Liquid chromatography mass spectrometry LC-MS/MS

LC-MS/MS

1. LC-MS/MS

2. 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.

3. 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)

4. Brief Introduction of LC-MS/MS

・ Sensitive analytical system.
・ LC separation + MS/MS identification.

・ Suitable for wild range of compound-matrix combinations
analysis.

ㆍ 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).

5. Applications of MS

* Drug Development

Y Determination of drugs and metabolites in plasma or
other biological fluids.

・ Food Safety

Y Melamine dosing. Pesticides residue, myotoxins,
additives.

・ Life Science
Y Proteomics, metabolomics, polysaccharides
* Clinical Science

Y Neonatal Screening, Therapeutic Drug Monitoring,
Occupational Bio-monitoring

・ Forensic Science
Y” Drug Abuse

6. Components of Mass Spectrometer

Gas Phase fons lon Sorting Jon Detection | Mass Spectrum

Sample Vacuum Pumps Data Output
Introduction sá

・ Mass Spectrometer operates at very high vacuum
condition........... To avoid the collision between ¡ons inside the
ion path.

7. General setup of a Triple Quad System

lon Production
+

lon Transport
+

7 lon Filtering
y

lon Fragmentation
+

\] lon Filtering
+

lon Detection

8. Tandem Mass Spectrometry

을
ionisation fragmentation
M Mt > m + m
radical cation radical
a cation
em
molecule molecular fragment

ion ion

9. Tandem Mass Spectrometry

‘Collision:
a Photon
ESI 4 Surface
4 >
E) * +) や =
de Pa
MS1 MS2
Precursor Product
ion in

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).

lons 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)

10. LC-MS/MS System

* lon Source (ESI/APCI/APPI/MALDI)

、 く く 4

11. Electrospray lonization (si)

LC | Gas1- Nebulizer Gas

Gas2- Heater Gas

lonization Capillary lonizer: Charged Capillary

Chamber
Gas
Heater

12. lon Source: Overview

Nebulizer Gas (GS1)

Heater Gas (GS2)

lon Spray Capillary Voltage (IS)
Temperature (TEM)

Curtain Gas (CUR)

Interface Heater (ihe)

Orifice Plate

13. Electro-Spray lonization (ESI)

14. Electro-Spray lonization (ESI)

PROS CONS

Soft ionization technique Lower flow rates

Suitable for Thermo Labile

Compounds lon Suppression

Suitable for wide range of analytes

Highly efficient ion production

15. ESI: lon Supression

~90%reduction

16. Atmospheric Pressure Chemical lonization (APCI)

ue |

MS

—

[10% torr

lonization
Chamber

Sample
Cone

Capillary

Spray

Gas

Nebulized

Corona
discharge

needle
760 torr

Gas1- Nebulizer Gas

Gas2・ Heater Gas

lonizer: Charged Corona Needle

17. Atmospheric Pressure Chemical lonization (APCI)

ATMOSPHERIC PRESSURE cutsinGas VACUUM
1. Tonisation of

solvent molecules fi. Mass Analyser

E — a 그 ey A a 6) + ne: {0 QE 一 一

analyte molecules;

ご ㅣ 3, Declustering
(4) Sample Needle Curtain Gas

： Formation of
(X) Solvent molecules Corona Discharge clusters

(120°C) 2, Reaction with JE

18. Atmospheric Pressure Chemical lonization (APCI)

PROS CONS

Hard ionization technique in Not suitable for Thermo Labile
comparison with ESI Compounds

Reduced effect of lon Suppression High temperature is required

Compatible with high mobile phase Fragmentation takes place for
flow rate weak bond molecules

19. Atmospheric Pressure Photo lonization (APPI)

==
4
:

Heater

Curtain Gas

20. Atmospheric Pressure Photoionization (APPI)

Lc |

MS

——

10 torr

lonization
Chamber

Sample

Cone

Capillary

Gas
Heater

uv
lamp

Nebulized
Spray

760 torr

Gas1- Nebulizer Gas
Gas2- Heater Gas

lonizer: UV Lamp

21. Matrix Assisted Laser Desorption lonization (MALDI)

Laser

Analyte
lon

SO Lie

Sanatyte/Matrix
Mixture

e

Use of Matrix

Intermediate for charge transfer
Protect fragile analyte molecule getting broken

22. Matrix-Assisted Laser Desorption/lonization (MALDI)

Applied for

- High MW, ionization not

vane possible using ESI, APCI,
APPI

ー High MW separates these
compounds from the
background signal of the
matrix

23. Matrix Assisted Laser Desorption lonization (MALDI)

PROS CONS

Fast and Highly Accurate Low Resolution in Mass

Not suitable for Photosensitive

Can be used for large mass range
9 9 analytes

Soft ionization with High Sensitivity Frequent cleaning is necessary

24. LC-MS lonization Application

10,000 ーー

nonpolar ionic

Polarity

25. LC-MS/MS System

+ Analyzer (Quad/TOF/Trap)

li

26. Triple Quadrupole

lon filtration based on their m/z ratio using DC/RF voltage ratio applied on quadrupole.

