LC-MS INSTURMENTATION & APPLICATIONS

1

LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY (LC-MS)

Presented by

S.NAVEEN JAIN

11AB1R0078

UNDER THE ESTEEMED GUIDANCE OF

Mr. Ch. DEVADASU M.Pharm

Assistant professor

Department of PA & QA

VIGNAN PHARMACY COLLEGE (Approved by AICTE, PCI &

Affiliated to JNTU-K)VADLAMUDI, 522213.

VIGNAN PHARMACY COLLEGE 2

CONTENTSHISTORY

INTRODUCTION

THEORY

MASS SPECTRUM

LC-MS

ION INTERFACE SOURCES

IONIZATION SOURCES

MASS ANALYZERS

DETECTORS

APPLICATIONS

CONCLUSION

REFERENCES


A Brief History of Mass SpectrometryIn 1897, Modern mass spectrometry (MS) is credited to the cathode-

ray-tube experiments of J.J. Thomson of Manchester, England.

In 1953, Wolfgang Paul’s invention of the quadrupole and

quadrupole ion trap earned him the Nobel Prize in physics.

In 1968, Malcolm Dole developed electrospray ionization (ESI).

In 1974, Atmospheric pressure chemical ionization (APCI) was

developed by Horning.

In 1983, Vestal and Blakely’s work with heating a liquid stream

known as thermospray was developed.


The concept of mass spectrometry was first put forth by Sir J.J Thomson, English Physicist Who discovered the

electron in 1887.He got 1906 Nobel Laureate in Physics.

DEMPSTER

Sir J.J Thomson


INTRODUCTION


What does a mass spectrometer do?

Mass –spec or simply MS is a super important technique

Mass spec is easy technique to give you Molecular weight

(from molecular ion (M+)

You can get Molecular formula (Elements present).

Nearly ALL ELEMENTS in the periodic table can be determined by

mass spectrometry.

MS is incredibly valuable in getting structure (from fragments) of

Bio molecules such as peptides and proteins and also

natural products and also organic structures.

It can give information about chemical structures.

InletIon

SourceMassFilter Detector

DataSyste

m

High Vacuum System

Sample PlateTargetHPLCGC

Solids probe

MALDIESI

Ion SprayFAB

LSIMSEI/CI

TOFQuadrupol

eIon Trap

Mag. SectorFTMS

Electron Multiplier, Faraday

cup

PC’sUNIXMac

Basic components in mass spectrometer


Mass Sor

Illustration of the basic components of a mass spectrometry system.

Solid• Liquid• Vapor

IonizationSource

MassAnalyzer Detector

Inlet

Form ions

charged molecules

selectedions

DataSystem

Sort or separates ions by M/Z

When ions strikeDetector it Detect ions

The inlet transfers the sample into the vacuum of the mass spectrometer. In the

source region, neutral sample molecules are ionized and then accelerated into

the mass analyzer. The mass analyzer is the heart of the mass spectrometer.

This section separates ions, either in space or in time, according to their mass to

charge ratio. After the ions are separated, they are detected and the signal is

transferred to a data system for analysis..

Neutral samplemolecules


The mass spectrometer is an instrument which help in separating the individual

atoms or molecules because of difference in their masses.

Consider a molecule M, Which is bombarded with a beam of electrons

M + e- M+. +2e-

where, M+. is molecular ion or radical ion

2e- is electron

Now voltage “v” is applied in an electric field then ions are accelerated. In this

condition the energy given to each particle is zV and this is equal to kinetic

energy which is equal to 1/2mv2 .

THEORY


i.e. potential energy=kinetic energy

zV = 1/2mv2

2zV = mv2

2zV/m= v2

= v

Where V = Velocity of particle

m = mass

z = charge of an electron

V = Acceleration voltage

All the particles posses some energy zV with some kinetic energy

1/2mv2, but m value changes from molecule to molecule with respect velocity

‘v’ also changes. i.e. ½ mv=zV


When all charged particles have been accelerated by an applied voltage, they

enter into a magnetic field “H”. Then attractive force is HzV. And balancing

force of particle is mv2 /r.

Centripetal = Centrifugal

HzV=mv2/r

Hz=mv/r

From the above equation v=

Hz=m / r

By squaring on both sides

H2 z2 = m2 (2zV/m) / r2

H2 z = 2v m/ r2

m/z = H2 r2 / 2v


MASS SPECTRUM

The mass spectrum is the plot of mass to charge ratio of positively charged

ions against their relative abundance. The m/z ratio are taken along the

abscissa, while relative abundance is taken on ordinate.

BASE PEAK:

The most intense peak in the mass spectrum is called the base peak. Base

peak is the highest peak it is assigned a relative intensity of 100%.


MOLECULAR ION PEAK:

The ion formed from a molecule by removal of one electron of lowest

ionization potential is known as molecular ion.

