Selected fragment passed through Q3 and reaches to detector.

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LC-MS/MS System

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SiO
2
Layer

12-24 dynodes
Coated with BeO
and MgO on SS
plate
Channel Electron Multiplier (CEM)
Photo Multiplier Tube (PMT) / (ETP)

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How it works…??
•An ion strikes on internal surface of device and typically produces 2-3
secondary electron.
•These electrons are accelerated down the channel by positive
bias.
•The electron strikes on the channel walls and produces additional
electrons.
•The emitted electron will be recorded as a signal on computer system
in the form of peak or intensity.

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Voltage Parameters in Quad MS
Q0 Q1 Q2 Q3
EP CEP CXPDP
CE
CEM
•De-clustering Potential (DP) – On Orifice Plate
•Entrance Potential (EP) – On Skimmer and Q0
•Collision Cell Entrance Potential ( CEP) – On Entrance of Collision Cell
•Collision Energy( CE) – In Collision Cell
•Collision Cell Exit Potential ( CEP) – On Exit of Collision Cell

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Recommended Solvents & Buffers suitable for ESI-MS
•Acetonitrile
•Methanol
•Water
•Formic Acid
•Acetic Acid
•Ammonium Acetate
•Ammonium Formate
•Ammonium Triflouroacetate
Only volatile buffers can be used. Strictly avoid the Sticky and
Corrosive item.
Also solid buffer strength should be minimum (<20-25mM).

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Sulfasalazine
C
18
H
14
N
4
O
5
S
MW 398.40
N N
H
S NN
OO
OH
COH
O
Case Study

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Max. 7.4e6 cps.
396.5 397.0 397.5 398.0 398.5 399.0 399.5 400.0 400.5 401.0 401.5
m/z, amu
0.0
5.0e5
1.0e6
1.5e6
2.0e6
2.5e6
3.0e6
3.5e6
4.0e6
4.5e6
5.0e6
5.5e6
6.0e6
6.5e6
7.0e6
7.4e6
In
t
e
n
s
it
y
, c
p
s
399.0
400.0
400.9401.1
Q1 scan

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+Product (399.0): Experiment 3, 0.345 to 1.047 min from 049-IS Sulfasalazine MSMS.wiffMax. 9.3e5 cps.
6080100120140160180200220240260280300320340360380400
m/z, amu
0.0
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
3.0e5
3.5e5
4.0e5
4.5e5
5.0e5
5.5e5
6.0e5
6.5e5
7.0e5
7.5e5
8.0e5
8.5e5
9.0e5
I
n
t
e
n
s
it
y
,

c
p
s
223.2
119.0
213.1
94.0 147.1
239.0
165.3
137.1
381.1
240.8
316.9
287.2157.1169.0185.391.0 257.0224.3
333.2
111.1
121.1
N N
H
S NN
OO
OH
COH
O
m/z 137
m/z 165
m/z 94
m/z 119
m/z 223
Q3 Scan

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Multiple Reaction Monitoring
+
+ ES Ionization
+
+
+
+
+
++
+
+
+
+
+
+
Collision
gas
11.5 12 12.5 13 13.5 14 14.5
MRM Transition
m/z 339 – m/z 223
Q1 – Fixed
precursor m/z
(m/z 399)
Q3 – Fixed
product m/z
(m/z 223)
Q2 – Collision
chamber
Ar
Ar
+
+
+

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79
Br :
81
Br, intensity 1:1 and
35
Cl :
37
Cl, intensity 3:1

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