MBAT LC MS-MS

Subject : Modern bio analytical techniques
Topic : LC MS / MS
Presented By,
VENKATESAN R
(Reg No : 22PA023) (2nd Sem)
M. Pharm Pharmaceutical analysis,
Presented to,
Dr. Harsha K Tripathy,
Department of Pharmaceutical analysis
KARNATAKA COLLEGE OF
PHARMACY

KARNATAKA COLLEGE OF PHARMACY
BANGALORE
Content:
• Hyphenated technique
• LC MS/MS
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1. Hyphenated Technique
Definition:
Hyphenated technique are those techniques in which a
chromatographic technique and spectroscopic technique are coupled for
the analysis of various biologic, chemical, and toxicological samples.
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The Liquid Chromatography-Mass Spectrometry (LC-MS)is hyphenated analytical technique
which is combination of Liquid Chromatography (LC) and Mass Spectrometry (MS).
HPLC is one of most common analytical technique used in pharmaceutical industry for
determination and quantification of drug substances and its related substances. Due to high
reproducibility and accuracy.
Mass Spectrometry is also used for identification of unknown compounds, their relative
abundance and to elucidate the structure.
LC-MS is more significantly used in , bioavailability studies, bioequivalence and Pharma co-
dynamics studies.
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LC-MS

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Qualitative and Quantitative testing
Liquid chromatography
• Separates mixture of components
• Based on polarity of the analyte
Tandem mass spectrometry
• Detector
• Identification & Quantification of components
• Based on compound mass
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Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS) is a powerful
analytical technique used for the separation, identification, and quantification of
biomolecules, especially in complex mixtures. It combines the separation capabilities of
liquid chromatography with the sensitive and specific detection of mass spectrometry,
allowing for detailed characterization of biomolecules in terms of their mass, structure, and
abundance. In this technique, selected ions from the first mass analyzer are further
fragmented, and the resulting fragment ions are analyzed in the second mass analyzer.
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2. LC-MS/MS

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LC- MS/MS
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QUAD MS
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The process of biomolecule separation using LC-
MS/MS involves several steps:
• Sample preparation
• Liquid Chromatography (LC)
• Mass Spectroscopy (MS)
• Tendem Mass Spectrometry (MS/MS)
• Data Analysis
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1. Sample preparation
 Similar to other analytical techniques, proper sample preparation is crucial. It
involves extracting, purifying, and concentrating the target biomolecules from
the biological matrix. Sample preparation methods depend on the type of
biomolecules being analyzed.
 This include following technique:
Dilution, Protein precipitation, Liquid- Liquid extraction, Solid Phase
Extraction, Phospholipid Removal etc.
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2. Liquid Chromatography (LC):
 In this step, the prepared sample is injected into a liquid chromatography system.
The LC system separates the mixture into its individual components based on their
chemical properties, such as polarity, hydrophobicity, or size. The choice of
column and mobile phase conditions depends on the specific biomolecules of
interest.
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Instrumentation
HPLC instrumentation is made up of basic components such as
1) Solvent reservoir and degassing system
2) Pumps
3) Pre-columns
4) Sample injection systems
5) Columns
6) Temperature controller ( thermostat)
7) Detectors.
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3. Mass Spectrometry (MS):
• The eluted components from the LC system are directed into the mass
spectrometer. The mass spectrometer ionizes the molecules and separates them
based on their mass-to-charge ratio (m/z). The ions are then detected and
quantified.
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4. Tandem Mass Spectrometry:
In LC-MS/MS, tandem mass spectrometry is used to provide additional
structural information about the separated biomolecules. In this technique, selected
ions from the first mass analyzer are further fragmented, and the resulting fragment
ions are analyzed in the second mass analyzer. This fragmentation pattern can be
used to identify the biomolecule's structure and sequence.
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5. Data Analysis
• The data generated from LC-MS/MS experiments are typically presented as mass spectra,
chromatograms, and fragmentation patterns. Specialized software is used to analyze the
data, identify the biomolecules based on their mass and fragmentation patterns, and
quantify their abundance.
• LC-MS/MS is particularly useful for identifying and quantifying proteins, peptides,
metabolites, lipids, and other biomolecules in complex biological samples. It is widely
used in proteomics, metabolomics, lipidomics, and other omics disciplines. The technique
offers high sensitivity, specificity, and the ability to analyze a wide range of biomolecules
in a single experiment.
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• One notable advantage of LC-MS/MS is its ability to analyze multiple analytes
simultaneously, making it a versatile tool for biomarker discovery, drug
development, and understanding the molecular mechanisms underlying various
biological processes and diseases.
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Application of LC-MS/MS
• Scanning of bioactive constituents present in the natural plant drugs.
• Discovery of lead compounds from plants and natural products.
• LC-MS guided isolation of the bio active constituents.
• Quantification of the bioactive constituents in various formulations or
Nutraceuticals.
• Identification of synthetic drugs and drug adulteration in natural products.
• Monitoring and quantification of bio active constituents and their metabolites in
biological fluids.
• Differentiation of natural and synthetic variant of various nutraceuticals available
in the market.
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MBAT LC MS-MS

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