Lc ms 2003

PRESENTED BY

Nilesh S. Jawalkar
(T.Y.B.PHARM)

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INTRODUCTION
 Liquid chromatography-mass spectrometry (LC-MS) is an analytical
chemistry technique that combines the physical separation capabilities of
liquid chromatography with the mass analysis capabilities of mass

spectrometry.
 The combination of these two powerful techniques gives the chemical analyst
the ability to analyze virtually any molecular species; including, thermally
labile, non-volatile, and high molecular weight species.
 It has been said that over 80% of known organic species are amenable to
separation with liquid chromatography.
 Mass spectrometry is capable of providing structure, molecular weight,
empirical formula, and quantitative information about a specific analyte so,
LCMS in recent years, liquid chromatography/mass spectrometry (LC/MS) has
become one of the most powerful analytical techniques for qualitative and
quantitative analysis . 3

AIMS
To identify the different proteins,
peptides drugs in various samples also
to study the bioequivalence in future.

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ADVANTAGES OF LC-MS
Provides compound identity.
Provides sensitive response to most analytes .
Provides compound class information.
Provides compound structure.
Provides sequence information.
Provides molecular weight information.
Provides the five 5s
Speed
Selectivity
Specificity
Sensitivity
$...Low Cost per Information Content.

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SAMPLE CONCIDERATION FOR LC-MS

The analyte must have ionizable groups such as
Amines, Carboxylic Acids, Ketones and Aldehydes.

For best sensitivity, work at a pH where the analyte is
ionized.

i.e. for acid, Neutral to basic pH (7-9) and Acidic pH
(3-4) for bases`

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FLOW CHART OF LC-MS
Sample Column Detector Eluent
Port
collector

Ionization Source

Mass analyzer Vacuum

Detector

Read out device

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LIQUID CHROMATOGRAPHY
 Liquid chromatography is type of chromatography in which analyte
molecule get partitioned between moving mobile phase and
stationary phase
 In liquid chromatography mobile phase is always liquid while
stationary phase is either liquid or solid.
 Liquid chromatography includes following chromatographic
techniques:
Paper chromatography
Thin layer chromatography
Adsorption column chromatography
High performance liquid chromatography
Ion exchange chromatography
Liquid-liquid chromatography

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HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
High performance (pressure) liquid chromatography is a
of column chromatography used frequently in
biochemistry and analytical chemistry to separate,
identify, and quantify compounds. HPLC utilizes a column
that holds chromatographic packing material (stationary
phase), a pump that moves the mobile phase(s) through
the column, and a detector that shows the retention times
of the molecules.
Retention time varies depending on the interactions
between the stationary phase, the molecules being
analyzed, and the solvent(s) used .

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TERMS RELATED TO LC:
1) Retention time
2)Isocratic flow
3)Gradient elution

TYPES OF HPLC:
1) Partition chromatography
2) Normal phase chromatography
3) Displacement chromatography
4)Reverse phase chromatography
5) Size exclusion chromatography
6) Ion exchange chromatography
7) Bioaffinity chromatography

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Parameters:
1. Internal diameter
2. Particle size
3. Pore size
4. Pump pressure
Instrumentation of HPLC:
1. Detectors
2. Pumps
4. Column heaters
5. Auto samplers
Applications of LC:
1. Separation
2. Purification
3. Quantification
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MASS SPECTROSCOPY:
Mass spectroscopy is an analytical technique used to measure the
mass-to-charge-ratio of ions . It is most generally used to find the
composition of a physical sample by generating a mass spectrum
representing the masses of sample components.

Sample Ionization Mass Read
Detector
Inlet source Analyzer out

Vacuum

The stages within the mass spectrometer are:
1. Producing ions from the sample.
2. Separating ions of differing masses.
3. Detecting the number of ions of each mass produced.
4. Collecting the data and generating the mass spectrum.

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APPLICATION OF MS:

1. Identifying unknown compounds by the mass of the
compound molecules or their fragments.
2. Determining the isotopic composition of elements in a
compound.
3. Determining the structure of a compound by observing
its fragmentation.
4. Quantifying the amount of a compound in a sample
using carefully designed methods.
5. Determining other physical, chemical, or even biological
properties of compounds with a variety of other
approaches.
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INTERFACES USED IN LC-MS:
1. Direct Liquid Introduction –

The first attempts to introduce a liquid into an MS using the classic
electron impact ionization (EI)/chemical ionization (CI) source were
based on the simple principle that by minimizing the amount of
liquid, the vacuum system would remove the solvent leaving the
analyte in the gas phase for ionization.

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2. Moving belt/wire interface-
The moving-belt interface separates the condensed liquid-phase
side of the LC from the high vacuum of the MS and uses a belt to
transport the analytes from one to the other. The mobile phase of the
LC is deposited on a band and evaporated. The analytes remain on
the continuously cycling belt and are transported from atmospheric
pressure into the vacuum of the ion source through two differentially
pumped vacuum locks. A heater in the ion source evaporates the
sample from the belt allowing MS analysis.

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3.Thermospray Interface-

As the name thermospray implies, heating the liquid flow leaving an
LC system creates a spray of superheated mist containing small
liquid droplets.
The most successful method involves directing the liquid flow
through an electrically heated capillary, which can be directly
introduced into the MS ion source. The droplets are further
vaporized as they collide against the walls of the heated ion
source.

