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Liquid chromatography–mass
spectrometry
• prof. P.Ravisankar
• Vignan Pharmacy college
• Valdlamudi
• Guntur Dist.
• Andhra Pradesh
• India.
• banuman35@gmail.com
• 00919059994000
Liquid chromatography–mass
spectrometry
• Liquid chromatography–mass spectrometry
(LC-MS, or alternatively HPLC-MS) is an
analytical chemistry technique that combines
the physical separation capabilities of liquid
chromatography (or HPLC) with the mass
analysis capabilities of mass spectrometry.
Spectral resolution is possible
Compound identification from spectral data
High degree of specificity
Mass spectrometers work by ionising the
molecules and identifying the ions according to
their mass-to-charge(m/z) ratios.
Two key components in this process are the
ion source,which generates the ions,and the
mass analyser which sorts the ions.
The basic LC/MS system consists of a HPLC
pump,an injector,and a column mated to a mass
spectrometer through an evaporative/ionising
interface.
Liquid chromatography–mass spectrometry (LC-MS) BY P. RAVISANKAR
MOBILE PHASE :-
The mobile phase is the solvent that moves the
solute 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.
Liquid chromatography–mass spectrometry (LC-MS) BY P. RAVISANKAR
COLUMN :-
The use of di-functional or tri-functional silanes
to create bonded groups with two or three
attachment points leading to phases with higher
stability.
Most widely used columns for LCMS are:-
(1) fast LC column- short column(15-50mm)
(2)Micro LC column-large column( 20-
150mm)
Sample preparation:-
Sample preparation generally consists of concentrating
the analyte and removing compounds that can cause
background ions or suppress ionization.
Example of sample preparation include:-
(1) on –column concentration to increase analyte
concentration.
(2) desalting to reduce the sodium and potassium adduct
formation that commonly occurs in electro spray
(3) filtration to separate a low molecular-weight drug
from proteins in plasma, milk, or tissue.
Common ionisation techniques are :
Electrospray ionisation(ESI)
Atmospheric pressure chemical
ionisation(APCI)
Atmospheric pressure photoionisation(APPI)
Matrix assisted laser desorption with time of
flight(MALDI-TOF)
Particle beam
Which is best?
•It depends on the exact application.
•Increasing polarity and molecular weight and
thermal instability favors electrospray.
•Most drugs of abuse are highly polar and are
easily analyzed using electrospray.
•High molecular weight proteins also require
electrospray
•Lower polarity and molecular weight favors APCI or
APPI.
•compounds must be more thermally stable.
Electrospray ionization:
(ESI) is a technique used in mass spectrometry to produce ions. It is
especially useful in producing ions from macromolecules because it
overcomes the propensity of these molecules to fragment when ionized.
The liquid containing the analyte(s) of interest is dispersed by
electrospray into a fine aerosol.
 Because the ion formation involves extensive solvent evaporation, the
typical solvents for electrospray ionization are prepared by mixing water
with volatile organic compounds (e.g. methanol, acetonitrile)
 To decrease the initial droplet size, compounds that increase the
conductivity (e.g. acetic acid) are customarily added to the solution.
 Large-flow electrosprays can benefit from additional nebulization by
an inert gas such as nitrogen.
 The aerosol is sampled into the first vacuum stage of a mass
spectrometer through a capillary, which can be heated to aid further
solvent evaporation from the charged droplets.
The solvent evaporates from a charged droplet until it becomes unstable
upon reaching its Rayleigh limit.
 At this point, the droplet deforms and emits charged jets in a process
known 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%).
Atmospheric pressure chemical ionization
• In APCI,the eluent is sprayed through a
heated vaporizer at atmospheric
pressure.The heat vaporises the liquid.
• The resulting gas-phase solvent molecules
are ionised by electrons discharged from a
corona needle.
• The solvent ions then transfer charge to
the analyte molecules through chemical
reactions.
• The analyte ions pass through a capillary
sampling orifice into the mass analyzer.
Atmospheric pressure photoionization
• As in APCI,a vaporizer converts the LC eluent to
the gas phase.
• A discharge lamp generates photons in a narrow
range of ionisation energies.
• The range of energies is carefully chosen to
ionize as many analyte molecules as possible
while minimising the ionisation of solvent
molecules.
• The resulting ions pass through a capillary
sampling orifice into the mass analyser.
