INSTRUMENTATION OF LC-MS

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INTRODUCTION:
Liquid chromatography-mass spectrometry (LC-MS), oralternatively 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 (MS).
LC-MS is a powerful technique that has very high sensitivity, making it useful in
many applications. Its application is oriented towards the separation, general detection
and potential identification of chemicals of particular masses in the pressure of other
chemicals (i.e., in complex mixtures)
e.g., Natural products fromnatural-products extracts, and pure substances from mixtures
of chemical intermediates. Preparative LC-MS systems can be used for rapid mass-
directed purification of specific substances from such mixtures that are important in
basic research, and pharmaceutical, agrochemical, food, and other industries.
INSTRUMENTATION OF LC-MS
 The mass spectrometer is an instrument designed to analysis gas phase ion
according to their m/z (mass charge ratio) value.
 Mass spectrometerinvolves the separation ofcharged species which are produced
by a variety of ionization methods in LC-MS. These includes:
 Electron spray ionization (EI)
 Atmospheric pressure chemical ionization (APCI)
 In all cases the charged species are produced as gas phaseions under atmospheric
pressure conditions.
 The separation of the gas phase ions is achieved within the mass spectrometer
using electrical and/or magnetic fields to different ions.
 In addition to the analyzer, the mass spectrometer also includes an atmospheric
ionization chamber, a vacuum systems and a detector.

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Solvent delivery system:
 Pneumatic pump
 Syringe pump
 Gas displacement pump
 Reciprocating pump
Sample injection system:
 Syringe injection
 Stop flow injection
 Micro volume sampling valves

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Columns:
 Porous polymeric beads
 Porous-layer beads
 Total porous silica particles
Analyzers:
 Quadrapole analyzer
 Triple quadrapole
 Time-of-flight
 Ion trap analyzer
Detector:
 The high energy dynode detector

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IONIZATION TECHNIQUE
IONISATION:
 Ionization is the process wrong are either produce or added to atom or
molecules to produce ions. In LC-MS charge may also be applied to the
molecule via association with other molecules-for example a proton.
 Such ions are produced in LC-MS system by the use of strong electrical field
in the vapor or condensed phase. Interfaces whereby the sample is ionized or
desolvated under atmospheric pressure conditions are termed atmospheric
pressure ionization (API).
 The most common ionization method in LC-MS include:
 Electrons pray ionization (ESI)-ionization in the condensed phase.
 Atmospheric pressure photo ionization-ionization in the gas phase.
 Atmospheric pressure photo ionization-ionization in the gas phase.

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ELECTRONSPRAYIONIZATION:
Electron spray ionization (ESI) uses condensed phase (liquid) charge separation
and ion evaporation techniques to producevapor phase analyte ions. Primarily-analyte
molecules of interest must be in the ionizer a form prior to spraying into the electron
sprayinterference in order to achieve a reasonable response. This indicates that analytes
are ionized by manipulation of the HPLC eluent PH either before or after separation in
the HPLC column.
In electrospray ionization there are three important processes that occur in order
to transfer sample ions from the HPLC eluent into the gas phase within the mass
spectrometer. These processes are:
 Production of charged droplets at the capillary tip.
 Desolvation of the droplets
 Production of gas phase ions from small/highly charged droplets.

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ATMOSPHERIC PRESSURECHEMICALIONIZATION;
Atmospheric pressure chemical ionization uses analyte desolvation and transfer
reactions in the vapor phase to produce vapor phase analyte ions.
In APCI the eluent is introduced into the interface using capillary of similar
design to the ESI source. However, no potential is applied to the capillary but instead
the liquid emerges from the capillary surrounded by a flow of inert, nebulizing gas into
a heated region.
The combination of nebulizing gas and heat forms an aerosol that begins to
rapidly evaporate. A pin is placed within the heated region that has a high potential
applied to it and produces an electrical discharge that ionizes eluent molecules, these
ionized molecules impart charge to the analyte molecules via charge transfer reactions
or molecular association.
Both ESI and APCI are termed “soft”ionization methods. This means that in the
process of producing ions there is negligible energy transferred to the ion.as a
consequence the ion formed does not fragment to small mass ion, the resultant mass
spectrum therefore consists predominantly ions, either
[M+H] + or [M-H]- or adduct ions like [M + Na]+