The molecular ion is detected as mass to charge ratio that corresponds to

molecular weight of molecule. The molecular ion peak gives the molecular

weight of compound . The molecular ion peak is highest mass number except

isotope peak. Molecular ion peakBase peak

Fragment ions


FRAGMENT IONS:

The ions produced from the molecular ion by cleavage of bonds are called

fragment ions

They have lower masses and used as building blocks to reconstruct the

molecular structure. Fragmentation of molecular ion cleavage bond occurs in

heterolytic and homolytic cleavage.

METASTABLE IONS:

Mass spectrum of molecule shows sharp peaks at m/z integrals. But some show

diffuse, broad low intensity peaks at non integral m/z values these are called

metastable ions

m1+ m2

++ neutral fragment


If in the reaction m1+--------->m2

++ takes place in source then the

daughter ion may be m2+. But m1

+----->m2++ if occurs after the source and

before arrival at collector at lower mass than m2+ and is said to be

metastable ion m*.The peak (m*) due to such fragmentation therefore

occurs at lower mass than m2+ and generally broad. The relation between

the m* with that of m1+ & m2+ can be written as

m*=(mz+)/m1


Rela

tive a

bu

nd

an

ce

m/z values

110108

8179

29 –C2 H5

M+2M+

CH3CH2Br


Liquid chromatography–Mass spectrometry

Liquid chromatography–mass spectrometry (LC-MS, or alternatively

HPLC-MS) is an ADVANCED ANALYTICAL INSTRUMNTAL

technique that combines the physical separation capabilities of LIQUID

CHROMATOGRAPHY (or HPLC) with the mass analysis capabilities

of MASS SPECTROMETER


It is the combination of liquid chromatography and the mass spectrometry.

In LC-MS we are removing the detector from the column of LC and fitting the column to interface of MS.

In the most of the cases the interface used in LC-MS are ionization source.

LC-MS


HPLC is a method for separating a complex mixture in to its individual

components.

High sensitivity of mass spectrometry provides the information for

identification of compounds or structural elucidation of compounds.

Combination of these two techniques is LC-MS


ION INTERFACE SOURCES

DIRECT CHEMICAL IONIZATION

MOVING WIRE OR BELT INTERFACE

THERMOSPRAY


DIRECT CHEMICAL IONIZATION:

The simplest way to introduce HPLC effluent into mass spectrometer is to

split the flow.

Chemical ionization is most suitable in this technique because under CI

pressure conditions, solvent rates as high as 10 micro lit / min can be

tolerated. This permits10-20 micro lit/ min (1-2%) eluate from the HPLC to

that of the ion source.


MOVING WIRE OR BELT INTERFACE:

The moving wire or belt interface consists of an auxiliary vacuum

chamber through which a continuous train carries the column eluate,

evaporates the solvent and subsequently vaporizes the solute. The

moving interface was developed by Mc Fadden et al84,85 which can

transfer upto 30-40% of solute from HPLC to ion source. The residual

solvent helps to maintain vaccum in the MS. The sample is finally

conducted into the ion source, where it vaporizes.


THERMOSPRAY:

The eluent from the column is vapourised and a portion of vapour is transferred

to the mass spectrometer and rest of the vapour is pumped to waste. As a result

a supersonic jet vapour, containing a mist of particles and solvent droplet is

created. There vaporization takes place in presence of an electrolyte the LC

buffer, the droplets are charged. And finally they enter into the ionization

chamber.


IONIZATION SOURCES

Electron spray Ionization [ESI]

Atmospheric pressure chemical ionization [APCI]

Atmospheric Pressure photo ionization [APPI]


Electron Spray Ionization:

The LC eluent is sprayed into chambers in presence of strong

electrostatic field and heated drying gas. Large molecules even acquires

more than one charge this process mathematically called deconvolution.

The heated gas causes solvent in the droplet evaporation. The repulsive

force b/w ions with like charge exceeds cohesive force and ions are eject

into gas phase and passed into the analyser.


Atmospheric Pressure Chemical Ionization[APCI]:

In APCI, the LC eluent is sprayed through a heated [250-400] vapourises at

atmospheric pressure. The heat vapourises liquid which results gas solvent

are ionized by corona needle by which electrons are discharged. Thereby

chemical reactions takes place and ions passes through a capillary orifice

into mass analyser.


Atmospheric Pressure Photo Ionization[APPI]:

Atmospheric pressure photo ionization is relatively never technique. Here a

discharge UV lamp is placed which generates photons in a narrow range of

ionization energies. It shows its ionization for highly non polar compounds

and low flow rates[<100m/min]


Mass analyzers

Quadrupole

Ion trap

Time of flight

Fourier transform ion

cyclotron resonance

Mass Analyzers

They deflects ions down a curved tubes in a magnetic fields based on

their kinetic energy determined by the mass, charge and velocity. The

magnetic field is scanned to measure different ions.


