Mass Spectrometry (MS).pptxnpharmaceutıcal analysıs

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1. Mass Spectrometry (MS) By Abrar Ahmad

2. Introduction to Mass Spectrometry Analytical chemistry technique that helps in determination of molecular mass of the compounds Accurate method in all other methods of spectroscopy. Sample is converted to rapidly moving positive ions by electron bombardment and charged particles are separated according to their masses.

3. Introduction to Mass Spectrometry Mass spectrum is a plot of relative abundance against the ratio of mass/charge (m/e). These spectra are used to determine Elemental or isotopic signature of a sample Masses of fragments and   molecules

4. Principle of Mass Spectrometry In this technique, molecules are bombarded with a beam of energetic electrons. The molecules are ionized and broken up into many fragments, some of which are positive ions. Each kind of ion has a particular ratio of mass to charge, i.e. m/e ratio (value).

5. Principle of Mass Spectrometry The ions pass through magnetic and electric fields to reach the detector where they are detected and signals are recorded to give mass spectra . All mass spectrometers also have a vacuum system to maintain the low pressure, which is also called high vacuum, required for operation.

6. Instrumentation and Steps of Mass Spectrometry (MS ) I nvolves a series of steps and the use of specific instrumentation to analyze the mass-to-charge ratio (m/z) of ions . Here are the key steps and components involved in a typical mass spectrometry experiment:

7. Sample Introduction The selection of a sample inlet depends upon the sample and the sample matrix. Direct Vapor Inlet Gas Chromatography Liquid Chromatography Direct Insertion Probe Direct Ionization of Sample

8. Ion Source The ion source is responsible for ionizing the sample molecules. Different ionization techniques are employed, including: Electron Ionization (EI): High-energy electrons bombard the sample. Electrospray Ionization (ESI): A spray of charged droplets is formed from a liquid sample. Atmospheric Pressure Chemical Ionization (APCI): Ionization occurs through chemical reactions in the gas phase. Matrix-Assisted Laser Desorption/Ionization (MALDI): A laser beam is used to ionize molecules embedded in a matrix.

9. Electron ionization Technique used in mass spectrometry to ionize gas-phase molecules for analysis. Unlike Electrospray Ionization (ESI), which is commonly used for large biomolecules and polar compounds, EI is more suitable for volatile and nonpolar compounds . E mployed in the analysis of small organic molecules.

10. Electron ionization Introduction of Sample: Introduced into the mass spectrometer as a gas-phase sample. This can be achieved by vaporizing a solid or liquid sample or by directly introducing a gaseous sample. Electron Beam Impact: In the ionization source, the gas-phase molecules are bombarded by high-energy electrons. These electrons typically have energies in the range of 70 to 150 electron volts (eV ).

11. Electron ionization Bond Cleavage: Absorbed energy can cause breaking of chemical bonds within the molecular ion by knocking off electron Fragment Ions: Result of bond cleavage is formation of smaller fragment ions. These fragment ions are detected in the mass spectrometer, and their mass-to-charge ratios (m/z) are recorded.

12. Electrospray Ionization (ESI ) Principle : In ESI, a liquid sample is sprayed through a fine needle or capillary at a high voltage. This generates a fine mist of charged droplets, and as solvent evaporates, highly charged ions are produced. Sample Type: ESI is particularly suitable for large and polar molecules such as peptides, proteins, and nucleic acids.

13. Electrospray Ionization (ESI ) Applications: Commonly used in Liquid Chromatography-Mass Spectrometry (LC-MS) for the analysis of biomolecules. Widely employed in proteomics and metabolomics. Advantages: Gentle ionization, making it suitable for fragile biomolecules. Allows for the analysis of large and complex molecules.

14. Mass Analyzer The mass analyzer separates ions based on their mass-to-charge ratio. Common types include: Time-of-Flight (TOF): Ions are accelerated into a field and their time of flight is measured. Quadrupole: Ions pass through a series of radiofrequency (RF) and direct current (DC) fields. Ion Trap: Ions are trapped in a three-dimensional space and manipulated using electric and magnetic fields .

15. Mass Detector The mass detector records the mass spectrum by detecting the ions reaching the detector after passing through the mass analyzer. Photomultiplier Tube (PMT): Common in TOF instruments. Electron Multiplier (EM): Used in quadrupole and ion trap instruments. Faraday Cup or Channel Tron Detector: Used in some older instruments.

16. Working of Mass Spectrometry (MS ) In a typical procedure, a sample, which may be solid, liquid, or gas , is ionized, for example by bombarding it with electrons . This may cause some of the sample’s molecules to break into charged fragments . + ve Charge These ions are then separated according to their mass-to-charge ratio, typically by accelerating them and subjecting them to an electric or magnetic field :

17. Working of Mass Spectrometry (MS ) Ions of the same mass-to-charge ratio will undergo the same amount of deflection. The ions are detected by a mechanism capable of detecting charged particles, such as an electron multiplier.

18. Working of Mass Spectrometry (MS ) Results are displayed as spectra of the relative abundance of detected ions as a function of the mass-to-charge ratio. I dentified by correlating known masses (e.g. an entire molecule) to the identified masses.

19. Data Analysis Software Specialized software is employed for data analysis. It helps interpret mass spectra, identify compounds, and quantify their concentrations.

20. Control System The operation of the mass spectrometer is controlled by a computerized system. Users can set parameters such as ionization conditions , mass analyzer settings , and data acquisition parameters through software.

21. Calibration Regular calibration is essential for accurate mass measurements. Calibration standards with known mass values are used to adjust the instrument.

22. Vacuum System Most mass spectrometers operate under vacuum conditions to ensure the free movement of ions Eliminate collision with gas molecules.

23. P harmaceutical A pplications of mass spectrometry

24. Drug Discovery and Development Target Identification and Validation : Mass spectrometry helps identify and validate potential drug targets by analyzing proteins and understanding their functions in biological systems. Lead Compound Identification: MS is used to identify and characterize lead compounds during the drug discovery process.

25. Protein and Peptide Analysis Protein Identification Identification of proteins in complex biological samples Aiding in understanding of drug interactions and mechanisms of action .

26. Metabolite Profiling Metabolomics Applied in metabolomics studies to profile and quantify small molecules (metabolites) in biological samples. This helps in understanding the metabolic pathways affected by drugs and their impact on the body.

27. Pharmacokinetics and Bioavailability Drug Metabolism Studies: Mass spectrometry is used to study the metabolism of drugs in vivo, providing insights into the formation of metabolites and their pharmacological activities. Bioavailability Assessment: MS helps determine the bioavailability of drugs by analyzing their concentrations in blood and other biological matrices over time.

28. Quality Control and Formulation Drug Purity and Impurity Analysis: Mass spectrometry is employed to assess the purity of drug substances and identify impurities, ensuring the quality and safety of pharmaceutical products. Formulation Analysis: MS is used to analyze pharmaceutical formulations to ensure the proper composition and stability of the drug product.

29. Doping Control in Sports Anti-Doping Testing: Mass spectrometry is a key tool in anti-doping testing, helping to detect prohibited substances and their metabolites in athletes' biological samples.

30. Forensic Toxicology Drug Overdose Investigations: Mass spectrometry is used in forensic toxicology to analyze biological samples Presence of drugs, helping in drug overdose investigations.