LIQUID CHROMATOGRAPHY-MASS CHROMATOGRAPHYSPECTROMETRYSPECTROMETRY-MASS SPECTROMETRY

Visit www.bpharmstuf.com For more ppt s

www.bpharmstuf.com

INTRODUCTION :
LC/MS/MS

is an analytical chemistry technique that combines the physical separation capabilities of LC with mass analysis capabilities of MS sensitivity & specificity

High

Specific

detection and potential identification of chemicals in presence of other chemicals

www.bpharmstuf.com 2

LC/LC-MS/MS-Tandem LC, Tandem MS

www.bpharmstuf.com

principle

Sample

+ _

Ionizer

Mass Analyzer

Detector

www.bpharmstuf.com

Mass Spectrometry (MS)

Introduce sample to the instrument Generate ions in the gas phase Separate ions on the basis of differences in m/z with a mass analyzer Detect ions

www.bpharmstuf.com

INSTRUMENTATION
COMPONENTS: Vacuum system Sample inlet Ion source Mass analyzer Detector

www.bpharmstuf.com

SCHEMATIC REPRESENTATION OF MASS SPECTROMETRY

High Vacuum System
Turbo pumps Diffusion pumps Rough pumps Rotary pumps

Inlet

Ion Source
MALDI ESI IonSpray FAB LSIMS EI/CI

Mass Filter
TOF Quadrupole Ion Trap Mag. Sector FTMS

Detector

Data System
PCs UNIX Mac

Sample Plate Target HPLC GC Solids probe

Microch plate Electron Mult. Hybrid Detec.

www.bpharmstuf.com

Vacuum system
Ion source, mass analyzer, detector are to be maintained under vacuum Vacuum system makes desirable ions not to collide with other ions and molecules High vacuum is achieved by connecting a low vacuum pump to output of a high vacuum pump

www.bpharmstuf.com

Inlet system
Sample is to be in vapour state and it should enter the ionization chamber at constant rate to achieve this system is usually heated. Less volatile liquids and solids may be directly vapourised within the ionization chamber. Liquids are handled by hypodermic needle injection through a silicone rubber dam.

www.bpharmstuf.com

ION SOURCE
Initial step for MS analysis is formation of gaseous analyte ions Scope of MS method depend upon ionization process These are categorised into two types 1. Gas phase source 2. Desorption source
www.bpharmstuf.com 10

In gas phase source sample is first vapourised and then ionised. Compounds of gaseous sources have molecular masses ranging 10 daltons. 10 In desorption source solid or liquid sample is converted directly into gaseous ions. It do not require volatilization.

www.bpharmstuf.com

11

Ion sources are also classified as: 1. hard ionization sources (EI, chemical ionization) 2. soft ionization sources (MALDI,ESI)  Hard ionization gives information about structural and functional groups of analyte molecule  Soft sources gives information about molecular mass of analyte molecule.

www.bpharmstuf.com

12

www.bpharmstuf.com

13

Electron Impact Ionization



Sample is brought to high temperatures to produce molecular vapour Resulting molecules ionised by bombarding with energetic molecules produced by tungsten filament Primary product is singly +ve ion

www.bpharmstuf.com

14

CHEMICAL IONIZATION
In these gaseous atoms of sample are ionised by collision with ions produced by electron bombardment of excess of reagent gas. Usually +ve ions are used. To carryout CI experiments ionization area is modified by adding vaccum pump and by reducing the width of the slit.
www.bpharmstuf.com 15

MALDI
It can be used to obtain accurate molecular mass information about polar bio polymers. ranging in molecular mass about 10 10 daltons. Low concentration analyte is dispersed in solid or liquid matrix deposited on the end of metal plate These plate placed in vaccum chamber & laser beam is focused on to the sample.

www.bpharmstuf.com

16

MatrixMatrix-Assisted Laser Desorption Ionization

337 nm UV laser

Nicotinic acid

MALDI
www.bpharmstuf.com 17

MALDI Ionization
+ + ++ + + + + + -+ +
+
+ + Matrix Laser Analyte

Laser beam is focused on to the sample Matrix must strongly absorb laser radiation Matrix and analyte are then desorbed and ionised by the proton transfer with protonated matrix ions. Eg of matrix is nicotinic acid.
www.bpharmstuf.com 18

ELECTROSPRAY IONIZATION
 It

is used for analyzing bio molecules having molecular weight 10 lakhs daltons or more  Liquid sample is made to pass through capillary stainless steel needle and drying gas is introduced into the apparatus and by applying vaccum liquid sample turns to droplets  ions evaporate from the surface of the droplets.
www.bpharmstuf.com 19

Electrospray (Detail)

www.bpharmstuf.com

20

ESI: Droplet size reduction & fission

Droplet size reduction occurs by the continual repetition of two processes:
1. Desolvation (evaporation of neutral solvent and volatile buffers) 2. Droplet fission caused by electric repulsion between like charges.
++ + + + + + + + + + + ++ +
+++++ + + + + + ++ ++ +

+++++ + + + + + ++ ++ +
www.bpharmstuf.com

+++++ + + + + + ++ ++ +

+ + + + +

+++++ + + + + + ++ ++ +

21

Electrospray: Overview
Orifice Plate

5kV
++ ++ + + + ++ + + + +++ ++

Nebulizing Gas

Desolvation ++ ++ & Fission + +

+

+ +

+++

+ +

+
++++ + + + + + ++

Droplet Formation

+
+ +

To MS

Gas Phase Ion Generation

Drying Gas

www.bpharmstuf.com

22

Positive or Negative Ion Mode?

