hypenated technique hplc mass spectroscopy application with example

1. LIQUID CHROMATOGRAPHY – MASS SPECTROMETRY
PRESENTED BY:
Bhavya K B
2nd Sem M pharma
Dept. of Pharm. Analysis
SAC College of pharmacy ,
B G Nagara .
PRESENTED TO:
Dr . T. Yunus pasha
Head of the department,
Dept. Of Pharm. Analysis
SAC College of pharmacy,
B G Nagara .
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2. LIQUID CHROMATOGRAPHY –
MASS SPECTROMETRY.
(LC-MS)
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3. CONTENT:
 Introduction
 Principle
 Instrumentation
 Applications
 Reference
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4. INTRODUCTION:
LIQUID CHROMATOGROPHY – 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
 Liquid chromatography tandem mass spectrometry (LC–MS), has led to major
breakthroughs in the field of quantitative bioanalysis since 1990s due to its
inherent specificity, sensitivity, and speed.
 It is now generally accepted as the preferred technique for quantitation of small
molecule drugs, metabolites in biological matrices (plasma, blood, serum, urine,
and tissue).
 In the most of the cases the interface used in LC-MS are ionization source. Which
provide structural information on the separated sample component.
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5. Principle of LC-MS
 LC/MS combines the separating power of High Pressure liquid
chromatography (HPLC), with the detection power of mass spectrometry.
 It is combining the liquid chromatography with the mass spectrometry using
several types of interfaces in between,(which remove the solvent from the
column effluent and then bring the non- volatile and even thermally liable
analytes in gaseous form).
 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.
Which provide structural information on the separated sample component.
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6. INSTRUMENTATION OF LC-MS
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7. Neutral
samples
Inlet
INLET Form ion charged
molecules
Solid
Liquid
vapor
DATA
SYSTEM
Ionization
Source
Mass
Analyzer
Detector
Sort or separates
ions by M/Z
When ions strike
Detector it Detect ions
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8. ION INTERFACE SOURCE:
The various interfaces differ among themselves in the means of
separating the analytes from the mobile phase and the method are
used for ionization of the analyte. The commonly used interfaces are :
(I ) Thermo spray ionization interfaces (TSI);
(ii) Particle beam ionization interface (PBI);
(ii) Atmospheric pressure chemical ionizations interface (APCI);
(iv) Electrospray ionization interface (ESI).
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9. THERMOSPRAY IONIZATION INTERFACE:
 The effluent from the column [(usually containing an ammonium acetate buffer is
passed through a heated tube (300-400°C)] and rapidly expands as a jet spray into a
heated vacuum chamber where it forms supersonically a mist of electrically
charged droplets.
 The solvent is rapidly pumped away from the droplets to leave molecular ions such
as MH+ and MNH4+ by an ammonia chemical ionization process.
 The analyte ions with a static charge enter through a skimmer into the mass
spectrometer.
 Thermospray provides soft ionization with very small or no fragmentation.
 Thus the nonvolatile thermally labile organic compounds undergo gentle ionization
and the spectrum produced usually exhibits the protonated molecular ions.
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10. Thermospray ionization interface:
1. Chromatographic effluent comes in
at - a,
2.The transfer line is suddenly heated
at -b
3.And the spray is formed under
vacuum at- c.
4.At - d the spray goes between a
pusher with a positive potential and a
negative cone for positive ions.
5. The ions are thus extracted from the
spray droplets and accelerated
towards the spectrometer.
6. At - e, a high-capacity pump
maintains the vacuum in HPLC-MS.
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11. THERMOSPRAY IONIZATION
INTERFACE:
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12. PARTICLE BEAM INTERFACE
 The mobile phase from the columns nebulized with helium gas to produce an aerosol in
a reduced pressure compartment at 70°C.
 A cone is provided with a small orifice at the end of the chamber and this leads the
mixture of He, solvent vapors, and analyte molecules after being accelerated into a low
pressure area (a two stage momentum separator)
 where it expands supersonically the He and solvent molecules are lighter than the solute
molecules and these diffuse out of the stream and are readily pumped away .
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13.  now the relatively heavier solute molecules (having much greater momentum)
through a second cone are passed into a yet lower pressure area and then pass
straight through two skimmer plates along a narrow tube into the heated ionization
compartment.
 The particle beam interface provides electron ionization (EI) spectra and hence a
wide knowledge of such spectra is required for the compound identification.
 At present the TSI pressure and PBI have largely been replaced by systems usable
at atmospheric pressure.
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14. PARTICLE BEAM INTERFACE
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15. ATMOSPHERIC PRESSURE CHEMICAL
IONIZATION (APCI)
 In APCI, the LC eluent is sprayed through a heated [250-400] vaporizes at
atmospheric pressure.
 The heat vaporizes 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 analyzer.
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16. ATMOSPHERIC PRESSURE CHEMICAL
IONIZATION (APCI)
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17. ELECTRO SPRAY IONIZATION INTERFACE
 This is most popular one and operates at the atmospheric pressure. This is also a
soft ionization technique.