Atmospheric pressure photoionization
HPLC inlet
Nebulising gas
UV
lamp
Nebuliser
heater
Drying gas
capillary
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 upper
layer (~micron) of the matrix material.
A hot plume produced during the ablation contains many species: neutral and
ionized 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) of
analyte molecules.
Matrix-assisted laser desorption/ionization
•Useful interface which is applicable to a wide range of
molecules.
•The heavier sample molecules enter the MS and can be ionised.
•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.
Four types of mass analysers commonly used are :
•Quadrupole
•Time-of –flight
•Ion trap
•Fourier transform-ion cyclotron reasonance(FT-ICR)
Ions entering the chamber are trapped in the circular orbits by
powerful electrical and magnetic fields.
When excited by a radio frequency electrical field,the ions
generate a time-dependent current.
This current is converted into orbital frequencies of the ions
which correspond to their mass-to-charge ratios.
Quadrupole
The quadrupole consists of four parallel metal rods.
 Each opposing rod pair is connected together electrically, and a
radio frequency (RF) voltage is applied between one pair of rods
and the other.
 A direct current voltage is then superimposed on the RF
voltage. Ions travel down the quadrupole between the rods.
 Only ions of a certain mass-to-charge ratio m/z will reach the
detector for a given ratio of voltages: other ions have unstable
trajectories and will collide with the rods.
This permits selection of an ion with a particular m/z or allows
the operator to scan for a range of m/z-values by continuously
varying the applied voltage.
Liquid chromatography–mass spectrometry (LC-MS) BY P. RAVISANKAR
Ring Electrode
Ring Electrode
Entrance
Endcap
Electrode
Exit Endcap
Electrode
Ion Traps:
Ions fill the space between a ring electrode and a pair of
end-cap electrodes.
Mass analysis and fragmentation occur in the same space.
In full scan mode:
-Ions fill and are trapped in space then masses are scanned out
of the trap sequentially.
-Ions are not lost, so full scan sensitivity is better, but filling/closing
cycles make them poorer at quantitation.
Mass resolution is controlled by the “speed” at which masses
are scanned out of the trap.
slower scanning = better mass resolution.
In MS/MS mode:
-Ions trapped,fragmentation occurs when the selected ion is
excited by a voltage and collides with bath gas (He).
-This process can occur recursively thus MS/MS/MS/MS….
•LC/MS is suitable for many applications,from
pharmaceutical development to environmental
analysis.
•It ability to detect a wide range of compounds
with great sensitivity and specificity has made it
popular in a variety of fields,
 Molecular weight determination e.g differentiation of
similar octapeptides,determinig the molecular weight of
green fluorescent protein.
 Structural determination e.g. structural determination of
ginsenoside.
 Pharmaceutical application e.g. rapid chromatography of
benzodiazepines,identification of bile acid metabolites.
Clinical application e.g. high sensitivity detection of
trimipramine and thioridazine.
Food application e.g. identification of aflatoxin in food,
determination of vitamin D 3 in poultry feed supplement
using MS 3
 Environmental application e.g. detection of phenyl urea
herbicides, detection of low level of carbaryl in food.
One fundamental application of LC/MS is the determination
of molecular weights.This information is key information to
determining 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 fragments
are identical in the two spectras ,indicating that large
portions of the two peptides are very similar.The larger
fragments contain the differentiating peptides.
Mass spectra differentiating two very similar
octapeptides
Figure 1
Structural determination of ginsenosides using MS
analysis:
•Ginseng root,a traditional chinese herbal
remedy,contains more than a dozen biologically active
saponins called ginsenosides.
•Since ginsenosides contain multiple oligosaccharide
chains at different positions in the molecule,stuctural
elucidation of these components can be quite
complicated.
•MSn analysis in an ion trap mass spectrometer permits
multiple stages of precursor ion isolation and
fragmentation.this stepwise fragmentation permits
individual fragmentation pathways to be followed and
provides a great deal of structural information.
Rapid chromatography of benzodiazepines:
•The information available in a mass spectrum
allows some compounds to be separated even
though they are unresolved.
•In this example , a series of benzodiazepines was
analysed using both UV and MS detectors.
•The UV trace could not be used for
quantitation,but the extracted ion chromatograms
from MS could be used.
IDENTIFICATION AND QUANTIFICATION OF INDIVIDUAL
BENZODIAZEPINES
Figure 2
High sensitivity detection of trimipramine and
thioridazine:
•Trimipramine is a tricyclic antidepressant with
sedative properties.