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ATMOSPHERIC PRESSUREPHOTO IONIZATION (APPI):
APPI is a compliment to ESI and APCI and has been developed to broaden the
range of ionizable analytes at atmospheric pressure.
APPI is important in the analysis of certain compounds that are not easily
ionizable by ESI or APCI like low and non-polar compounds (APPI has been used in
the analysis of polycyclic aromatic hydrocarbons).
In APPI, the ionization process is accomplished by exposing an aerosol of
droplets to photo irradiation. A molecular radical ion is formed when the molecule
absorbs a photon. This process is possible only when the irradiating photon (of energy)
exceeds the ionization potential (IP) of the molecule.

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MASS ANALYSERS
MASS ANALYSERS:
 The following are mass analyzers in mass spectroscopy
 Quadrapole mass analyzer
 Ion trap mass analyzer
 Time of flight
 Magnetic sectormass analyzer
Quadrapole mass analyzer:
In quadrapole mass analyzing devices electrical field are used to separate ions
according to their mass-to-charge ratio (m/z) as they pass along the central axis of
four parallel equidistant rods (or poles). Ion separation is performed by using
controlled voltages applied to the mass analyzer rods which impart an electrostatic
field inside the analyzing device.
Advantages:
 Reproducibility
 Low cost
Disadvantages:
 Low resolution
 Mass discrimination, peak height Vs mass responsemust be ”tuned”.

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ION TRAP MASS ANALYZER:
Ion trap mass analyzer work on the basis of strongions in a trap and manipulating
the ions by using applied DC and RF fields .The amplitude of the applied voltages
enable the analyzer to trap ions of specified mass to charge ratio within the analyzing
device. Non-selected ions are given a trajectory by the electrostatic field that causes
them to exit the trap.by filling the trap with an inert gas fragmentation of selected ions
is possible. This is useful when structural information is required.
The system has some unique capabilities including being able to perform,
multiple product ion scans with very good sensitivity (MS). it should be noted that the
spectra acquired with an ion trap mass analyzer may be significantly different to those
acquired from a triple quadrapole system due to the different collision regimes within
the systems (collision energy/gas).
Advantages:
 High sensitivity
 Multiple production scan capability (MS)
 High resolution
 Good for DNA analyses
Disadvantages:
 Produces very unusual spectraif the ions are stored in the trap too long.
 Easily saturated
 Poorfor low mass work ( below 100Da)

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TIME OF FLIGHT:
The basic principle of mass analysis using time-of-flight mass analyzer are
relatively straight forward in comparisonto many of the other typical analyzing devices.
Ions are extracted (or produced) in short bursts or packets within the ion source
and subjected to an accelerating voltage. The ions then “drift” or “fly” down an
evacuated tube of a set length (d). Once free from the region of accelerating voltage the
speed at which the ions travel down the tube is dependent upon their mass (m) and
charge (z). This mass analyzer is useful as all ions are detected (almost) simultaneously,
scanning the mass range of all ions is very rapid and as such the inherent sensitivity of
the instrument is increased.
Advantages:
 High ion transmission
 Highest practical mass range of all MS analyzers.
 Detection limit.
Disadvantages:
 Fast digitizers used in TOF can have limited dynamic range.