 The sample solution is sprayed across a 3-6 KV potential from a metal capillary
tube into an orifice in the interface.
 They charged and appears in the form of the highly charged droplets.
 When the solvent evaporates, the drop shrinks, charge density is increased giving
rise to their explosive break up into the still smaller charged droplets.
 The process is repeated many times so as to produce a spray of ever smaller
droplets.
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18.  As a consequence multiply-charged analyte species are produced. Such species
are passed through skimmers into the mass spectrometer and the uncharged
solvent molecules are pumped away.
 The characteristic and stability of such an spray depends upon the flow rate and
applied potential.
 Multiple charged sites are created on proteins and peptides and hence this permits
determination of a relatively large molecular weights > 3000 Dalton m/z ratio
limit of quadruple mass spectrometer ,
 as the m/z ratio decreases by a factor of 5, proteins of 50,000 Dalton or larger can
be monitored.
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19. ELECTRO SPRAY IONIZATION
INTERFACE
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20. MASS ANALYZER
 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.
Its task is to separate ions in terms of their mass-to- charge
ratio and to direct the beam of focused ions to the detector.
The key performance parameters of an analyzer
include;
(a) separation efficiency
(b) m/z measurement precision
(c) range of the m/z valuesmeasured
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21. Mass analyzers
Quadrupole Ion trap Time of flight
Fourier transformion
cyclotron resonance
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22. QUADRUPOLE
 In a quadrupole mass analyzer 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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23. TIME OF FLIGHT
 TOF mass analyzer 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 time-of-flight mass analyzer (TOF) consists of an ion accelerating grid and a flight tube
(about 1 m long), through which the ions travel to the detector.
 The analyzer separates ions accelerated by an electric field according to their velocity which
Depends on their mass and charges
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24. ION TRAP MASS ANALYZER
The ion trap mass analyzer 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
 One of the most popular ion trap analyzers (IT) is the
quadrupole ion trap consisting of a ring-shaped electrode and
two electrodes with a spherical cross-section, with the space
between them forming a trap. The ion trap analyzer traps
ions with a specific mass-to-charge ratio by means of an
electricfield.
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25. DETECTORS
 The detector is used to count the ions emergent from the mass analyzer, and may
also amplify the signal generatedfromeachion.Followingarethreedifferent kinds
of detectors are used in Mass Spectrometry
 Photographic plates
 Faraday cup
 Electron multiplier
 Channel electron multipliers
 Scintillation multiplier
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26. 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
providea reverse geometry analyzer.
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27. 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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28. Electron multiplier
 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 recordedwith electron
multiplier surface.
 Equation describe is 2n
 Where n= no of collisions with electron multiplier surface.
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29. 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 up to 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 up to 90 min
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31. APPLICATION:
 Determination of impurities in insulin-like growth factor with
electrospray-
 mass spectrometry (ES-MS)ES-MS provides an excellent means
for quality control of recombinant proteins, some of which are now
used as drugs,
 e g. human insulin, interferons, erythropoietin and tissue
plasminogen activating factor.
 variations in protein structure, such as degree of glycosylation, or
in the terminal amino acids of the protein can be seen quite clearly
 An example of how ES-MS can be used to determine minor
impurities in a recombinant protein is shown in Figure 9.38, where
some small additional ions in the mass spectrum of recombinant
insulin-like growth factor (IGF) can be seen.
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33. Characterization of a degradant of famotidine
 Tablets of famotidine, an anti-ulcer compound, were subjected to stress
conditioning pack.
 ' Figure 9.41 indicates the profile obtained from analysis of an extract from the
stressed tablets by LC atmospheric pressure chemical ionization mass
spectrometry (APCIMS).
 The structure of famotidine is shown in Figure 942.The mass spectra obtained for
famotidine and its degradant by APCIMS and APCIMS-MS are shown in Figures
9.43 and 9.44
 The degradant had a MW 12 amu higher than that of famotidine. The fragment at
m/z 189 was common to both spectra, indicating that the two molecules were
similar in structure.
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37. Profiling impurities and degradants in
butorphanol tartrate
HPLC coupled to an ES-MS was used to elucidate the structure of a number of
degradants in butorphanol following its storage in aqueous solution Figure 9.45shows
the LC-MS profile of the degradants which were detected in butorphanol.
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38. 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.(A)
 The second shows minor metabolite which is eluted at 9.41min.(B)
 Third graph show product spectrum from the ion at m/z 407 which confirms identity.(C)
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40. 4. Drug Development
Determinationof drugs and metabolites in plasma or other
biological fluids.
5.FOOD SCIENCE-melanin dosing. pesticide residues.
6. Life science.
7. Clinical science.
8. Forensic science.
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41. REFERENCE
 David G. Watson, Pharmaceutical analysis, second edition, page no -207
 P. C. Kamboj. Pharmaceutical analysis, page no –212
 Beckett –Practical pharmaceutical chemistry, fourth edition. Part two
 Vogel’s - text book of quantitative chemical analysis. J. Mendham,
R, C, Denney.
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