Quadrupole: In a quadrupole mass analyser a set of four rods are arranged parallel to

the direction. Here a DC current and radio frequency RF is applied to

generate oscillating electrostatic field in between the rods. Based on this

only m/z is been determined and stable oscillation takes place. And ion

travels in quadrupole axis with cork screw type of trajectory.


TIME OF FLIGHT:

TOF mass analyser is based on simple idea that the

velocities of two ions are created by uniform

electromagnetic force applied to all the ions at same

time, causing them to accelerate down a flight tube.

Lighter ions travels faster and strike the detector first so

that the m/z ratio of ions is detected.


The ion trap mass analyser operates by similar principles where it

consists of circular ring electrode

Plus two end caps that form a chamber. Here AC or DC power along RF

potential is applied between the cups and the ring electrode.

There the ions entering into the chamber are trapped by electromagnetic

fields and they oscillates in concentric trajectories. This process is

called resonant ejection.

ION TRAP MASS ANALYSER:


ION TRAP MASS ANALYSER:


FOURIER ION TRASNFORM ION CYCLOTRON RESONANCE:In this ions entering are trapped in circular well defined orbits for extended

periods by electrical and magnetic fields. These are excited by radio

frequency RF and generates the current. This current is converted to

Fourier transform into orbital frequencies. The angular frequency of

motion is called cyclotron frequency.


DETECTORS

Photo graphi

c plates

Faraday cup

Electron

multiplier

Channel

electron

multipliers

Scintillation

detectors


Photo graphic plates:

It is used as it is capable of higher resolution and speeder than

electronic devices. i.e. it can detect ions of all the masses and provide

a reverse geometry analyzer.


Faraday Cup:

It is a metal cup into which all the ions are directed and the signal

produced is very stable and reproducible. It is used on spectrometers

where quantitative data is very important


Electron multipliers:

In this the current can be measured so accurately by just one ion strikes the

detector can be measured i.e. when an ion strikes the surface of electron

multiplier two electron are ejected. This process continues until the end of

electro multiplier end is reached and electric current is analyzed and recorded

with electron multiplier surface. Equation describe is 2n

Where n= no of collisions with electron multiplier surface.


DATA HANDLING

All the mass spectrometers now employ computer control of same functions

and also use a computerised display and output.

The amount of data generated even by a fairly modest mass spectrometer is

very large indeed, a single run may store data for upto 100 fragments from

each type of molecule and if, LCMS analyses is being performed, a complete

mass spectrum is generated and stored every sec for upto 90 min

APPLICATIONS

MOLECULAR WEIGHT DETERMINATION

DIFFERENTIATION OF SIMILAR OCTAPEPTIDES:

The spectra of octa peptides whose m/z ratio differ only by 1m/z. The only

difference in sequence is at C-terminus that is one having threonine and other

having threonine amide.



DETERMINATION OF MOLECULAR WEIGHT OF GREEN

FLORESCENT PROTEIN:

GRF is 27000 Dalton protein with 238 amino acids. During electron spray

ionization GFP acquires multiple charges. This allows to be analyzed by mass

spectrometer by m/z range. Here the mass deconvolution is then used to

determine the weight of the protein GFP.


STRUCTURAL DETERMINATION:

LC/MS is also applied for the information about the molecular structure. this

can be in addition of mol wt if the identity of the compound is already known.

STRUCUTRAL DETERMINATION OF GINSENOSIDES USING

LC/MS ANALYSIS:

GINSENG root have dozens of biologically active saponins called

gensinosides. In mass spectrometer, ion trap mass analyser permits multiplier

stages of precursor ion isolation and fragmentation

The most prominent feature is sodium adduct ion corresponding to cleavage of

single bonded glycoside.

Subsequent isolation and fragmentation at m/z 789.7 yields two products.


1.A more abundant ion at m/z 365.1 losses oligosaccharide ion

2. Loss of less abundant ion at m/z 627.5 i.e. de-oxyhexose sugar


PHARMACEUTICAL APPLICATIONS

RAPID CHROMATOGRAPH OF BENZODIAZEPINES:

It allows compounds to be separated even they are chromatographically

unresolved. A series of benzodiazepines are analyzed using both UV and MS

detectors. In this chlorine cl- has a characteristic abundance of 2 most abundant

isotopes. By which TRAIZOLAM spectrum shows a two cl- ions and

DIAZEPAM shows only one cl- ion.


IDENTIFICATION OF BILE ACID METABOLITES:

The use of in-vitro incubation of bile acid deoxycholic with rat liver

microsomes to stimulate metabolism of drug candidate. These precursor ions

are were automatically fragmented and full scan ion spectra is collected.

