LC- MS

hypenated technique
hplc
mass spectroscopy
application with example

1. LIQUID CHROMATOGRAPHY – MASS SPECTROMETRY PRESENTED BY: Bhavya K B 2 nd Sem M pharma Dept. of Pharm. Analysis SAC College of pharmacy , B G Nagara . PRESENTED TO: Dr . T. Yunus pasha Head of the department, Dept. Of Pharm. Analysis SAC College of pharmacy, B G Nagara . 1

2. LIQUID CHROMATOGRAPHY – MASS SPECTROMETRY. (LC-MS) 2

3. CONTENT : Introduction Principle Instrumentation Applications Reference 3

4. INTRODUCTION: LIQUID CHROMATOGROPHY – MASS SPECTROMETRY Liquid chromatography–mass spectrometry (LC-MS, or alternatively HPLC-MS) is an ADVANCED ANALYTICAL INSTRUMNTAL technique that combines the physical separation capabilities of LIQUID CHROMATOGRAPHY (or HPLC) with the mass analysis capabilities of MASS SPECTROMETER Liquid chromatography tandem mass spectrometry (LC–MS), has led to major breakthroughs in the field of quantitative bioanalysis since 1990s due to its inherent specificity, sensitivity, and speed. It is now generally accepted as the preferred technique for quantitation of small molecule drugs, metabolites in biological matrices (plasma, blood, serum, urine, and tissue). In the most of the cases the interface used in LC-MS are ionization source. Which provide structural information on the separated sample component. 4

5. Principle of LC-MS LC/MS combines the separating power of High Pressure liquid chromatography (HPLC), with the detection power of mass spectrometry. It is combining the liquid chromatography with the mass spectrometry using several types of interfaces in between,(which remove the solvent from the column effluent and then bring the non- volatile and even thermally liable analytes in gaseous form). In LC-MS we are removing the detector from the column of LC and fitting the column to interface of MS. In the most of the cases the interface used in LC-MS are ionization source. Which provide structural information on the separated sample component. 5

6. INSTRUMENTATION OF LC-MS 6

7. Neutral samples Inlet INLET Form ion charged molecules Solid Liquid vapor DATA SYSTEM Ionization Source Mass Analyzer   Detector Sort or separates ions by M/Z When ions strike Detector it Detect ions 7

8. ION INTERFACE SOURCE: The various interfaces differ among themselves in the means of separating the analytes from the mobile phase and the method are used for ionization of the analyte. The commonly used interfaces are : (I ) Thermo spray ionization interfaces (TSI); (ii) Particle beam ionization interface (PBI); (ii) Atmospheric pressure chemical ionizations interface (APCI); (iv) Electrospray ionization interface (ESI). 8

9. THERMOSPRAY IONIZATION INTERFACE: The effluent from the column [(usually containing an ammonium acetate buffer is passed through a heated tube (300-400°C)] and rapidly expands as a jet spray into a heated vacuum chamber where it forms supersonically a mist of electrically charged droplets. The solvent is rapidly pumped away from the droplets to leave molecular ions such as MH+ and MNH4+ by an ammonia chemical ionization process. The analyte ions with a static charge enter through a skimmer into the mass spectrometer. Thermospray provides soft ionization with very small or no fragmentation. Thus the nonvolatile thermally labile organic compounds undergo gentle ionization and the spectrum produced usually exhibits the protonated molecular ions. 9

10. Thermospray ionization interface: 1. Chromatographic effluent comes in at - a, 2.The transfer line is suddenly heated at - b 3.And the spray is formed under vacuum at- c. 4.At - d the spray goes between a pusher with a positive potential and a negative cone for positive ions. 5. The ions are thus extracted from the spray droplets and accelerated towards the spectrometer. 6. At - e , a high-capacity pump maintains the vacuum in HPLC-MS. 10

