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PRESENTED BY

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

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 .

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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-MSProvides 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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Sample Column Detector Eluent Port collector Ionization Source

Mass analyzer Vacuum

Detector Read out device

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FLOW CHART OF LC-MS

LIQUID CHROMATOGRAPHYLiquid 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 chromatographyThin layer chromatographyAdsorption column chromatographyHigh performance liquid chromatographyIon exchange chromatographyLiquid-liquid chromatography

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HIGH PERFORMANCE LIQUID CHROMATOGRAPHYHigh 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 time2)Isocratic flow3)Gradient elution

TYPES OF HPLC:1) Partition chromatography2) Normal phase chromatography3) Displacement chromatography4)Reverse phase chromatography5) Size exclusion chromatography6) Ion exchange chromatography7) 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:

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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 Inlet

Ionizationsource

MassAnalyzer Detector Read

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.

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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APPLICATION OF MS:

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-

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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.

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.

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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.

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)-

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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.

5.2 Electrospray Ionization (ESI):

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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.

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)-

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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.

APPLICATIONS:

LC-MS USED IN FOLLOWING AREASDrug Discovery.Clinical Analysis. Proteomics.Forensic ChemistryDrug Metabolism study.Environmental chemistry.Diagnostic studies.

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CONCLUSIONThe 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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REFERENCES1. 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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