•Thioridazine is a tranquiliser.
•Figure 4 shows these compounds in urine extract at a
low level that could not be detected by UV.
•To get the maximum sensitivity from a MS,the analysis
was done by selected ion monitoring.
High sensitivity detection of trimipramine
and thioridazine
ffi
Figure 4
Identification of aflatoxins in food:
•Aflatoxins are toxic metabolites produced in foods by
certain fungi.
•Figure 3 shows the total ion chromatogram from a
mixture of four aflatoxins.
•Even though they are structurally similar,each
aflatoxin can be uniquely identified by its mass
spectrum.
Identification of aflatoxins in food
Figure 3
Detection of phenyl urea herbicides:
•Many of the phenyl urea herbicides are very similar and
difficult to distinguish with a UV detector.
•Monuron and diuron have one benzene ring and differ
by a single chlorine.Chloroxuron has two chlorines and a
second benzene ring attached to the first by an oxygen.
•The UV spectra are similar for monuron and diuron but
different for chloroxuron.
•When analysed using LC/MS system,each compound
has a uniquely identifiable mass spectrum.
Chromatogram of phenylurea herbicide
with UV and MS spectra
Figure 5
Pharmacokinetics
LC-MS is very commonly used in pharmacokinetic
studies of pharmaceuticals and is thus the most
frequently used technique in the field of bioanalysis.
 These studies give information about how quickly a
drug will be cleared from the hepatic blood flow, and
organs of the body.
•LC-MS is the method of choice for the study of
drug metabolism because of its sensitivity and
specificity.
•The metabolism of droloxifene,an analogue of
anti-breast cancer drug tamoxifen,by human
liver microsomes is an example .
Drug development
LC-MS is frequently used in drug development
at many different stages including Peptide
Mapping, Glycoprotein Mapping, Natural
Products Dereplication, Bioaffinity Screening, In
Vivo Drug Screening, Metabolic Stability
Screening, Metabolite Identification, Impurity
Identification, Degradant Identification,
Quantitative Bioanalysis, and Quality Control.
•LC-MS has proved to be an extremely
sensitive and specific technique for the
analysis of pharmaceuticals.
•It plays important roles 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.
Liquid chromatography-mass spectrometry-
Marcel Dekker
LC/MS A Practical user guide-Marvin C Mc
Master and Christopher Mc Master
Liquid chromatography–mass spectrometry (LC-MS) BY P. RAVISANKAR

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

  • 1. Liquid chromatography–mass spectrometry • prof. P.Ravisankar • Vignan Pharmacy college • Valdlamudi • Guntur Dist. • Andhra Pradesh • India. • banuman35@gmail.com • 00919059994000
  • 2. Liquid chromatography–mass spectrometry • Liquid chromatography–mass spectrometry (LC-MS, or alternatively HPLC-MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry.
  • 3. Spectral resolution is possible Compound identification from spectral data High degree of specificity
  • 4. Mass spectrometers work by ionising the molecules and identifying the ions according to their mass-to-charge(m/z) ratios. Two key components in this process are the ion source,which generates the ions,and the mass analyser which sorts the ions. The basic LC/MS system consists of a HPLC pump,an injector,and a column mated to a mass spectrometer through an evaporative/ionising interface.
  • 6. MOBILE PHASE :- The mobile phase is the solvent that moves the solute 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.
  • 8. COLUMN :- The use of di-functional or tri-functional silanes to create bonded groups with two or three attachment points leading to phases with higher stability. Most widely used columns for LCMS are:- (1) fast LC column- short column(15-50mm) (2)Micro LC column-large column( 20- 150mm)
  • 9. Sample preparation:- Sample preparation generally consists of concentrating the analyte and removing compounds that can cause background ions or suppress ionization. Example of sample preparation include:- (1) on –column concentration to increase analyte concentration. (2) desalting to reduce the sodium and potassium adduct formation that commonly occurs in electro spray (3) filtration to separate a low molecular-weight drug from proteins in plasma, milk, or tissue.
