1. LIQUID CHROMATOGRAPHY MASS SPECTROMETRY
(LC MS)
Prepared by:
MAHENDRA G S
M pharma
Department of Pharmaceutical chemistry
JSS College of pharmacy
Mysuru.
2. Liquid Chromatography Mass Spectrometry.
• It refers to the combination of liquid chromatographic separation with mass spectrometric
detection for non polar, low volatility, thermally unstable compounds.
• LC differentiates compounds by their physico-chemical properties and MS differentiates
compounds by mass (specifically their mass-to-charge ratio).
• The mass spectrometer acts not only as the “LC detector” but, it provides the capability to
identify the species corresponding to each chromatographic peak through its unique mass
spectrum.
3. Principle:
Thermospray method:
• The thermospray method depends on the thermal generation of a spray and further
separate heat treatment of that spray to yield desolvated ions.
• The heat creates a supersonic, expanding aerosol jet that contains a mist of fine droplets of
solvent vapour and sample molecules., the droplets vaporize.
• The excess vapor is pumped away by an added mechanical pump, which is directly coupled
to the ion source.
• Ions of the sample molecules are formed in the spray either by direct desorption or by
chemical ionization when used with polar mobile phases that contain appropriate buffers.
• A conventional electron beam is used to provide gas phase reagent, ions for the chemical
ionization of solute molecules.
4. • The ions are led into a quadrupole or magnetic sector mass spectrometer, flow rates efluent
up to 2ml/min are permissible.
Monodisperse Aerosol generation interface:
The interface is configured in three sections:
1. Aerosol generator
2. Desolvation chamber
3. Two stage aerosol beam pressure reducer.
• In the aerosol generates the high pressure effluent from the chromatographic column, passes
through a small diameter orifice to form a fine liquid jet. The jet breaks up under natural
forces to form uniform drops, which are immediately dispersed with gas stream introduced at
right angles to the liquid flow direction.
5. • The solvent evaporates in the desolvation chamber. The chamber is maintained near room
temperature by heating gently to replace the latent heat of vaporization necessary for solvent
evaporation.
• The two stages aerosol beam separator consists of two nozzle and skimmer devices.
• These reduces the pressure from an initial value close to atmospheric pressure in the
desolvation chamber to a final value close to the pressure in the ion source.
• The separator also allows solute particles to be preferentially transferred through the system,
while dispersion of the gas and solvent vapor are pumped away.
6. Instrumentation:
• HPLC system
• Ion source: used in the vaporisation ,
ionization of target
Molecule.
• Mass analyser: used to separate
the gas phase ions by
mass to-charge ratio(m/z).
• Ion detector: detection of the mass
separated.
• Data system
7. Instrumentation of HPLC system:
• Solvent reservoir
• Tubing
• Pump
• Injection device
• Column
• Detector
Solvent Reservoir:
Composition of these reservoirs should be inert.
Should not be reactive with solvents.
Capacity of the reservoir should be greater than 500ml.
8. TUBING:
• It is used to connect all parts of systems.
• Inside diameter of the tubing prior to injection device is not critical but tubing should have
ability to withstand pressure and able to carry sufficient volume of solvents.
• Usually tubing of very small diameter (less than 0.5 mm inner diameter) are used.
PUMPS:
• The function of pumps in HPLC is to pass mobile phase through the column at high pressure
and at controlled flow rate.
• Pumps must be constructed from material that are inert to mobile phase.
• Materials commonly used are glass, stainless steel, Teflon.
• HPLC is high pressure chromatography the pump must be capable of generating pressure of
up to 5000 psi at a flow rate up to 3ml/min for analytical conditions.
10. The efficiency of a chromatographic separation is a measure of the dispersion of the analyte
band as it travels through the LC system and column.
Column
• Column forms the heart of any HPLC system.
• Particle size less than 2 to 5 micrometre
• Internal diameter (2.1, 3.0 or 4.6 mm )
• Length (20-150 mm)
• The quality of its packing materials are essential for good separation and minimum
dispersion.
Materials Used:
• Stainless steel
• Glass
• Poly ether ether ketone(PEEK)
11. Mobile phase:
• Reversed-phase (RP)
• Flow rate 0.3-1.0 mL/min (depending on column dimensions) at 40-60 C.
