MBAT LC MS-MS

SEPARATION, BIOANALYTICAL TECHNIQUES, EXTRACTION, LC MS, LIQUID CHROMATOGRAPHY AND MASS SPECTROSCOPY, MBAT, M.PHARM, PHARMACEUTICAL ANALYSIS, 1 ST YEAR

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

2. Content: Hyphenated technique LC MS/MS 2

3. 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. 3

4. 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. 4 LC-MS

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6. 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 6

7. 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. 7 2. LC-MS/MS

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9. LC- MS/MS 9

10. QUAD MS 10

11. 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 11

12. 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. 12

13. 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. 13

14. Instrumentation HPLC instrumentation is made up of basic components such as 1) S olvent reservoir and degassing system 2) P umps  3) P re-columns 4) S ample injection systems 5) C olumns 6) T emperature controller ( thermostat) 7) D etectors . 14

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17. 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. 17

18. 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. 18

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20. 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. 20

21. 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. 21

22. 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. 22