If the sample has functional groups that readily accept H+ (such as amide and amino groups found in peptides and proteins) then positive ion detection is used-PROTEINS usedIf a sample has functional groups that readily lose a proton (such as carboxylic acids and hydroxyls as found in nucleic acids and sugars) then negative ion detection is used-DNA usedwww.bpharmstuf.com 23

Different Mass Analyzers

Magnetic Sector Analyzer (MSA) (MSA)

Quadrupole Analyzer (Q) (Q

Time-ofTime-of-Flight Analyzer (TOF) (TOF)

www.bpharmstuf.com

24

Magnetic Sector Analyzer

www.bpharmstuf.com

25

MAGNETIC SECTOR ANALYZER

Permanent magnet or electro magnet to cause beam from ion source travel in curved path Ions enter in to the mass analyzer tube Ions of different masses can be scanned Then ions fall on detector

www.bpharmstuf.com

26

TIME OF FLIGHT MASS ANALYZER :

Ions

enter into field-free drift fieldtube by an electric field pulse of 1000 to 10000 volts Ions are separated based on their mass Lighter particles arrive at the detector earlier than the heavier ones
www.bpharmstuf.com 27

Quadrupole Mass Analyzer

www.bpharmstuf.com

28

Quadruple Mass Analyzer

A quadrupole mass filter consists of four parallel metal rods with different charges Two opposite rods have an applied + potential and the other two rods have a potential The applied voltages affect the trajectory of ions traveling down the flight path For given dc and ac voltages, only ions of a voltages, certain mass-to-charge ratio pass through the mass-toquadrupole filter and all other ions are thrown out of their original path
www.bpharmstuf.com 29

DETECTORS
The detector counts the ions and signal is generated that is proportional to the ions Once an ionized sample has been analyzed by m/e ratio in the analyzer The ions are detected and reported to the data system

www.bpharmstuf.com

30

Mass Detectors

Electron Multiplier (Dynode)

www.bpharmstuf.com 31

TANDEM MASS SPECTROMETRY

It

is also called as mass spectrometryspectrometry-mass spectrometry Ions formed enter mass analyzer1 Precursor ions enter interaction cell Product ions are formed They are analyzed by mass analyzer2 They are detected by detectors
www.bpharmstuf.com 32

BLOCK DIAGRAM OF TANDEM SPECTROMETER

Sample Precursor ion
ABCD+

Original ions Ion source

ABC+ , ABCD+, ABCDE+

Mass Analyzer 1

Interaction cell Product ions

A+ AB+ AABC+

Detectors

Mass analyzer 2

www.bpharmstuf.com

33

Tandem Mass Spectrometry

Purpose is to fragment ions from parent ion to provide structural information about a molecule Also allows mass separation and AA identification of compounds in complex mixtures Uses two or more mass analyzers/filters separated by a collision cell filled with Argon or Xenon Collision cell is where selected ions are sent for further fragmentation
www.bpharmstuf.com 34

LC/MS/MS
The spectra are collected for compounds as they exit from a chromatography column These spectra are then stored in a computer for subsequent processing and is introduced to ion source from which ions are formed

www.bpharmstuf.com

35

SCHEMATIC REPRESENTATION OF LC/MS/MS

HPLC system Ion source Mass analyzer1 Interaction cell

detectors

Mass analyzer2

www.bpharmstuf.com

36

Applications of MS
elucidation of organic & biological molecules Identification of components in TLC&PC Determination of AA sequences in proteins Analysis of aerosol particles Determination of molecular mass of peptides, proteins
Structural
www.bpharmstuf.com 37

Different compounds can be uniquely identified by their mass

Butorphanol
N -CH2OH HO

L-dopa
COOH -CH2CH-NH2 HO

Ethanol

CH3CH2OH

HO

MW = 327.1

MW = 197.2
www.bpharmstuf.com

MW = 46.1
38

Analytical method to measure the molecular or atomic weight of samples

www.bpharmstuf.com

39

Pharmacokinetics: It is used in pharmacokinetic studies of pharmaceuticals and employed in bioanalysis Drug development: impurity identification, invivo drug screening, quality control Determine isotopic composition of elements within a sample
www.bpharmstuf.com 40

Peptide Mass Fingerprinting (PMF)

Characteristic pattern of peptides is used for the identification of protein the method is called peptide mass finger printing (PMF)

www.bpharmstuf.com

41

REFERENCES
Skoog, Principles of Instrumental Analysis. B.K.Sharma, Instrumental and Chemical Analysis. K.A. Connors, A Textbook of pharmaceutical analysis. Gurdeep R. Chatwal Sham k. Anand. Robert D. Brown, Introduction to Instrumental Analysis. Y. Anjaneyulu and Maraiah, Quality Assurance and Quality Management in Pharmaceutical Industry.
www.bpharmstuf.com 42

Visit www.bpharmstuf.com For more ppt s

www.bpharmstuf.com

43