WORKING AND APPLICATION OF LC-MS/MS 2025

LC-MS/MS (Liquid Chromatography-Tandem Mass Spectrometry) is a powerful analytical tool for comparing innovator and biosimilar drugs. It ensures precise characterization, detecting structural variations, impurities, and post-translational modifications, ensuring biosimilar quality, efficacy, and regulatory compliance in pharmaceutical development.

1. LC-MS/MS (LIQUID CHROMATOGRAPHY - TANDEM MASS SPECTROMETRY) PHARMACEUTICAL TECHNOLOGY ASHWATH B P ( 22B205) ASHWATH B P ( 22B205) PSG COLLEGE OF TECHNOLOGY ( 2022-2026) 3 rd B.Tech BIOTECHNOLOGY

2. DEFINITION Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) is an advanced analytical technique that combines liquid chromatography (LC) with tandem mass spectrometry (MS/MS) for highly sensitive and selective detection of compounds.

3. PARTS FUNCTION LIQUID CHROMATOGRAPHY Separates the sample mixture into individual components based on their chemical properties. FIRST MS Detects and selects ions based on their mass-to-charge ratio (m/z). SECOND MS Further fragments the selected ions for detailed structural analysis and improved specificity.

4. LC-MS V/S LC-MS/MS LC-MS LC-MS/MS Selects intact ions Selects precursor ions Lower selectivity and Moderate sensitivity Higher selectivity & sensitivity Limited structural elucidation Provides detailed structural analysis of compounds simpler sample analysis complex samples, metabolite identification, and trace analysis

5. PRINCIPLE SCHEMATIC

6. COMPONENTS OF LC Pump Mobile Phase Injection Port Column Detector Display (Chromatogram Output)

7. COMPONENTS IN TANDEM MS

8. IONIZATION TECHNIQUES Electrospray Ionization (ESI) Matrix-Assisted Laser Desorption/Ionization (MALDI) Atmospheric Pressure Chemical Ionization (APCI) Nano-Electrospray Ionization (Nano-ESI) Desorption Electrospray Ionization (DESI)

9. ELECTROSPRAY IONIZATION WHY ESI? Soft Ionization Compatibility with LC High Sensitivity Wide Mass Range Aqueous Compatibility

10. MASS ANALYZER TECHNIQUES Quadrupole (QqQ) Time-of-Flight (TOF) Orbitrap Ion Trap (IT) Fourier Transform Ion Cyclotron Resonance (FT-ICR)

11. QUADRUPOLE MASS ANALYZER WHY QqQ? High selectivity Superior quantification High sensitivity Fast scanning speed Cost-effective

12. COLLISION INDUCED DISSOCIATION Collision-Induced Dissociation (CID) works by selecting a precursor ion in the first mass analyzer, which then enters a collision cell filled with an inert gas like nitrogen or argon. The ion collides with the gas molecules, transferring kinetic energy, which excites and weakens its chemical bonds. This leads to fragmentation, generating smaller product ions based on the molecular structure.

13. DETECTORS USED PHOTOMULTIPLIER

14. Thermo Fisher Xcalibur SCIEX Analyst Agilent MassHunter Waters MassLynx Bruker Compass DataAnalysis SOFTWARES FOR DATA ANALYSIS

15. WORKFLOW OF PEPTIDE MAPPING

16. GOALS The goal of biosimilar product development is to elucidate the similarities and differences between innovator and biosimilar drugs LC/MS‑based peptide mapping serves as a primary tool for protein identification as well as monitoring PTMs. Critical quality attributes (CQAs) of biologics define their efficacy and safety. PTMs such as glycosylation, oxidation, and deamidation variants are amongst the CQAs that can significantly impact biological function

17. LC/MS PARAMETERS

18. PTM ANALYSIS( OXIDATION)