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4. Particle Beam Interface (MAGIC)-

MAGIC, an acronym for monodisperse aerosol generation
interface for chromatography. The LC eluent is forced through a
small nebulizer using a He gas flow to form a stream of uniform
droplets. These droplets move through a desolvation chamber and
evaporate to a solid particle. These particles are separated from the
gas and transported into the MS source using a differentially pumped
momentum separator.

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5.Atmospheric Pressure Ionization (API)-
Widely used, sensitive and rugged technique.
Ions are formed at atmospheric
pressure.

5.1 Atmospheric Pressure Chemical Ionization (APCI):
The "corona effect" term describes the partial discharge around a
conductor placed at a high potential. This leads to ionisation and
electrical breakdown of the atmosphere immediately surrounding
the conductor. This effect is known as corona discharge.

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5.2 Electrospray Ionization (ESI):
The analyte solution flow passes through the electrospray needle that
has a high potential difference (with respect to the counter electrode)
applied to it. This forces the spraying of charged droplets from the
needle with a surface charge of the same polarity to the charge on the
needle. The droplets are repelled from the needle towards the source
sampling cone on the counter electrode. As the droplets traverse the
space between the needle tip and the cone and solvent evaporation
occurs.

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5.3 Matrix-assisted Laser Desorption/Ionisation
(MALDI)-
The use of a matrix (a small organic molecule) in LD to circumvent
the mass limitation. The matrix had a strong absorbance at the laser
wavelength and was highly sublimable . A low concentration of the
analyte was mixed with this matrix onto a probe or metal plate and
introduced into a pulsed laser beam. The mechanism of MALDI is
believed to consist of three basic steps :
(i) Formation of a 'Solid Solution':
(ii) Matrix Excitation:
(iii) Analyte Ionisation:

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5.4 Surface Enhanced Laser Desorption
/ionization (SELDI)-
Surface-enhanced laser desorption ionization - time of flight (SELDI-
TOF) system is an extremely versatile and convenient proteomic tool
that facilitates the screening of tissue or body fluids. It rapidly
identifies proteins that alter as a consequence of a particular disease,
toxin or treatment. It is very complementary to genomic studies.
A whole series of samples and a ELISA-plate holding device (the
"Bioprosessor") facilitates the analysis of 96 samples
simultaneously.

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APPLICATIONS:
LC-MS USED IN FOLLOWING AREAS
Drug Discovery.
Clinical Analysis.
Proteomics.
Forensic Chemistry
Drug Metabolism study.
Environmental chemistry.
Diagnostic studies.

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CONCLUSION
The study has been shown that the LC-MS is the valuable tool
for analysis of various biological samples.

 The LCMS provides accuracy, specificity, selectivity & rapid less
time consuming.

Instrumentation of LC-MS is quite complicated but very
efficient for their accuracy, sensitivity.

It is mostly useful in pharmaceutical industries finds application
in pharmcokinetic study, proteomics, drug development,
radiopharmaceutics and clinical analysis & in toxicological
studies.

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FUTURE SCOPE
LC-MS is the technique which have application in
future in bioequivalence study.
It can be use for blood analysis, for detection of drugs
whose identity is not known till now.

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REFERENCES
1. Willard, Merritt, Dean, Settle ‘Instrumental methods of analysis’Seventh edition, CBS Publishers &
Distributers New Delhi 110002.
2. Ning Ma, Bi-Kui hang, Huan-De Li et. al . Journal of Clinica Chimica Acta. 1-2, 380, 2007, 100-105.
3. Hiren N. Mistri, Arvind G. Jangid, Mallika Sanyal et.al. Journal of Chromatography B, 1-2, 850, 2007, 318-
326.

4. Willard H.H, Lyne L.M, John A.D, Fran S, Instrumenal methods of analysis, CBS publication, Ed 7th , p

608-610.

5. Ardrey, R. E.; Ardrey, Robert (2003). Liquid chromatography-mass spectrometry: an introduction.

London: J. Wiley. ISBN 0-471-49801-7.

6. McMaster, Marvin C. (2005). LC/MS: a practical user's guide. New York: John Wiley. ISBN 0-471-
65531-7

7. Wilfried M.A. Niessen, Wilfried M. Niessen (2006). Liquid Chromatography-Mass Spectrometry, Third

Edition (Chromatographic Science). Boca Raton: CRC. ISBN 0-8247-4082-3.

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1. Yergey, Alfred L. (1990). Liquid chromatography/mass spectrometry:
techniques and a Displacement Chromatography 101. [1] Sachem,
Inc. Austin, TX 78737
2. Xiang, Y.; Liu Y. and Lee M.L. (2006)."Ultrahigh pressure liquid
chromatography using elevated temperature". Journal of
Chromatography A 1104 (1-2): 198–202.
doi:10.1016/j.chroma.2005.11.118.
 www.biocompare.com
 www.chm.bris.ac.uk/ms/theory/esi-ionisation.html
 www.bdal.com
 www.waters.com/watersdivision/ContentD.asp?wa.
 www.opdac.com/lc/contents/api.htm
 www.jir.com
 www.lcms.com
 www.proteomicsnijmegen.nl/selditof.htm
 www.proteomesci.com/content/4/1/5/figure/F3
 www.wikipedia.com
 www.sciencedirect.com
 www.lcgceurope.com

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Lc ms 2003

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Lc ms 2003