LC-MS INSTURMENTATION & APPLICATIONS

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LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY (LC-MS)
UNDER THE ESTEEMED GUIDANCE OF Mr. Ch. DEVADASU
M.Pharm Assistant professor
Department of PA QA
Presented by S.NAVEEN JAIN 11AB1R0078
VIGNAN PHARMACY COLLEGE (Approved by AICTE, PCI
Affiliated to JNTU-K) VADLAMUDI, 522213.
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CONTENTS

• HISTORY
• 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 Spectrometry
• In 1897, Modern mass spectrometry (MS) is
  credited to the cathode-ray-tube experiments of
  J.J. Thomson of Manchester, England.
• In 1953, Wolfgang Pauls 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 Blakelys 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.

Sir J.J Thomson
DEMPSTER
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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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Basic components in mass spectrometer
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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..
Illustration of the basic components of a mass
spectrometry system.
Mass Sor
Neutral sample molecules
Mass Analyzer
Ionization Source
Detector
Solid Liquid Vapor
Data System
selected ions
When ions strike Detector it Detect ions
Inlet
Sort or separates ions by M/Z
Form ions charged molecules
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THEORY
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 .

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• i.e. potential energykinetic 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 mvzV

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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 HzVmv2/r Hzmv/r From
the above equation v Hzm / 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 peak
Base 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---------gtm2 takes place
in source then the daughter ion may be m2. But
m1-----gtm2 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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CH3CH2Br
29 C2 H5
110
108
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Relative abundance
M2
M
m/z values
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Liquid chromatographyMass spectrometry

• Liquid chromatographymass 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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LC-MS

• 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.

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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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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
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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 IonizationAPCI

• 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 IonizationAPPI

• 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 rateslt100m/min

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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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ION TRAP MASS ANALYSER

• 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.

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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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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
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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 APPLICATIONS INDENTIFICATION 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
  dioxymethamphetamineMDMA, 3,4-methylenedioxyeth
  yl amphetamineMDEA and 3,4-methylenedioxy
  amphetamineMDA 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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Conclusion

• 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.

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REFERENCES

• 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.

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• 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

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• Organic Spectroscopy Principles Applications.
  By-JAG MOHAN
• Page no 443-446
• Vogels 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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ACKNOWLEDGEMENT

• I sincerely thank my guide Ch. DEVADASU sir for
  giving support.
• I thank our principal Dr. P. SRINIVASA BABU sir
  seminar committee for giving me this
  opportunity.

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