JSPM’SCharak college of Pharmacy and Reaserch,Gate No.720/1&2 Punenagar Road,Wagholi,Pune 412207 Liquid chromatography and Mass Spectrometry Presented By, Guided By, Miss. Mayuri Shitre Dr. Rajesh J Oswal Prof.Sandip Kshirsagar Department of Pharmaceutical Chemistry
Content Introduction Liquid chromatography Mass spectrometry Liquid chromatography mass spectrometry (LCMS) Interface Application of LCMS
Introduction Definition: Liquid chromatography–mass spectrometry (LC- MS, or alternatively 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 LC-MS is a powerful technique used for many applications which has very high sensitivity and selectivity.
Liquid Chromatography Scale: A major difference between traditional HPLC and the chromatography used in LC-MS is that in the latter case the scale is usually much smaller, both with respect to the internal diameter of the column and even more so with respect to flow rate since it scales as the square of the diameter. 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. The mass spectrometry on the other hand will give improved sensitivity at flow rates of 200 μL/m
Mass spectrometryInstrumental Requirement: Ionisation source Electron impact ioniser Field ionisation technique Thermal ionisation Matrix assisted ionisation Ion collector: Faraday cup collector Electron multiplier
Analysers: Double focousing Quadrapole mass analyser Time of flight spectrometer Plasma desorption.
Instrumentation of LCMS Sample Introduction Ion Source Detector (LC) Read out System
Interface in LCMS Interface: The problem encounter when interface HPLC with mass is mismatch between mass flow in HPLC. The methods to overcome problems are: Thermospray method Monodisperse aerosol generation interface Moving Belt interface
Application of LCMS1.Pharmacokinetics LC-MS is very commonly used in pharmacokinetics studies of pharmaceuticals and is thus the most frequently used technique in the field of bioanalysis . These studies give information about how quickly a drug will be cleared from the hepatic blood flow, and organs of the body. MS is used for this due to high sensitivity and exceptional specificity compared to UV (as long as the analyte can be suitably ionised), and short analysis time.
• The major advantage MS has is the use of tandem MS-MS.The detector may be programmed to select certain ions tofragment. The process is essentially a selectiontechnique, but is in fact more complex.•The measured quantity is the sum of molecule fragmentschosen by the operator. As long as there are nointerferences or ion suppression, the LC separation can bequite quick.•It is common now to have analysis times of 1 minute or lessby MS-MS detection, compared to over 10 mins with UVdetection
2.Proteomics/ metabolomics LC-MS is also used in the study of proteomics where again components of a complex mixture must be detected and identified in some manner. Samples of complex biological fluids like human serum may be run in a modern LC-MS/MS system and result in over 1000 proteins being identified, provided that the sample was first separated on an SDS-PAGE gel or HPLC-SCX.
• The bottom-proteomics LC-MS approach toproteomics generally involves protease digestionand denaturation (usually trypsin as aprotease, urea to denature tertiary structure andiodoacetamide to cap cystesine residues) followedby LC-MS with peptide mass fingerprinting or LC-MS/MS (tandem MS) to derive sequence ofindividual peptides.LC-MS/MS is most commonlyused for proteomic analysis of complex sampleswhere peptide masses may overlap even with ahigh-resolution mass spectrometer.
3.Drug developmentLC-MS is frequently used in drug development at many different stages including Peptide Mapping, GlycoproteinMapping, Natural Products Dereplication, BioaffinityScreening, In Vivo Drug Screening, Metabolic StabilityScreening, Metabolite Identification, Impurity Identification, Degradant Identification, Quantitative Bioanalysis, and Quality Control.