Guard column/pre-column filters:
• Guard column acts as a trap for particulates and strongly retained components arising from
samples.
• Use same particle size as analytical columns.
• Installed immediately before the analytical column.
• A pre-column filter protects the column from particulates (from samples, pump seals, and
injector valve wear)
• Filter must be smaller in size than column, Installed immediately before the guard column.
12. Injection mode :
Full/filled loop or flow through needle.
Elution methods:
• Isocratic conditions may not be appropriate if the requirement is to separate multiple
analytes of different physiochemical properties
• Program a rapid gradient elution, changing the solvent composition with time.
• Start at a high proportion of aqueous and change the composition linearly to high organic
over a period of 5 minutes.
• It is prudent to hold the elution at the high organic composition for a fixed period (e.g. 2
minutes) to ensure all analytes and co-extractives have eluted off the column before
returning to the starting conditions.
• Include an appropriate amount of time for re-equilibration of the column.
13. Different Ionization Methods
Electron Impact:
small molecules, 1-1000 Daltons
Fast Atom Bombardment:
peptides, sugars, up to 6000 Daltons
Electrospray Ionization:
peptides, proteins, up to 200,000 Daltons
Matrix Assisted Laser Desorption Ionization:
peptides, proteins, DNA, up to 500 Daltons
14. The ion source:
• The direct coupling of LC and MS (LC-MS) was solving the problem with the incompatibility
of introducing the flow of liquid mobile phase from the LC column into the vacuum required
in the mass spectrometer by the use of atmospheric pressure ionisation (API) interfaces.
• Electrospray ionisation (ESI) and atmospheric pressure chemical ionisation (APCI) are the
most common API techniques in routine use for quantitation of small molecules by LC-MS.
• Atmospheric pressure photoionization (APPI) was developed to increase ionisation
efficiencies of non-polar compounds such as polyaromatic hydrocarbons and steroids.
• Atmospheric pressure ionization: The API interface is a critical part of the mass
spectrometer and needs to produce ions from the molecules of interest whilst coping with
typical LC flow rates (0.2-2 mL/min).
1. Pressure reduction.
2. Evaporative processes called desolvation.
3. Ionisation.
4. Removal of excess solvent and un-ionised material.
15. Electrospray ionisation (ESI):
• The LC eluent flows through a metal capillary.
• A charge is transferred onto the droplets by applying a large (2-5 kV) potential difference
between the electrospray capillary and counter electrode.
• The droplet size reduces by evaporating the mobile phase by the use of a heated drying gas.
• This desolvation increases charge density on the surface of the smaller droplets.
• Electric repulsion due to the charge density results in droplet fission.
• When this exceeds the surface tension of the droplet it results in coulombic fission.
• The main advantage of the use of ESI for quantitative LC-MS is the formation of protonated or
de-protonated molecules with little fragmentation, ideal for selection of precursor ions and for
maximising sensitivity.
16. Atmospheric pressure chemical ionisation (APCI) :
• APCI is more suited to the analysis of relatively non-polar molecules.
• The LC eluent flows through a silica capillary.
• Droplets are produced by nebulisation of the LC flow into a spray.
• Solvent is evaporated by the use of a heater in the probe to produce gas-phase molecules.
• A corona discharge needle placed in the ion source generates electrons and ionises air and
gaseous solvent molecules forming reactive species (N2 + , H2O+ , O2 + ), which quickly
react to form H3O+.
• Proton transfer from/to the ionised solvent results in chemical ionisation of analyte [M+H] .
17. Mass analyser:
• Quadra pole Mass Analyzer
• Time of Flight Mass Analyzer
• Magnetic Sector Mass Analyzer
• Electrostatic Sector Mass Analyzer
• Quadra pole Ion Trap Mass Analyzers
• Ion Cyclotron Resonance
18. Magnetic Sector Mass Analyzer
• The magnetic field is used to separate the ions. As
moving charges enter a magnetic field, the charge is
deflected to a circular motion of a unique radius in a
direction perpendicular to the applied magnetic field.
• Ions in the magnetic field experience two equal forces;
force due to the magnetic field and centripetal force.