The first graph shows the base peak chromatogram.

The second shows minor metabolite which is eluted at 9.41min.

Third graph show product spectrum from the ion at m/z 407 which confirms

identity.


Graph-1

Graph-2

Graph-3


CLINICAL APPLICATIONS

HIGH SENSITIVITY DETECTION OF TRIMIPRAMINE AND

THIORIDAZINE:

Triminpramine is a tricyclic anti depressant, Thioridazine is a tranquilizer

when these compounds are detected by UV there is no extract level. For this

single quadrupole of LCMS was been used for analysis by ion monitoring.


GENERAL CONSIDERATION:

Immunoassays can be detected as it is more sensitive and very easily allocated.

Similarly many drug and other small molecules assay are readily performed by

immunoassay, LC/MS, GLC etc.

Lc/ms is best suited for either problems i.e. those which have tedious specimen

preparation protocols in which more than one compound must be measured

simultaneously.

BIOCHEMICAL GENETICS:

One of the seminal applications of Lc/ms was multiple analyte screening for

inborn errors of metabolism.

Few of inherited metabolic disorders detected by newborn screening with

Lc/ms.


AMINO ACIDOPATHIES

Phenyl ketoxuria

Tyrosinemia

Non ketotic hyperglycemia

Argininemia

ORGANIC ACIDEMIAS

Glutaric academia type I

Propionic academia

Methyl malonic academia

DEFECTS OF FATTY ACID OXIDATION

Short chain acyl-CoA dehydrogenase deficiency

Ethylmalonic academia

Carnitine transport defect


THERAPEUTIC DRUG MONITORING:

A direct injection of HPLC/MS method has been developed for the rapid

identification and quantitation of seven tricyclic antidepressants in human

plasma can be done.

A sensitive and specific method is determined for phenprocoumon, warfarin in

human plasma by Lc/ms has been developed for monitoring of anticoagulant

therapy.


FOOD APPLICATIONSINDENTIFICATION OF ALFATOXINS IN FOOD:

Aflatoxins are fungi produced in food. By total in chromatogram we can detect

4 different aflatoxins. Even though they are structurally very similar to each

other but can be uniquely identified by LC/ms.


PHARMACOLOGY:

Since most drugs have a core chemical structure, which is retained by

their metabolites, the determination of drug metabolites in pharmacology

is now dominated by Lc/ms. The core structure produces charged or

neutral fragment. The charged can be detected using ion scanning

strategy. Unchanged can be recognized by neutral loss scanning.


TOXICOLOGY

•An even simpler strategy solid phase extraction and direct flow

injection with no HPLC has been shown to be viable using clinical

specimens.

• Determination of designer drugs 3,4-methylene

dioxymethamphetamine[MDMA], 3,4-methylenedioxyethyl

amphetamine[MDEA] and 3,4-methylenedioxy

amphetamine[MDA] by Lc/ms has been showing using fluorescent

detection.

•The method was found suitable for determination for whole blood,

serum, vitreous humor and urine.


IN DRUG DISCOVERY

Drug discovery involves a number of phases, including target

identification, lead identification, small molecule optimization and pre-

clinical and clinical development.

Target identification has been speeded up as result of genomics but the

measurement of gene transcription through detection of RNA does not

necessarily indicate proteins produced are, which are translated by

glycosylation or phosphorycation.

With advances in Lc/ms, identification of translated proteins is

possible.


LC-MS has proved to be an extremely sensitive and specific

technique for the analysis of pharmaceuticals.

It plays important role in the studies of drug metabolism,

discovery of new drug candidates and the analysis,

identification and characterisation of impurities and

degradants in drug substances and products.

Conclusion


Pharmaceutical Analysis -A Text book for pharmacy students &

chemical applications By- DAVID G. WATSON.

Page no: 206-213.

Instrumental analysis By- SKOOG, HOLLER,CROUCH.

Page no- 607-631.

Spectroscopy By- PAVIA, LAMPMAN,KRIZ, VYVYAM

Page no- 401-417.

REFERENCES


Elementary Organic Spectroscopy principles &

chemical applications. By- Y.R.SHARMA

Page No- 280-289

Instrumental Methods Of Chemical Analysis. By- B.K

SHARMA

Page No- C286-C291

Instrumental Methods Of Chemical Analysis By-

GURDEEP R.CHATWAL,SHAM K.ANAND

Page No 2.272-2.286


Organic Spectroscopy Principles & Applications. By-

JAG MOHAN

Page no 443-446

Vogel’s Text Book Of Quantitative Chemical Analysis.

By- J.MENDHAN, R C DENNY, J.D. BARNES, M.THOMAS,

B. SIVA SANKAR.

Page no 720-741

AGILENT TECHNOLOGIES.

Page no 6-30

LC-MS INSTURMENTATION & APPLICATIONS

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