11. THERMOSPRAY IONIZATION INTERFACE: 11

12. PARTICLE BEAM INTERFACE The mobile phase from the columns nebulized with helium gas to produce an aerosol in a reduced pressure compartment at 70°C. A cone is provided with a small orifice at the end of the chamber and this leads the mixture of He, solvent vapors, and analyte molecules after being accelerated into a low pressure area (a two stage momentum separator) where it expands supersonically the He and solvent molecules are lighter than the solute molecules and these diffuse out of the stream and are readily pumped away . 12

13. now the relatively heavier solute molecules (having much greater momentum) through a second cone are passed into a yet lower pressure area and then pass straight through two skimmer plates along a narrow tube into the heated ionization compartment. The particle beam interface provides electron ionization (EI) spectra and hence a wide knowledge of such spectra is required for the compound identification. At present the TSI pressure and PBI have largely been replaced by systems usable at atmospheric pressure. 13

14. PARTICLE BEAM INTERFACE 14

15. ATMOSPHERIC PRESSURE CHEMICAL IONIZATION (APCI) In APCI, the LC eluent is sprayed through a heated [250-400] vaporizes at atmospheric pressure. The heat vaporizes liquid which results gas solvent are ionized by corona needle by which electrons are discharged. Thereby chemical reactions takes place and ions passes through a capillary orifice into mass analyzer. 15

16. ATMOSPHERIC PRESSURE CHEMICAL IONIZATION (APCI) 16

17. ELECTRO SPRAY IONIZATION INTERFACE This is most popular one and operates at the atmospheric pressure. This is also a soft ionization technique. The sample solution is sprayed across a 3-6 KV potential from a metal capillary tube into an orifice in the interface. They charged and appears in the form of the highly charged droplets. When the solvent evaporates, the drop shrinks, charge density is increased giving rise to their explosive break up into the still smaller charged droplets. The process is repeated many times so as to produce a spray of ever smaller droplets. 17

18. As a consequence multiply-charged analyte species are produced. Such species are passed through skimmers into the mass spectrometer and the uncharged solvent molecules are pumped away. The characteristic and stability of such an spray depends upon the flow rate and applied potential. Multiple charged sites are created on proteins and peptides and hence this permits determination of a relatively large molecular weights > 3000 Dalton m/z ratio limit of quadruple mass spectrometer , as the m/z ratio decreases by a factor of 5, proteins of 50,000 Dalton or larger can be monitored. 18

19. ELECTRO SPRAY IONIZATION INTERFACE 19

20. MASS ANALYZER They deflects ions down a curved tubes in a magnetic fields based on their kinetic energy determined by the mass, charge and velocity . The magnetic field is scanned to measure different ions. Its task is to separate ions in terms of their mass-to- charge ratio and to direct the beam of focused ions to the detector. The key performance parameters of an analyzer include; separation efficiency m/z measurement precision range of the m / z values measured 20

21. Mass analyzers Quadrupole Ion trap Time of flight Fourier transform ion cyclotron resonance 21

22. QUADRUPOLE In a quadrupole mass analyzer a set of four rods are arranged parallel to the direction . Here a DC current and radio frequency RF is applied to generate oscillating electrostatic field in between the rods . Based on this only m/z is been determined and stable oscillation takes place. And ion travels in quadrupole axis with cork screw type of trajectory 22

23. TIME OF FLIGHT TOF mass analyzer is based on simple idea that the velocities of two ions are created by uniform electromagnetic force applied to all the ions at same time, causing them to accelerate down a flight tube. Lighter ions travels faster and strike the detector first so that the m/z ratio of ions is detected. The time-of-flight mass analyzer (TOF) consists of an ion accelerating grid and a flight tube (about 1 m long), through which the ions travel to the detector. The analyzer separates ions accelerated by an electric field according to their velocity which Depends on their mass and charges 23

24. ION TRAP MASS ANALYZER The ion trap mass analyzer operates by similar principles where it consists of circular ring electrode Plus two end caps that form a chamber. Here AC or DC power along RF potential is applied between the cups and the ring electrode. There the ions entering into the chamber are trapped by electromagnetic fields and they oscillates in concentric trajectories. This process is called resonant ejection One of the most popular ion trap analyzers (IT) is the quadrupole ion trap consisting of a ring-shaped electrode and two electrodes with a spherical cross-section, with the space between them forming a trap. The ion trap analyzer traps ions with a specific mass-to-charge ratio by means of an electric field. 24