/ curain Gas” flow
Q2
LINAC®
Collision Exit m
00 ST Qi ST Cell 573

ラー | 3 Los 一
I LS 日 198 비리! iz AD

À\ Ne jo oo

NS

\\ | Skimmer 102 103 Deflector
\ ~ Orifice

\ 101

レー curtain Plate

27. Basic of Quadrupole

A quadrupole is

formed by four (2 set) + 1 support

paralel round rods

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.

28. Single/Selected lon Monitoring (SIM)

Exit lens
lon accumulation
sp ee & 5 es a
A
Passing of

Precursor ion No Fragmentation
selection Argon 아리 Precursor ion
CAD Gas

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

29. Multiple Reaction Monitoring (MRM)

Exit lens
fon accumulation À

Precursor ion Fragmentation Daughter jon
selection ‘Argon or N, Selection
CAD Gas

Q0 Allows all ions to pass through
Q1 Selects an [M+H]* or [M-HT ion
Q2 fragments the selected ion.

Q3 monitors only one daughter ion

30. Time of Flight (TOF) \

lon 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.

31. Schematic Diagram of Q-TOF >

Capillary voltage — Cone voltage Collision gas
Voltage adjusted to maintain | Desolvation of protein Activation and detergent
stable 100 current (1.4-1.B kVI | complexes (50-200 V) removal (1-4 MPa; Ar or SF)

Microchan
on guide Collision cell s plate detect

Nanotlow pressure
Pressure appbed to
initiate spray, but best
with none
(0-0.3 nl min)

Collision voltage
Source pressure Activation and

Pressure adjusted for optimum detergent removal
11 ol protein complexes (100-200 V)
Refiec

32. Time of Flight (TOF)

PROS CONS

Lesser Sensitivity as compared to

Higher Resolution and Accuracy Quadrupole

Specially used in Protein & Peptide Not suitable for Quantitative
analysis Analysis

Best for Qualitative Analysis

33. lon Trap

ton Source

Conventional lon Trap Ring Blectrod

Sich Mm

ーー pues
Capiliary Octopote
x 0
O 를 neuen

Depending on different voltage settings, ions at a specific m/z is

trapped, ejected and detected
lontrap provides possibilities to fragment trapped ions further.

lontrap combined with quadrupole provide sensitivity.

34. vv

vw

v

Quadrupole- lon Trap

Exit lens
lon accumulation À à |

Precursorion Fragmentation

selection N. CAD Gas linear ion trap 3x10* Torr
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.

35. LC-MS/MS System

* Fragmentation (CAD)

:

36. Fragmentation
(Collision Associated Dissociation)

a Q2 03
== A o
o | ore | MRM |
7 Ger =| Signal
e, o. に 4 に 2 o. eee +
e “9 시 No: E
ゴキ mm Time

Parent Ion Selection Fragmentation Daughter Ton Selection

37. CAD gas (NJAr) CXP
me y Deflector

CEP CE CEM or PEM

Mechanism:

1.CEP guides parent ion into Q2 (Collision cell).
2.lons enters into Q2 cell (Collision cell).
3.Collide with inert collision gas (N,/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.

38. LC-MS/MS System

<< < 4

* Detector (CEM/PMT) |

39. Channel Electron Multiplier (CEM)
Photo Multiplier Tube (PMT) / (ETP)

CEM ィ ler

__ HIGH SECONDARY EMSSIVE

A SEMICONDUCTIVE SURFACE
_ CASCADING

/ BLECTRONS

ETP

JON

First
Dynode

12-24 dynodes
Coated with Be
and MgO on SS
plate

40. 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.

41. Voltage Parameters in Quad MS
A | 09501

DE CR me)
一 一 一

DP EP CEP CXP
CE CEM

De-clustering Potential (DP) - On Orifice Plate
Entrance Potential (EP) - On Skimmer and QO

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

42. Recommended Solvents & Buffers suitable for ESI-MS

+ Acetonitrile
+ Methanol
+ Water

+ Formic Acid
ㆍ 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).

43. Case Study

Sulfasalazine
Ci, Hig N, 9; 5
MW 398.40 9
CーOH

H =
Ns Vs PONEN 애
CT &

44. 7.406 399.0
ru | Q1 scan

0.508

5006

5.46

5.106

4506

4006

3.586

sé Aseo

3.006
2.506

2.006
400.0

1.506

1.006
400.9 4014

5.065
og?
396,5 3970 397.5 390 3985 399.0 3995 400.0 4005 4010 401,5
miz, amu

Pharmaceutical Technology Center

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ass Q3 Scan
ans
755
705
ces
sas
sos
ans
405
ass
205
2506
206
Less
105
som
ao

oe 0 10 LD 10 tm #0 20 20 20 an m am an m ap 39 an
CN

Pharmaceuti

Technology Ce

46. Multiple Reaction Monitoring

+ ES Ionization

(©) +) Collision
- gas 들
Ú = ©
fis SIG 2 =
Q1 - Fixed Q2 - Collision Q3 - Fixed
precursor m/z chamber product m/z

(m/z 399) - (m/z 223)

MRM Transition |
m/z 339 - m/z 223 |

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