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Liquid chromatography–massspectrometry• prof. P.Ravisankar• Vignan Pharmacy college• Valdlamudi• Guntur Dist.• Andhra Prad...
Liquid chromatography–massspectrometry• Liquid chromatography–mass spectrometry(LC-MS, or alternatively HPLC-MS) is ananal...
Spectral resolution is possibleCompound identification from spectral dataHigh degree of specificity
Mass spectrometers work by ionising themolecules and identifying the ions according totheir mass-to-charge(m/z) ratios.T...
MOBILE PHASE :-The mobile phase is the solvent that moves thesolute through out column.General requirements:-(1)low cost, ...
COLUMN :-The use of di-functional or tri-functional silanesto create bonded groups with two or threeattachment points lead...
Sample preparation:-Sample preparation generally consists of concentratingthe analyte and removing compounds that can caus...
Common ionisation techniques are :Electrospray ionisation(ESI)Atmospheric pressure chemicalionisation(APCI)Atmospheric ...
Which is best?•It depends on the exact application.•Increasing polarity and molecular weight andthermal instability favors...
Electrospray ionization:(ESI) is a technique used in mass spectrometry to produce ions. It isespecially useful in producin...
The solvent evaporates from a charged droplet until it becomes unstableupon reaching its Rayleigh limit. At this point, ...
Atmospheric pressure chemical ionization• In APCI,the eluent is sprayed through aheated vaporizer at atmosphericpressure.T...
Atmospheric pressure photoionization• As in APCI,a vaporizer converts the LC eluent tothe gas phase.• A discharge lamp gen...
Atmospheric pressure photoionizationHPLC inletNebulising gasUVlampNebuliserheaterDrying gascapillary
Matrix-assisted laser desorption/ionization(MALDI)The MALDI is a two step process. First, desorption is triggered by a U...
Matrix-assisted laser desorption/ionization
•Useful interface which is applicable to a wide range ofmolecules.•The heavier sample molecules enter the MS and can be io...
•They deflects ions down a curved tubes in a magneticfields based on their kinetic energy determined by themass, charge an...
Ions entering the chamber are trapped in the circular orbits bypowerful electrical and magnetic fields.When excited by a...
QuadrupoleThe quadrupole consists of four parallel metal rods. Each opposing rod pair is connected together electrically...
Ring ElectrodeRing ElectrodeEntranceEndcapElectrodeExit EndcapElectrodeIon Traps:Ions fill the space between a ring electr...
In full scan mode:-Ions fill and are trapped in space then masses are scanned outof the trap sequentially.-Ions are not lo...
•LC/MS is suitable for many applications,frompharmaceutical development to environmentalanalysis.•It ability to detect a w...
 Molecular weight determination e.g differentiation ofsimilar octapeptides,determinig the molecular weight ofgreen fluore...
One fundamental application of LC/MS is the determinationof molecular weights.This information is key information todeterm...
Mass spectra differentiating two very similaroctapeptidesFigure 1
Structural determination of ginsenosides using MSanalysis:•Ginseng root,a traditional chinese herbalremedy,contains more t...
Rapid chromatography of benzodiazepines:•The information available in a mass spectrumallows some compounds to be separated...
IDENTIFICATION AND QUANTIFICATION OF INDIVIDUALBENZODIAZEPINESFigure 2
High sensitivity detection of trimipramine andthioridazine:•Trimipramine is a tricyclic antidepressant withsedative proper...
High sensitivity detection of trimipramineand thioridazineffiFigure 4
Identification of aflatoxins in food:•Aflatoxins are toxic metabolites produced in foods bycertain fungi.•Figure 3 shows t...
Identification of aflatoxins in foodFigure 3
Detection of phenyl urea herbicides:•Many of the phenyl urea herbicides are very similar anddifficult to distinguish with ...
Chromatogram of phenylurea herbicidewith UV and MS spectraFigure 5
PharmacokineticsLC-MS is very commonly used in pharmacokineticstudies of pharmaceuticals and is thus the mostfrequently u...
•LC-MS is the method of choice for the study ofdrug metabolism because of its sensitivity andspecificity.•The metabolism o...
Drug developmentLC-MS is frequently used in drug developmentat many different stages including PeptideMapping, Glycoprote...
•LC-MS has proved to be an extremelysensitive and specific technique for theanalysis of pharmaceuticals.•It plays importan...
