this will help to know about the advance technique to analysis the biological sample in cancer diagnosis and general separation of proteins based upon the molecular weight and helps to analysis the new drug synthesis level

1. BY
S.BOOBASH RAJ
3RD
M.TECH
BIOTECHNOLOGY
LIQUID CHROMATOGRAPHY/MASS SPECTROSCOPY/MASS
SPECTROSCOPY (LC/MS/MS)

2. CONTENT
HYPHENATED TECHNIQUE
INTRODUCTION TO LC/MS/MS
PRINCIPLES OF LC/MS/MS
MASS SPECTROMETRY
INSTRUMENTATION OF LC/MS/MS
METHOD OF WORKING
SAMPLE PREPARATION
ION SOURCES
MASS ANALYZERS
APPLICATIONS& ADVANTAGES

3. HYPHENATED TECHNIQUE
THE TERM “HYPHENATION” WAS FIRST ADAPTED BY “HIRSCHFELD”
IN 1980. THE TECHNIQUE DEVELOPED FROM THE COUPLING OF A
SEPARATION TECHNIQUE AND AN ON-LINE SPECTROSCOPIC
DETECTION TECHNOLOGY.
ADVANTAGE OF HYPHENATED TECHNIQUES:
1. FAST AND ACCURATE ANALYSIS
2. HIGHER DEGREE OF AUTOMATION
3. HIGHER SAMPLE THROUGHPUT
4. BETTER REPRODUCIBILITY
5. REDUCTION OF CONTAMINATION DUE TO ITS CLOSED SYSTEM
6. SEPARATION AND QUANTIFICATION ACHIEVED AT SAME TIME
LIST OF HYPHENATED TECHNIQUES:
1. GC-MS, 2. LC/MS, 3. GC-IR, 4. LC/MS/MS, 5. GC-MS-MS

4. 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.

5. PRINCIPLES OF LC/MS/MS
LIQUID CHROMATOGRAPHY GENERALLY UTILIZES VERY
SMALL PARTICLES AND OPERATING AT RELATIVELY
HIGH PRESSURE, AND IS REFERRED TO AS HIGH
PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
IN HPLC, THE SAMPLE IS FORCED BY A LIQUID AT HIGH
PRESSURE (THE MOBILE PHASE) THROUGH A COLUMN
THAT IS PACKED WITH A STATIONARY PHASE
GENERALLY COMPOSED OF IRREGULARLY OR
SPHERICALLY SHAPE PARTICLES DERIVATIZED TO
ACCOMPLISH PARTICULAR TYPES OF SEPARATIONS.

6. MASS SPECTROMETRY
(MS)
MASS SPECTROMETRY (MS) IS AN ANALYTICAL
TECHNIQUE THAT MEASURES THE MASS-TO-CHARGE
RATIO OF CHARGED PARTICLES.
MS WORKS BY IONIZING CHEMICAL COMPOUNDS TO
GENERATE CHARGED MOLECULES OR MOLECULE
FRAGMENTS AND MEASURING THEIR MASS-TO-CHARGE
RATIOS.

7. INSTRUMENTATION OF LC/MS/MS
 1) HPLC CONSTITUTEES THE LC PART:
a) SOLVENT SYSTEM (MOBILE PHASE)
b) PUMPS
c) MIXER
d) INJECTOR
e) COLUMN
 2) MAS SPECTROMETER
A. ION SOURCES
i. ELECTROSPRAY IONIZATION.
ii. ATMOSPHERIC PRESSURE
CHEMICAL IONIZATION.
ATMOSPHERIC PRESSURE PHOTOIONIZATION

8. SAMPLE PREPARATION:
 SAMPLE PREPARATION CONSIST OF
CONCENTRATING THE ANALYTE AND
REMOVING COMPOUNDS THAT CAN CAUSE
BACKGROUND ION OR SUPPRESS IONIZATION.
 DESALTING TO REDUCE THE SODIUM AND
POTASSIUM ADDUCT FORMATION THAT
COMMONLY OCCURS IN ELECTROSPRAY.

10. ION SOURCES
 ELECTROSPRAY IONIZATION (ESI)
ATMOSPHERIC PRESSURE CHEMICAL IONIZATION(APCI)
ATMOSPHERIC PRESSURE PHOTOIONIZATION(APPI)

11. MASS ANALYSER
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)

12. QUADRA POLE MASS FILTER
 IT IS ALSO KNOWN AS A TRANSMISSION QUADRUPOLE MASS
SPECTROMETER
 IT CONSISTS OF FOUR CYLINDRICAL RODS, SET PARALLEL TO EACH OTHER.
 EACH OPPOSING ROD PAIR IS CONNECTED TOGETHER ELECTRICALLY, AND
A RADIO FREQUENCY (RF) VOLTAGE WITH A DC OFFSET VOLTAGE IS APPLIED
BETWEEN ONE PAIR OF RODS AND THE OTHER
 A LINEAR SERIES OF THREE QUADRUPOLES IS KNOWN AS A TRIPLE
QUADRUPOLE MASS SPECTROMETER.
 THE FIRST (Q1) AND THIRD (Q3) QUADRUPOLES ACT AS MASS FILTERS, AND THE
MIDDLE (Q2) QUADRUPOLE IS EMPLOYED AS A COLLISION CELL.
.

13. TIME OF FLIGHT
 TIME OF FLIGHT (TOF) IS THE MEASUREMENT OF THE TIME
TAKEN BY AN OBJECT, PARTICLE OR WAVE (BE IT ACOUSTIC,
ELECTROMAGNETIC, ETC.) TO TRAVEL A DISTANCE THROUGH A
MEDIUM.
 IONS ARE ACCELERATED BY AN ELECTRIC FIELD OF KNOWN
STRENGTH.
 THIS ACCELERATION RESULTS IN AN ION HAVING THE
SAME KINETIC ENERGY AS ANY OTHER ION THAT HAS THE
SAME CHARGE.
 THE TIME THAT IT SUBSEQUENTLY TAKES FOR THE PARTICLE
TO REACH A DETECTOR AT A KNOWN DISTANCE IS MEASURED.
 THIS TIME WILL DEPEND ON THE MASS-TO-CHARGE RATIO OF
THE PARTICLE (HEAVIER PARTICLES REACH LOWER SPEEDS).

14. ION TRAP
AN ION TRAP IS A COMBINATION OF ELECTRIC OR MAGNETIC
FIELDS USED TO CAPTURE CHARGED PARTICLES, OFTEN IN A
SYSTEM ISOLATED FROM AN EXTERNAL ENVIRONMENT.
PAUL TRAPS ARE COMMONLY USED AS A COMPONENTS OF
A MASS SPECTROMETER.

16. Work flow of lc/ms /ms in cancer diagnostic

17. Just I wanna say
Thank you