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Mass Spectrometry Applications and spectral interpretation: Basics


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Mass Spectrometry Applications and spectral interpretation: Basics

  1. 1. Mass Spectrometry applications in Medicinal Chemistry Shreekant Deshpande
  2. 2. Mass spectrometry is a very powerful and versatile analytical tool. Extensively used in every fields to detect one or the other things. Here I try to give some outlines about HOW TO extract a INFORMATION FROM MASS SPECTRA.
  3. 3. Overview What is mass spectrometry? Where are mass spectrometers used? How does a mass spectrometer work? What information can it provide? Interpretation of spectra. and Applications in medicinal chemistry.
  4. 4. What is Mass Spectrometry? Analytical tool measuring molecular weight (MW) of sample. Only picomolar concentrations required. With an high accuracy and within 5 ppm of std. Error for small organic molecules. For a mass of 40 kDa, there is a 4 Da error. This means it can detect amino acid substitutions / post-translational modifications in biological systems.
  5. 5. Where are they used? Biotechnology: Analysis of proteins, peptides, oligonucleotides Pharmaceutical: Drugs discovery, combinatorial chemistry, pharmokinetics, Drug metabolism Clinical: Neonatal screening, haemoglobin analysis, drug testing Environmental: Water, food, air quality Geological: Oil composition
  6. 6. Mass Spectrometry is also used to... Detect and identify the use of steroids in athletes(Doping) Ex. Sunita Rani’s case!!! Monitor the breath of patients by anesthesiologists during surgery. Determine the composition of molecular species found in space Adulteration in food stuff Ex. Presence of pesticide in …..
  7. 7. How does a Mass Spectrometer work? 3 fundamental parts: the ionisation source, the analyser, the detector Samples easier to manipulate if ionised Separation in analyser according to mass-tocharge ratios (m/z) Detection of separated ions with their relative abundance Signals sent to data system and formatted in a m/z spectrum
  8. 8. Either directly or via chromatography for component separation (HPLC, GC, capillary electrophoresis) Ionisation can be positively charged (for proteins; Org. molecules) or negatively charged (for saccharides and oligonucleotides)
  9. 9. Electron Impact (EI) Chemical Ionisation (CI) Atmospheric Pressure Chemical Ionisation (APCI) Fast Atom Bombardment (FAB) Field Desorption / Field Ionisation (FD/FI) Electrospray Ionisation (ESI) Matrix Assisted Laser Desorption Ionisation (MALDI)
  10. 10. • Magnetic Sector • Time of flight (tof) • Quadrupole • Quadrupole ion trap • Fourier Transform Ion Cyclotron Resonance
  11. 11. The resolving power of analysers The respective resolving powers correspond to the resolution typically obtained for time-of-flight, quadrupole, and Fourier-transform mass analyzers. Notice that the individual isotope peaks can be distinguished at the higher resolution.
  12. 12. • Photo Multiplier • Faraday Cup Detector monitors ion current, amplifies it and then transmits signal to data system
  13. 13. Mass spectrometry is a very powerful method to analyse the structure of organic compounds, but suffers from 3 major limitations: 1 It is a destructive method 2 Compounds cannot be characterised without clean samples 3 The dynamic properties of molecules can not be detected.
  14. 14. Spectrum speaks What Molecular ion peak tells !! Most of the times chem. Strs. of the products are known are presumed. Sometime unexpected peaks will come then we’ll scratch our head. And we try to see +23/ +39 peaks(Na/ K adducts) If MI is observed what else we can see for further confirmations. Isotope peaks mass no- odd/even
  15. 15. m/z spectrum example
  16. 16. Natural abundance of isotopes Elements Carbon Nitrogen Oxygen Sulfur Chlorine Bromine Relative abundance C 14 N 16 O 32 S 35 Cl 79 Br 12 100 100 100 100 100 100 C 15 N 17 O 33 S 13 1.08 0.38 0.04 O 34 S 37 Cl 79 Br 18 0.78 3:1 1:1 0.20 3.35 32.5 98.0
  17. 17. Remember and Review! The Rule of Thirteen – Molecular Formulas from Molecular Mass When a molecular mass, M+, is known, a base formula can be generated from the following equation: M = n + r 13 the base formula being: CnHn 13 +r For this formula, the HDI can be calculated from the following formula: HDI = ( n – r + 2 ) 2
  18. 18. Interpretation How to interpret our spectra? • Check the quality of the data; does the total ion spectrum look ok? • Does the mass spectrum show significant peaks above the baseline? • Identify any major peaks. • Look for telltale isotope patterns. • Remember the Nitrogen Rule. (The Nitrogen Rule: A neutral compound containing an odd number of nitrogen atoms will always have an odd molecular mass.) • Do the masses fit with the proposed structure? • Have you used the appropriate masses in your calculations?
  19. 19. Interpretation How do I interpret my spectra? Identify the MI Check isotope pattern - halogens, sulphur etc. Odd/Even Mass - Nitrogen Rule If FAB check intensity of MI - aromatic or aliphatic Accurate mass - calculate r + db See neutral losses out of your mass.  Amine protecting groups CxHyNzOn  r + db = x - 0.5y + 0.5z + 1 
  20. 20. In Med. Chemistry Accurate molecular weight measurements: Unknown sample, Purity of sample, About other side products. Reaction monitoring: enzyme activity, chemical modification, protein digestion. Structural elucidations: Natural products, unknown products through fragmentations. Identification of Mechanism of actions: Both covalent n noncovalent interactions. Active site Identifications: Quantifying the amount of a compound in a sample using carefully designed methods. Impurity profiling for the bulk drugs.
  21. 21. AccuTOF-DART Technology Its an atmospheric pressure source capable of ionizing liquid, solid, and gas samples in real time. Principle: on an atmospheric pressure interaction of a sample with longlived electronic excited-state atoms or molecules and atmospheric gases. The system uses helium or nitrogen to produce metastable atoms or molecules that interact with the analytes. -lends to the analysis of samples without any preparation and provides data with accurate mass measurement. A complete analysis can be done within minutes by simply holding a sample in the open gas stream.
  22. 22. The DART can even analyze samples directly on surfaces such as concrete, currency, food, pills and clothing. The revolutionary DART ion source gives an entirely new meaning to the phrase "open access".
  23. 23. Conclusion MS is a highly applicable analytical tool in Medicinal Chemistry. The full structural information can get out of single mass spectrum with min. qty. of sample. Elemental analysis can b carried by using HRMS.
  24. 24. In his book, “Rays of Positive Electricity” (1913), J.J.Thomson remarked: “I feel sure that there are many problems in chemistry which could be solved with far greater ease by this than by any other method. The method is surprisingly sensitive,……..requires an infinitesimal amount of material and does not require this to be specially purified.” Thank you very much