  • 10. Common ionisation techniques are : Electrospray ionisation(ESI) Atmospheric pressure chemical ionisation(APCI) Atmospheric pressure photoionisation(APPI) Matrix assisted laser desorption with time of flight(MALDI-TOF) Particle beam
  • 11. Which is best? •It depends on the exact application. •Increasing polarity and molecular weight and thermal instability favors electrospray. •Most drugs of abuse are highly polar and are easily analyzed using electrospray. •High molecular weight proteins also require electrospray •Lower polarity and molecular weight favors APCI or APPI. •compounds must be more thermally stable.
  • 12. Electrospray ionization: (ESI) is a technique used in mass spectrometry to produce ions. It is especially useful in producing ions from macromolecules because it overcomes the propensity of these molecules to fragment when ionized. The liquid containing the analyte(s) of interest is dispersed by electrospray into a fine aerosol.  Because the ion formation involves extensive solvent evaporation, the typical solvents for electrospray ionization are prepared by mixing water with volatile organic compounds (e.g. methanol, acetonitrile)  To decrease the initial droplet size, compounds that increase the conductivity (e.g. acetic acid) are customarily added to the solution.  Large-flow electrosprays can benefit from additional nebulization by an inert gas such as nitrogen.  The aerosol is sampled into the first vacuum stage of a mass spectrometer through a capillary, which can be heated to aid further solvent evaporation from the charged droplets.
  • 13. The solvent evaporates from a charged droplet until it becomes unstable upon reaching its Rayleigh limit.  At this point, the droplet deforms and emits charged jets in a process known 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%).
  • 14. Atmospheric pressure chemical ionization • In APCI,the eluent is sprayed through a heated vaporizer at atmospheric pressure.The heat vaporises the liquid. • The resulting gas-phase solvent molecules are ionised by electrons discharged from a corona needle. • The solvent ions then transfer charge to the analyte molecules through chemical reactions. • The analyte ions pass through a capillary sampling orifice into the mass analyzer.
  • 15. Atmospheric pressure photoionization • As in APCI,a vaporizer converts the LC eluent to the gas phase. • A discharge lamp generates photons in a narrow range of ionisation energies. • The range of energies is carefully chosen to ionize as many analyte molecules as possible while minimising the ionisation of solvent molecules. • The resulting ions pass through a capillary sampling orifice into the mass analyser.
  • 16. Atmospheric pressure photoionization HPLC inlet Nebulising gas UV lamp Nebuliser heater Drying gas capillary
  • 17. 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 upper layer (~micron) of the matrix material. A hot plume produced during the ablation contains many species: neutral and ionized 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) of analyte molecules.
  • 19. •Useful interface which is applicable to a wide range of molecules. •The heavier sample molecules enter the MS and can be ionised.
  • 20. •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. Four types of mass analysers commonly used are : •Quadrupole •Time-of –flight •Ion trap •Fourier transform-ion cyclotron reasonance(FT-ICR)
  • 21. Ions entering the chamber are trapped in the circular orbits by powerful electrical and magnetic fields. When excited by a radio frequency electrical field,the ions generate a time-dependent current. This current is converted into orbital frequencies of the ions which correspond to their mass-to-charge ratios.
  • 22. Quadrupole The quadrupole consists of four parallel metal rods.  Each opposing rod pair is connected together electrically, and a radio frequency (RF) voltage is applied between one pair of rods and the other.  A direct current voltage is then superimposed on the RF voltage. Ions travel down the quadrupole between the rods.  Only ions of a certain mass-to-charge ratio m/z will reach the detector for a given ratio of voltages: other ions have unstable trajectories and will collide with the rods. This permits selection of an ion with a particular m/z or allows the operator to scan for a range of m/z-values by continuously varying the applied voltage.
  • 24. Ring Electrode Ring Electrode Entrance Endcap Electrode Exit Endcap Electrode Ion Traps: Ions fill the space between a ring electrode and a pair of end-cap electrodes. Mass analysis and fragmentation occur in the same space.
  • 25. In full scan mode: -Ions fill and are trapped in space then masses are scanned out of the trap sequentially. -Ions are not lost, so full scan sensitivity is better, but filling/closing cycles make them poorer at quantitation. Mass resolution is controlled by the “speed” at which masses are scanned out of the trap. slower scanning = better mass resolution. In MS/MS mode: -Ions trapped,fragmentation occurs when the selected ion is excited by a voltage and collides with bath gas (He). -This process can occur recursively thus MS/MS/MS/MS….
