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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.
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CONTENTSHISTORY
INTRODUCTION
THEORY
MASS SPECTRUM
LC-MS
ION INTERFACE SOURCES
IONIZATION SOURCES
MASS ANALYZERS
DETECTORS
APPLICATIONS
CONCLUSION
REFERENCES
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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.
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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
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INTRODUCTION
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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.
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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
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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
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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
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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
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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
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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%.
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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
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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
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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
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Rela
tive a
bu
nd
an
ce
m/z values
110108
8179
29 –C2 H5
M+2M+
CH3CH2Br
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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
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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
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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
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ION INTERFACE SOURCES
DIRECT CHEMICAL IONIZATION
MOVING WIRE OR BELT INTERFACE
THERMOSPRAY
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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.
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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.
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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.
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IONIZATION SOURCES
Electron spray Ionization [ESI]
Atmospheric pressure chemical ionization [APCI]
Atmospheric Pressure photo ionization [APPI]
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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.
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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.
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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]
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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.
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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.
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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.
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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:
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ION TRAP MASS ANALYSER:
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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.
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DETECTORS
Photo graphi
c plates
Faraday cup
Electron
multiplier
Channel
electron
multipliers
Scintillation
detectors
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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.
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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
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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.
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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.
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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.
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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.
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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
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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.
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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.
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Graph-1
Graph-2
Graph-3
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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.
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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.
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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
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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.
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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.
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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.
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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.
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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.
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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
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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
VIGNAN PHARMACY COLLEGE 63
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
VIGNAN PHARMACY COLLEGE 64
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
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