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MBAT S2 GOKULRAJ BIOMOLECULE SEPARATION OF LC-MSMS.pptx
1. SUBJECT: MBAT
TOPIC: BIOMOLECULE SEPARATION OF LC-
MS/MS
SUBMITTED TO:
DR.HARSHA K. TRIPATHY
PROFESSOR
DEPT. OF PHARMACEUTICAL ANALYSIS
KARNATAKA COLLEGE OF PHARMACY
BANGALORE
SUBMITTED BY:
S.GOKULRAJ
M PHARM 2nd SEMESTER
DEPT.OF PHARMACEUTICAL
ANALYSIS
KARNATAKA COLLEGE OF
PHARMACY
BANGALORE
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2. Definition
Liquid Chromatography with tandem mass
spectrometry (LC-MS-MS) is a powerful analytical
technique that combines the separating power of
liquid chromatography with the highly sensitive and
selective mass analysis capability of triple
quadrupole mass spectrometry.
The LC/MS/MS data may be used to provide
information about the molecular weight, structure,
identity and quantity of specific sample components.
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3. Brief Introduction of LC-MS/MS
Sensitive analytical system.
LC separation + MS/MS identification and
detection.
It is suitable for wild range of compound-matrix
combinations analysis.
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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4. Principle
Liquid chromatography-mass spectrometry is the
technique which performs separation by liquid
chromatography and mass analysis with the help of
the mass spectrometry.
Liquid chromatography tandem mass spectrometry
(LC-MS/MS)
Liquid Chromatography
Separates mixture components
Based on polarity
Tandem Mass Spectrometry
Detector
Identification & Quantification of components
Based on compound mass
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5. LC-MS/MS is mainly separated into the three parts:
1.Chromatography :
In liquid chromatography separation is performed
which is detected with the help of Photo diode Array.
These separated components then transferred to the
interface.
2. Interface:
In interface the liquid is volatilized and transferred
to the MS.
3. Spectrometry:
With the help of various ionization techniques the
compound is ionized and then it is analyzed by mass
analyzer.
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10. TANDEM MASS
SPECTROMETRY
KARNATAKA COLLEGE OF PHARMACY
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MS/MS
MS1: precursor ions are formed separate by m/z ratio
Daughter ions are created by fragmentation.
MS2: Ions are separated and detected
12/21/2023
14. Mass analyzer
In mass analyzer the magnetic field is scanned to
measure the different ions.
Types of mass analyzer:
1. Quadra pole mass filter
2. Time of flight
3. Ion trap
4. Fourier transform ion cyclotron resonance(FT-
ICR)
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15. Interfaces for LC-MS/MS
In LC-MS there are two key components, ionization source
and Interfaces.
The Liquid chromatography separates mixture of
components which are in liquid form, usually contains
methanol, acetonitrile and water .
This liquid containing mixture of components is transferred
into the ion source of mass spectrometer. As ion source is
under high vacuum.
Due to the difference in the pressure it is difficult to mass to
vaporize the liquid drops without losing mixture of
components.
Hence interfaces are used to resolve this problem.
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16. Various interfaces for LC-MS were developed, but issues
with sensitivity, stability and user-friendliness were faced.
After further improvements and developments, API, a type
of soft ionization technique, proved to be well-suited for
use in LC-MS.
It ionizes compounds under atmospheric pressure
conditions, which makes it especially useful for removing
solvents outside a vacuum.
API serves as both the ionization source and the
Interface in a LC-MS system.
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18. Various Interface are used in LC-
MS/MS
Moving Belt Interface
Particle Beam Interface
Direct Liquid Introduction Interface
Flow-FAB interfaces
Thermospray (TSP)
APCI Interface
APPI Interface
Electrospray (ESI)
Ion Spray
MALDI
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19. PROBLEMS IS COMBINING HPLC AND MS
HPLC
Liquid Phase operation
25-50 degree C temp.
No mass range limitation
Inorganic buffers are used.
1ml/min eluent flow is
equivalent to 500ml/min of gas.
MS
Vacuum operation.
200-300 degree C temp.
Upto 4000 Da
Required volatile buffers
Accepts 10ml/min gas flow
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22. Advantages of LC-MS/MS
Accuracy
Precision
Robustness
Sensitivity
Allows multi- analyte panels
Requires less sample preparations.
Compatible with generic sample preparations.
Versatility, can easily add new compounds
Speed
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23. Disadvantages
MS/MS capabilities cost more due to additional
hardware and software requirements
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24. Applications
LC-MS used to detect compounds from
polyaromatic (non-polar) to peptide and proteins.
LC-MS used for compounds identification and
purity.
Used for determination of pesticides, herbicides &
organic pollutant for environmental monitoring.
Proteome analysis is done by this technique.
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25. Applications
Molecular weight determination
2D gel spots
Protein complex (after primary fractionation)
Proteome separation and identification
Multi-dimensional liquid chromatography MS-based differential
proteomics
Quantitative proteomics
Analysis of post translational modification
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