Liquid chromatography-mass spectrometry-Marcel DekkerLC/MS A Practical user guide-Marvin C McMaster and Christopher Mc M...
Liquid chromatography–mass spectrometry (LC-MS) BY P. RAVISANKAR
Liquid chromatography–mass spectrometry (LC-MS) BY P. RAVISANKAR
Liquid chromatography–mass spectrometry (LC-MS) BY P. RAVISANKAR
Liquid chromatography–mass spectrometry (LC-MS) BY P. RAVISANKAR
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Liquid chromatography–mass spectrometry (LC-MS) BY P. RAVISANKAR

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Liquid chromatography–mass spectrometry (LC-MS) BY
P. RAVISANKAR
VIGNAN PHARMACY COLLEGE
VADLAMUDI
GUNTUR
A.P
INDIA

Published in: Education, Technology

Liquid chromatography–mass spectrometry (LC-MS) BY P. RAVISANKAR

  1. 1. Liquid chromatography–massspectrometry• prof. P.Ravisankar• Vignan Pharmacy college• Valdlamudi• Guntur Dist.• Andhra Pradesh• India.• banuman35@gmail.com• 00919059994000
  2. 2. Liquid chromatography–massspectrometry• Liquid chromatography–mass spectrometry(LC-MS, or alternatively HPLC-MS) is ananalytical chemistry technique that combinesthe physical separation capabilities of liquidchromatography (or HPLC) with the massanalysis capabilities of mass spectrometry.
  3. 3. Spectral resolution is possibleCompound identification from spectral dataHigh degree of specificity
  4. 4. Mass spectrometers work by ionising themolecules and identifying the ions according totheir mass-to-charge(m/z) ratios.Two key components in this process are theion source,which generates the ions,and themass analyser which sorts the ions.The basic LC/MS system consists of a HPLCpump,an injector,and a column mated to a massspectrometer through an evaporative/ionisinginterface.
  5. 5. MOBILE PHASE :-The mobile phase is the solvent that moves thesolute through out column.General requirements:-(1)low cost, uv transperancy,high purity.(2)low viscosity, low toxicity, non flammability.(3)non corrosive to LC system component.
  6. 6. COLUMN :-The use of di-functional or tri-functional silanesto create bonded groups with two or threeattachment points leading to phases with higherstability.Most widely used columns for LCMS are:-(1) fast LC column- short column(15-50mm)(2)Micro LC column-large column( 20-150mm)
  7. 7. Sample preparation:-Sample preparation generally consists of concentratingthe analyte and removing compounds that can causebackground ions or suppress ionization.Example of sample preparation include:-(1) on –column concentration to increase analyteconcentration.(2) desalting to reduce the sodium and potassium adductformation that commonly occurs in electro spray(3) filtration to separate a low molecular-weight drugfrom proteins in plasma, milk, or tissue.
  8. 8. Common ionisation techniques are :Electrospray ionisation(ESI)Atmospheric pressure chemicalionisation(APCI)Atmospheric pressure photoionisation(APPI)Matrix assisted laser desorption with time offlight(MALDI-TOF)Particle beam
  9. 9. Which is best?•It depends on the exact application.•Increasing polarity and molecular weight andthermal instability favors electrospray.•Most drugs of abuse are highly polar and areeasily analyzed using electrospray.•High molecular weight proteins also requireelectrospray•Lower polarity and molecular weight favors APCI orAPPI.•compounds must be more thermally stable.
  10. 10. Electrospray ionization:(ESI) is a technique used in mass spectrometry to produce ions. It isespecially useful in producing ions from macromolecules because itovercomes the propensity of these molecules to fragment when ionized.The liquid containing the analyte(s) of interest is dispersed byelectrospray into a fine aerosol. Because the ion formation involves extensive solvent evaporation, thetypical solvents for electrospray ionization are prepared by mixing waterwith volatile organic compounds (e.g. methanol, acetonitrile) To decrease the initial droplet size, compounds that increase theconductivity (e.g. acetic acid) are customarily added to the solution. Large-flow electrosprays can benefit from additional nebulization byan inert gas such as nitrogen. The aerosol is sampled into the first vacuum stage of a massspectrometer through a capillary, which can be heated to aid furthersolvent evaporation from the charged droplets.