  • 26. •LC/MS is suitable for many applications,from pharmaceutical development to environmental analysis. •It ability to detect a wide range of compounds with great sensitivity and specificity has made it popular in a variety of fields,
  • 27.  Molecular weight determination e.g differentiation of similar octapeptides,determinig the molecular weight of green fluorescent protein.  Structural determination e.g. structural determination of ginsenoside.  Pharmaceutical application e.g. rapid chromatography of benzodiazepines,identification of bile acid metabolites. Clinical application e.g. high sensitivity detection of trimipramine and thioridazine. Food application e.g. identification of aflatoxin in food, determination of vitamin D 3 in poultry feed supplement using MS 3  Environmental application e.g. detection of phenyl urea herbicides, detection of low level of carbaryl in food.
  • 28. One fundamental application of LC/MS is the determination of molecular weights.This information is key information to determining 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 fragments are identical in the two spectras ,indicating that large portions of the two peptides are very similar.The larger fragments contain the differentiating peptides.
  • 29. Mass spectra differentiating two very similar octapeptides Figure 1
  • 30. Structural determination of ginsenosides using MS analysis: •Ginseng root,a traditional chinese herbal remedy,contains more than a dozen biologically active saponins called ginsenosides. •Since ginsenosides contain multiple oligosaccharide chains at different positions in the molecule,stuctural elucidation of these components can be quite complicated. •MSn analysis in an ion trap mass spectrometer permits multiple stages of precursor ion isolation and fragmentation.this stepwise fragmentation permits individual fragmentation pathways to be followed and provides a great deal of structural information.
  • 31. Rapid chromatography of benzodiazepines: •The information available in a mass spectrum allows some compounds to be separated even though they are unresolved. •In this example , a series of benzodiazepines was analysed using both UV and MS detectors. •The UV trace could not be used for quantitation,but the extracted ion chromatograms from MS could be used.
  • 32. IDENTIFICATION AND QUANTIFICATION OF INDIVIDUAL BENZODIAZEPINES Figure 2
  • 33. High sensitivity detection of trimipramine and thioridazine: •Trimipramine is a tricyclic antidepressant with sedative properties. •Thioridazine is a tranquiliser. •Figure 4 shows these compounds in urine extract at a low level that could not be detected by UV. •To get the maximum sensitivity from a MS,the analysis was done by selected ion monitoring.
  • 34. High sensitivity detection of trimipramine and thioridazine ffi Figure 4
  • 35. Identification of aflatoxins in food: •Aflatoxins are toxic metabolites produced in foods by certain fungi. •Figure 3 shows the total ion chromatogram from a mixture of four aflatoxins. •Even though they are structurally similar,each aflatoxin can be uniquely identified by its mass spectrum.
  • 36. Identification of aflatoxins in food Figure 3
  • 37. Detection of phenyl urea herbicides: •Many of the phenyl urea herbicides are very similar and difficult to distinguish with a UV detector. •Monuron and diuron have one benzene ring and differ by a single chlorine.Chloroxuron has two chlorines and a second benzene ring attached to the first by an oxygen. •The UV spectra are similar for monuron and diuron but different for chloroxuron. •When analysed using LC/MS system,each compound has a uniquely identifiable mass spectrum.
  • 38. Chromatogram of phenylurea herbicide with UV and MS spectra Figure 5
  • 39. Pharmacokinetics LC-MS is very commonly used in pharmacokinetic studies of pharmaceuticals and is thus the most frequently used technique in the field of bioanalysis.  These studies give information about how quickly a drug will be cleared from the hepatic blood flow, and organs of the body.
  • 40. •LC-MS is the method of choice for the study of drug metabolism because of its sensitivity and specificity. •The metabolism of droloxifene,an analogue of anti-breast cancer drug tamoxifen,by human liver microsomes is an example .
  • 41. Drug development LC-MS is frequently used in drug development at many different stages including Peptide Mapping, Glycoprotein Mapping, Natural Products Dereplication, Bioaffinity Screening, In Vivo Drug Screening, Metabolic Stability Screening, Metabolite Identification, Impurity Identification, Degradant Identification, Quantitative Bioanalysis, and Quality Control.
  • 42. •LC-MS has proved to be an extremely sensitive and specific technique for the analysis of pharmaceuticals. •It plays important roles 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.
  • 43. Liquid chromatography-mass spectrometry- Marcel Dekker LC/MS A Practical user guide-Marvin C Mc Master and Christopher Mc Master