Electrostatic Sector Mass Analyzer
• Electrostatic sector analyzer consists of two curved
plates of equal and opposite potential.
• As the ion travels through the electric field, it is
deflected and the force on the ion due to the electric
field is equal to the centripetal force on the ion. Here
the ions of the same kinetic energy are focused, and
ions of different kinetic energies are dispersed.
19. Quadra pole Ion Trap Mass Analyzers:
• The analyzer is made with a ring electrode of a specific voltage and grounded end cap
electrodes. The ions enter the area between the electrodes through one of the end caps. After
entry, the electric field in the cavity due to the electrodes causes the ions of certain m/z values
to orbit in the space.
• As the radio frequency voltage increases, heavier mass ion orbits become more stabilized and
the light mass ions become less stabilized, causing them to collide with the wall, and
eliminating the possibility of traveling to and being detected by the detector.
20. Ion Cyclotron Resonance (ICR):
• ICR is an ion trap that uses a magnetic field in order to trap ions into an orbit inside of it.
• In this analyzer there is no separation that occurs rather all the ions of a particular range are
trapped inside, and an applied external electric field helps to generate a signal.
21. Application of LC MS
Biomedical Applications:
• LC-MS technique is useful for the detection of steroid drugs in body fluids and in profiling
endogenous steroids.
• Steroid sulphates have been detected with high sensitivity using this method.
• Plasma spray has been used to test saliva for steroid hormones in patients suffering from
congenital adrenal hyperplasia.
• Amino acids were one of the first compounds analysed using LC-MS coupled with laser
desorption and thermospray.
• Nucleosides, nucleotides, saccharides, peptides, and proteins were all analysed and their
molecular weights were determined using LC-MS coupled with electrospray.
• Bile acids have also been determined using LC-MS and thermospray.
22. Environmental Applications:
• LC-MS is used in the analysis of diverse samples such as soil, drinking water or waste water,
air, and sludge.
• Several pesticides and herbicides including triazine derivatives, chlorophenols,
phenoxyalkanoic acids, and sulfonylurea herbicides can be analysed using LC-MS.
• Separation of polycyclic aromatic hydrocarbons and organometallic compounds is also
possible using the technique.
Biochemical Screening for Genetic Disorders:
• Blood samples of new born babies are analysed using LC-MS to detect metabolic disorders.
23. Pharmaceuticals:
• LC-MS is widely used in the determination of pharmaceutical compounds and especially in
the separation of optically active drugs.
• Antibiotics and potential antimalarial have been studied using thermospray. The use of LC-
MS in the identification of bromazepam and similar drugs in case of intoxication has been
successfully demonstrated.
• Detection, isolation, and purification of drug metabolites is another major application of LC-
MS, as they are chemically or thermally labile, and need liquid chromatography.
• Separation and characterization of components in a crude mixture of natural products such
as complex lipids, alkaloids, and hydroxylated or unsaturated fatty acids has been achieved
using LC-MS.
24. Therapeutic Drug Monitoring and Toxicology:
• In drug monitoring, LC-MS assays have been developed for immunosuppressant including
tacrolimus, cyclosporin, everolimus, sirolimus, and mycophenolic acid. Similar assays for
aminoglycosides, anticancer drugs, and antiretroviral have also been described.
Vitamins and Related Metabolites:
• LC-MS is a preferred method for the measurement of vitamin D and its metabolites. LC-MS
assays have been developed for 25-hydroxyvitamin D2 and D3 in plasma and serum. Similar
assays are also available for fat-soluble vitamins such as vitamin K15 and Vitamin E13,15.
25. References:
• Instrumental methods of analysis
Hobart H Willard Page no: 608 to 610.
• Fundamentals of Analytical chemistry
Skoog , West, Holler, Crouch Page no: 980 – 982.
• http://www.lcms.com/lcms_information/refer_app_rev_pharm.html
• http://www.rsc.org/images/AMC%20LCMS%20Guide_tcm
• http://www.news-medical.net/life-sciences/Liquid-Chromatography-Mass-Spectrometry-
(LC-MS)-Applications.aspx
• https://chem.libretexts.org/Core/Analytical_Chemistry/Instrumental_Analysis/Mass_Spectr
ometry/Mass_Spectrometers