  11. 11. The solvent evaporates from a charged droplet until it becomes unstableupon reaching its Rayleigh limit. At this point, the droplet deforms and emits charged jets in a processknown as Coulomb fission.During the fission, the droplet loses a small percentage of its mass (1.0-2.3%) along with a relatively large percentage of charge (10-18%).
  12. 12. Atmospheric pressure chemical ionization• In APCI,the eluent is sprayed through aheated vaporizer at atmosphericpressure.The heat vaporises the liquid.• The resulting gas-phase solvent moleculesare ionised by electrons discharged from acorona needle.• The solvent ions then transfer charge tothe analyte molecules through chemicalreactions.• The analyte ions pass through a capillarysampling orifice into the mass analyzer.
  13. 13. Atmospheric pressure photoionization• As in APCI,a vaporizer converts the LC eluent tothe gas phase.• A discharge lamp generates photons in a narrowrange of ionisation energies.• The range of energies is carefully chosen toionize as many analyte molecules as possiblewhile minimising the ionisation of solventmolecules.• The resulting ions pass through a capillarysampling orifice into the mass analyser.
  14. 14. Atmospheric pressure photoionizationHPLC inletNebulising gasUVlampNebuliserheaterDrying gascapillary
  15. 15. Matrix-assisted laser desorption/ionization(MALDI)The MALDI is a two step process. First, desorption is triggered by a UV laser beam.Matrix material heavily absorbs UV laser light, leading to the ablation of upperlayer (~micron) of the matrix material.A hot plume produced during the ablation contains many species: neutral andionized matrix molecules, protonated and deprotonated matrix molecules,matrix clusters and nanodroplets.The second step is ionization (more accurately protonation or deprotonation).Protonation (deprotonation) of analyte molecules takes place in the hot plume.Some of the ablated species participate in protonation (deprotonation) ofanalyte molecules.
  16. 16. Matrix-assisted laser desorption/ionization
  17. 17. •Useful interface which is applicable to a wide range ofmolecules.•The heavier sample molecules enter the MS and can be ionised.
  18. 18. •They deflects ions down a curved tubes in a magneticfields based on their kinetic energy determined by themass, charge and velocity.• The magnetic field is scanned to measure differentions.Four types of mass analysers commonly used are :•Quadrupole•Time-of –flight•Ion trap•Fourier transform-ion cyclotron reasonance(FT-ICR)
  19. 19. Ions entering the chamber are trapped in the circular orbits bypowerful electrical and magnetic fields.When excited by a radio frequency electrical field,the ionsgenerate a time-dependent current.This current is converted into orbital frequencies of the ionswhich correspond to their mass-to-charge ratios.
  20. 20. QuadrupoleThe quadrupole consists of four parallel metal rods. Each opposing rod pair is connected together electrically, and aradio frequency (RF) voltage is applied between one pair of rodsand the other. A direct current voltage is then superimposed on the RFvoltage. Ions travel down the quadrupole between the rods. Only ions of a certain mass-to-charge ratio m/z will reach thedetector for a given ratio of voltages: other ions have unstabletrajectories and will collide with the rods.This permits selection of an ion with a particular m/z or allowsthe operator to scan for a range of m/z-values by continuouslyvarying the applied voltage.
  21. 21. Ring ElectrodeRing ElectrodeEntranceEndcapElectrodeExit EndcapElectrodeIon Traps:Ions fill the space between a ring electrode and a pair ofend-cap electrodes.Mass analysis and fragmentation occur in the same space.
  22. 22. In full scan mode:-Ions fill and are trapped in space then masses are scanned outof the trap sequentially.-Ions are not lost, so full scan sensitivity is better, but filling/closingcycles make them poorer at quantitation.Mass resolution is controlled by the “speed” at which massesare scanned out of the trap.slower scanning = better mass resolution.In MS/MS mode:-Ions trapped,fragmentation occurs when the selected ion isexcited by a voltage and collides with bath gas (He).-This process can occur recursively thus MS/MS/MS/MS….
  23. 23. •LC/MS is suitable for many applications,frompharmaceutical development to environmentalanalysis.•It ability to detect a wide range of compoundswith great sensitivity and specificity has made itpopular in a variety of fields,
  24. 24.  Molecular weight determination e.g differentiation ofsimilar octapeptides,determinig the molecular weight ofgreen fluorescent protein. Structural determination e.g. structural determination ofginsenoside. Pharmaceutical application e.g. rapid chromatography ofbenzodiazepines,identification of bile acid metabolites.Clinical application e.g. high sensitivity detection oftrimipramine and thioridazine.Food application e.g. identification of aflatoxin in food,determination of vitamin D 3 in poultry feed supplementusing MS 3 Environmental application e.g. detection of phenyl ureaherbicides, detection of low level of carbaryl in food.
