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LIQUID CHROMATOGRAPHY – MASS SPECTROSCOPY LC-MS Prepared & presented by:- Shraddha Patel Mpharm 1st SEM (QA) Roll no: 16 Guided by:- Dr. Nehal.J.Shah Dharmaj Degree Pharmacy College Dharmaj


Lc -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 . LC-MS is a powerful technique used for many applications which has very high sensitivity and specificity. It is application is oriented towards the specific detection and potential identification of chemicals in the presence of other chemicals (in a complex mixture).

Principles of LC/MS:   : 

Principles of LC/MS: LC/MS is a hyphenated technique, combining the separation power of HPLC, with the detection power of mass spectrometry. Even with a very sophisticated MS instrument, HPLC is still useful to remove the interferences from the sample that would impact the ionization.

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PARTS OF LC-MS HPLC Constitutes the LC part Mode of injection the sample a. Direct infusion b. Flow injection analysis FIA C. Column SOURCE FOR THE PRODUCTION OF IONS ION SPARY HEATED NEBULIZER

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INTERFACE : Most important and critical. Ions are transferred from LC to MS region MS Analyzer : Quadrupole consists of four rods and acts as mass filter DETECTOR : Channel electron multiplier (CEM) generates a current pulse for every ion that strikes the surface. Number of pulses detected at a given time reflects abundance of the ion in the spectrum

Liquid chromatography: : 

Liquid chromatography: Scale A major difference between traditional HPLC and the chromatography used in LC-MS is that the scale is much smaller , with respect to the internal diameter of the column and with respect to flow rate since it scales as the square of the diameter For a long time, 1 mm columns were typical for LC-MS work (as opposed to 4.6 mm for HPLC). 300 µm and even 75 µm capillary columns have become more prevalent. At the low end of these column diameters the flow rates approach 100 nL /min and are generally used with nanospray sources

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Flow splitting When standard bore (4.6 mm) columns are used The flow is often split ~10:1. This can be beneficial by allowing the use of other techniques in tandem such as MS and UV. However splitting the flow to UV will decrease the sensitivity of spectrophotometric detectors. The mass spectrometry on the other hand will give improved sensitivity at flow rates of 200 μL /min or less

Mass spectrometry: : 

Mass spectrometry: Mass analyzer There are a lot of mass analyzers that can be used in LC/MS . Single Quadrupole , Triple Quadrupole , Ion Trap , TOF ( time of Flight ) and Quadrupole -time of flight (Q-TOF).

Interface : 

Interface The interface between a liquid phase technique which continuously flows liquid, and a gas phase technique carried out in a vacuum was difficult for a long time Moving-belt interface Direct-liquid-introduction interface Thermospray interface Frit FAB/continuous-flow FAB interface Atmospheric-pressure chemical ionization interface Particle-beam interface Electrospray interface


APPLICATIONS OF LC-MS Today toxicologists are faced with expectation of finding drugs and toxins in a variety of materials, with smaller sample volume and at low concentrations. A case of finding the cause of children with mental retardation in 1934, Asbjorn Foling , a Norwegian doctor of medicine reported the cause mental retardation in two children. Among the samples of urine tested from children the chemical Phenylpyruvate was in excess. Doping agents and drug of abuse (Steroids, Diuretics

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Impurity Profiling Metabolite studies. Bio equivalence and Bio-Availability studies. Molecular weights. Assays of Drugs, intermediates. etc…………. QUALITATIVE STUDIES LC –MS provides online molecular masses without need for isolation of trace components LC –MS/MS PROVIDES ADDITIONAL STRUCTURAL INFORMATION

References:   : 

References: Robert E. Ardrey “ LIQUID CHROMATOGRAPHY– MASS SPECTROMETRY: AN INTRODUCTION” University of Huddersfield , Huddersfield , UK