25. DETECTORS The detector is used to count the ions emergent from the mass analyzer, and may also amplify the signal generated from each ion. Following are three different kinds of detectors are used in Mass Spectrometry Photographic plates Faraday cup Electron multiplier Channel electron multipliers Scintillation multiplier 25

26. Photo graphic plates It is used as it is capable of higher resolution and speeder than electronic devices. i.e. it can detect ions of all the masses and provide a reverse geometry analyzer. 26

27. FARADAY CUP It is a metal cup into which all the ions are directed and the signal produced is very stable and reproducible. It is used on spectrometers where quantitative data is very important 27

28. Electron multiplier In this the current can be measured so accurately by just one ion strikes the detector can be measured i.e. when an ion strikes the surface of electron multiplier two electron are ejected . This process continues until the end of electro multiplier end is reached and electric current is analyzed and recorded with electron multiplier surface. Equation describe is 2n Where n= no of collisions with electron multiplier surface. 28

29. DATA HANDLING All the mass spectrometers now employ computer control of same functions and also use a computerised display and output. The amount of data generated even by a fairly modest mass spectrometer is very large indeed, a single run may store data for up to 100 fragments from each type of molecule and if, LCMS analyses is being performed, a complete mass spectrum is generated and stored every sec for up to 90 min 29

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31. APPLICATION: Determination of impurities in insulin-like growth factor with electrospray- mass spectrometry (ES-MS)ES-MS provides an excellent means for quality control of recombinant proteins, some of which are now used as drugs, e g. human insulin, interferons, erythropoietin and tissue plasminogen activating factor. variations in protein structure, such as degree of glycosylation, or in the terminal amino acids of the protein can be seen quite clearly An example of how ES-MS can be used to determine minor impurities in a recombinant protein is shown in Figure 9.38, where some small additional ions in the mass spectrum of recombinant insulin-like growth factor (IGF) can be seen. 31

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33. Characterization of a degradant of famotidine Tablets of famotidine, an anti-ulcer compound, were subjected to stress conditioning pack. ' Figure 9.41 indicates the profile obtained from analysis of an extract from the stressed tablets by LC atmospheric pressure chemical ionization mass spectrometry (APCIMS). The structure of famotidine is shown in Figure 942.The mass spectra obtained for famotidine and its degradant by APCIMS and APCIMS-MS are shown in Figures 9.43 and 9.44 The degradant had a MW 12 amu higher than that of famotidine. The fragment at m/z 189 was common to both spectra, indicating that the two molecules were similar in structure. 33

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37. Profiling impurities and degradants in butorphanol tartrate HPLC coupled to an ES-MS was used to elucidate the structure of a number of degradants in butorphanol following its storage in aqueous solution Figure 9.45shows the LC-MS profile of the degradants which were detected in butorphanol. 37

38. IDENTIFICATION OF BILE ACID METABOLITES: The use of in-vitro incubation of bile acid deoxycholic with rat liver microsomes to stimulate metabolism of drug candidate. These precursor ions are were automatically fragmented and full scan ion spectra is collected. The first graph shows the base peak chromatogram .(A) The second shows minor metabolite which is eluted at 9.41min.(B) Third graph show product spectrum from the ion at m/z 407 which confirms identity .(C) 38

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40. 4. Drug Development Dete r m i nat i o n o f dr u g s a nd m e ta b o l ite s i n p la s m a o r other biological fluids. 5.FOOD SCIENCE- melanin dosing. pesticide residues. 6. Life science. 7. Clinical science. 8. Forensic science. 40

41. REFERENCE David G. Watson, Pharmaceutical analysis, second edition, page no -207 P. C. Kamboj. Pharmaceutical analysis, page no –212 Beckett –Practical pharmaceutical chemistry, fourth edition. Part two Vogel’s - text book of quantitative chemical analysis. J. Mendham, R, C, Denney. 41

42. THANK YOU 42