  25. 25. One fundamental application of LC/MS is the determinationof molecular weights.This information is key information todetermining identity.Determination of similar octapeptides:Figure 1 shows the spectra of two peptides whose mass-to-charge ratio differs by only 1 m/z .The smaller fragmentsare identical in the two spectras ,indicating that largeportions of the two peptides are very similar.The largerfragments contain the differentiating peptides.
  26. 26. Mass spectra differentiating two very similaroctapeptidesFigure 1
  27. 27. Structural determination of ginsenosides using MSanalysis:•Ginseng root,a traditional chinese herbalremedy,contains more than a dozen biologically activesaponins called ginsenosides.•Since ginsenosides contain multiple oligosaccharidechains at different positions in the molecule,stucturalelucidation of these components can be quitecomplicated.•MSn analysis in an ion trap mass spectrometer permitsmultiple stages of precursor ion isolation andfragmentation.this stepwise fragmentation permitsindividual fragmentation pathways to be followed andprovides a great deal of structural information.
  28. 28. Rapid chromatography of benzodiazepines:•The information available in a mass spectrumallows some compounds to be separated eventhough they are unresolved.•In this example , a series of benzodiazepines wasanalysed using both UV and MS detectors.•The UV trace could not be used forquantitation,but the extracted ion chromatogramsfrom MS could be used.
  29. 29. IDENTIFICATION AND QUANTIFICATION OF INDIVIDUALBENZODIAZEPINESFigure 2
  30. 30. High sensitivity detection of trimipramine andthioridazine:•Trimipramine is a tricyclic antidepressant withsedative properties.•Thioridazine is a tranquiliser.•Figure 4 shows these compounds in urine extract at alow level that could not be detected by UV.•To get the maximum sensitivity from a MS,the analysiswas done by selected ion monitoring.
  31. 31. High sensitivity detection of trimipramineand thioridazineffiFigure 4
  32. 32. Identification of aflatoxins in food:•Aflatoxins are toxic metabolites produced in foods bycertain fungi.•Figure 3 shows the total ion chromatogram from amixture of four aflatoxins.•Even though they are structurally similar,eachaflatoxin can be uniquely identified by its massspectrum.
  33. 33. Identification of aflatoxins in foodFigure 3
  34. 34. Detection of phenyl urea herbicides:•Many of the phenyl urea herbicides are very similar anddifficult to distinguish with a UV detector.•Monuron and diuron have one benzene ring and differby a single chlorine.Chloroxuron has two chlorines and asecond benzene ring attached to the first by an oxygen.•The UV spectra are similar for monuron and diuron butdifferent for chloroxuron.•When analysed using LC/MS system,each compoundhas a uniquely identifiable mass spectrum.
  35. 35. Chromatogram of phenylurea herbicidewith UV and MS spectraFigure 5
  36. 36. PharmacokineticsLC-MS is very commonly used in pharmacokineticstudies of pharmaceuticals and is thus the mostfrequently used technique in the field of bioanalysis. These studies give information about how quickly adrug will be cleared from the hepatic blood flow, andorgans of the body.
  37. 37. •LC-MS is the method of choice for the study ofdrug metabolism because of its sensitivity andspecificity.•The metabolism of droloxifene,an analogue ofanti-breast cancer drug tamoxifen,by humanliver microsomes is an example .
  38. 38. Drug developmentLC-MS is frequently used in drug developmentat many different stages including PeptideMapping, Glycoprotein Mapping, NaturalProducts Dereplication, Bioaffinity Screening, InVivo Drug Screening, Metabolic StabilityScreening, Metabolite Identification, ImpurityIdentification, Degradant Identification,Quantitative Bioanalysis, and Quality Control.
  39. 39. •LC-MS has proved to be an extremelysensitive and specific technique for theanalysis of pharmaceuticals.•It plays important roles in the studies ofdrug metabolism,discovery of new drugcandidates and the analysis,identificationand characterisation of impurities anddegradants in drug substances andproducts.
  40. 40. Liquid chromatography-mass spectrometry-Marcel DekkerLC/MS A Practical user guide-Marvin C McMaster and Christopher Mc Master

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