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DETECTORS
Once the ions have passed the mass analyzer they have to be detected and
transformed into a usable signal. The detector is an important element of the mass
spectrometer that generates a signal from the incident ions by either generating
secondaryelectrons, which are further amplified or by inducing a current (generated by
moving charges). Ion detector systems fall into two main classes.
 Point detector
 Array detector.
APPLICATIONS:
 LC-MS provide information about the molecular weight structure, identify &
quantity of specific sample components.
 Suitable for the analysis of large, polar ionic, thermally unstable and in volatile
compounds.
 LC-MS is most commonly used for proteomic analysis of complex and samples
where peptide masses may overlap even with a high-resolution mass
spectrometer.
 Molecular weight determination for example: the only difference in the sequence
is at the C-terminus where one peptide has threonine and the other has threonine
amide. The smaller fragments are identical in the two spectra, indicating that large
portions of the two peptides are very similar. The larger fragments contain the
differentiating peptides.
Trace gas analysis
Several techniques use ions created in a dedicated ion source injected into a flow
tube or a drift tube: selected ion flow tube (SIFT-MS), and proton transfer reaction (
PTR-MS), are variants of chemical ionization dedicated for trace gas allowing
calculations ofanalyte concentrations from the known reaction kinetics without the need
for internal standard or calibration.

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Pharmacokinetics:
Pharmacokinetics is often studied using mass spectrometry because of the
complex nature of the matrix (often blood or urine) and the need for high sensitivity to
observe low dose and longtime point data. The most common instrumentation used in
this application is LC-MS with a triple quadrapole mass spectrometer. Tandem mass
spectrometry is usually employed for added specificity, standard curves and internal
standards are used for quantification of usually a single pharmaceutical in the samples.
The samples represent different time points as a pharmaceutical is administered and then
metabolized or cleared from the body. Blank ort=0 samples taken beforeadministration
are important in determining background and ensuring data integrity with suchcomplex
sample matrices. Much attention is paid to the linearity of the standard curve; however
it is not uncommon to use curve fitting with more complexes functions such as
quadratics since the responseofmostmass spectrometers is less than linear across large
concentration ranges.
There is currently considerable interest in the use of very high sensitivity mass
spectrometry for micro dosing studies, which are seen as a promising alternative to
animal experimentation.
Glycan analysis
Mass spectrometry (MS), with its low sample requirement and high sensitivity,
has been predominantly used in glycol biology for characterization and elucidation of
glycan structures. Mass spectrometry provides a complementary method to HPLC for
the analysis of glycan. Intact glycans may be detected directly as singly charged ions
by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) or,
following permethylation or peracetylation, by fast atom bombardment mass
spectrometry (FAB-MS). Electrospray ionization mass spectrometry (ESI-MS) also
gives good signals for the smaller glycan. Various free and commercial software are
now available which interpret MS date and aid in glycan structure characterization.

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Protein characterization
Mass spectrometry is an important method for the characterization and
sequencing of proteins. The two primary methods for ionization of whole proteins are
electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI).
In keeping with the performance and mass range of available mass spectrometers, two
approaches are used for characterizing proteins. In the first, intact proteins are ionized
by either ofthe two techniques described above, and then introduced to a mass analyzer.
This approach is referred to as “top-down” strategy of protein analysis. In the second,
proteins are enzymatically digested into smaller peptides using proteasesuch as trypsin
or pepsin, either solution or in gel after electrophoretic separation. Other proteolytic
agents are also used. The collection of peptide products are then introduced to the mass
analyzer. When the characteristic pattern of peptides is used for identification of the
protein the method is called protein mass fingerprinting (PMF), if the identification is
performed using the sequence data determined in tandem MS analysis it is called de
novo peptide sequencing. These procedures of protein analysis are also referred to as
the “bottom-up” approach.
Drug development:
LC-MS is used in drug development at many stages including peptide and
glycoprotein in mapping, natural products de-replication, bio-affinity screening. In vivo
drug screening, metabolic stability screening, metabolic identification, impurity
identification, degradant identification, quantitative bio-analysis and quality control.
LC-MS in drug discovery:
Drug discovery involves number of phases including target identification, lead
identification, and small molecular optimization, pre-clinical & clinical development.
With advances in MS, identification oftranslated protein is possible. Protein may signal
disease processes in which case their regulation by a potential drug might indicates its
efficacy.

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REFERENCES:-
http://www.scribd.com/doc/28973479/gcms
http://huygemnsgcms.gsfc.nasa.gov/MS Detector
liquid chromatography-Mass spectrometry by W.M.A