LC-MS INSTURMENTATION & APPLICATIONS

A full view of LC-MS; history,mass spectrum,mass spectrometer,HPLC,interface of LC-MS,mass spec instrumentation,applications.

1. 1 LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY (LC-MS) Presented by S.NAVEEN JAIN 11AB1R0078 UNDER THE ESTEEMED GUIDANCE OF Mr. Ch. DEVADASU M.Pharm Assistant professor Department of PA & QA VIGNAN PHARMACY COLLEGE (Approved by AICTE, PCI & Affiliated to JNTU-K) VADLAMUDI, 522213.

2. CONTENTS HISTORY INTRODUCTION THEORY MASS SPECTRUM LC-MS ION INTERFACE SOURCES IONIZATION SOURCES MASS ANALYZERS DETECTORS APPLICATIONS CONCLUSION REFERENCES VIGNAN PHARMACY COLLEGE 2

3. A Brief History of Mass Spectrometry In 1897, Modern mass spectrometry (MS) is credited to the cathode-ray-tube experiments of J.J. Thomson of Manchester, England. In 1953, Wolfgang Paul’s invention of the quadrupole and quadrupole ion trap earned him the Nobel Prize in physics. In 1968, Malcolm Dole developed electrospray ionization (ESI). I n 1 974, Atmospheric pressure chemical ionization (APCI) was developed by Horning. In 1983, Vestal and Blakely’s work with heating a liquid stream known as thermospray was developed. VIGNAN PHARMACY COLLEGE 3

4. The concept of mass spectrometry was first put forth by Sir J.J Thomson, English Physicist Who discovered the electron in 1887. He got 1906 Nobel Laureate in Physics. DEMPSTER Sir J.J Thomson 4 VIGNAN PHARMACY COLLEGE

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6. What does a mass spectrometer do? Mass –spec or simply MS is a super important technique Mass spec is easy technique to give you Molecular weight (from molecular ion (M + ) You can get Molecular formula (Elements present) . Nearly ALL ELEMENTS in the periodic table can be determined by mass spectrometry . MS is incredibly valuable in getting structure (from fragments) of Bio molecules such as peptides and proteins and also natural products and also organic structures. It can give information about chemical structures. 6 VIGNAN PHARMACY COLLEGE

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8. Inlet Ion Source Mass Filter Detector Data System High Vacuum System Sample Plate Target HPLC GC Solids probe MALDI ESI Ion Spray FAB LSIMS EI/CI TOF Quadrupole Ion Trap Mag. Sector FTMS Electron Multiplier, Faraday cup PC’s UNIX Mac Basic components in mass spectrometer

9. Mass Sor Illustration of the basic components of a mass spectrometry system. Solid • Liquid • Vapor Ionization Source Mass Analyzer Detector Inlet Form ions charged molecules selected ions Data System Sort or separates ions by M/Z When ions strike Detector it Detect ions The inlet transfers the sample into the vacuum of the mass spectrometer. In the source region, neutral sample molecules are ionized and then accelerated into the mass analyzer . The mass analyzer is the heart of the mass spectrometer. This section separates ions, either in space or in time, according to their mass to charge ratio. After the ions are separated, they are detected and the signal is transferred to a data system for analysis.. Neutral sample molecules 9 VIGNAN PHARMACY COLLEGE

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11. VIGNAN PHARMACY COLLEGE 11 The mass spectrometer is an instrument which help in separating the individual atoms or molecules because of difference in their masses. Consider a molecule M, Which is bombarded with a beam of electrons M + e - M +. +2e - where, M +. is molecular ion or radical ion 2e - is electron Now voltage “v” is applied in an electric field then ions are accelerated. In this condition the energy given to each particle is zV and this is equal to kinetic energy which is equal to 1/2mv 2 . THEORY

12. i. e. potential energy=kinetic energy zV = 1/2mv 2 2zV = mv 2 2zV/m= v 2 = v Where V = Velocity of particle m = mass z = charge of an electron V = Acceleration voltage All the particles posses some energy zV with some kinetic energy 1/2mv 2 , but m value changes from molecule to molecule with respect velocity ‘v’ also changes. i.e. ½ mv=zV VIGNAN PHARMACY COLLEGE 12

13. When all charged particles have been accelerated by an applied voltage, they enter into a magnetic field “H”. Then attractive force is HzV. And balancing force of particle is mv 2 /r. Centripetal = Centrifugal HzV=mv 2 /r Hz=mv/r From the above equation v= Hz=m / r By squaring on both sides H 2 z 2 = m 2 (2zV/m) / r 2 H 2 z = 2v m/ r 2 m/z = H 2 r 2 / 2v VIGNAN PHARMACY COLLEGE 13

14. MASS SPECTRUM The mass spectrum is the plot of mass to charge ratio of positively charged ions against their relative abundance. T he m/z ratio are taken along the abscissa, while relative abundance is taken on ordinate. BASE PEAK: Th e most intense peak in the mass spectrum is called the base peak. Base peak is the highest peak it is assigned a relative intensity of 100%. VIGNAN PHARMACY COLLEGE 14

15. MOLECUL AR ION PEAK: The ion formed from a molecule by removal of one electron of lowest ionization potential is known as molecular ion. The molecular ion is detected as mass to charge ratio that corresponds to molecular weight of mole cule. T he molecular ion peak gives the molecular weight of compound . T he molecular ion peak is highest mass number except isotope peak. Molecular ion peak Base peak Fragment ions VIGNAN PHARMACY COLLEGE 15

16. FRAGMENT IONS: T he ions produced from the molecular ion by cleavage of bonds are called fragment ions T hey have lower masses and used as building blocks to reconstruct the molecular structure. Fragmentation of molecular ion cleavage bond occurs in heterolytic and homolytic cleavage. METASTABLE IONS: M ass spectrum of molecule shows sharp peaks at m/z integrals. But some show diffuse, broad low intensity peaks at non integral m/z values these are called metastable ions m 1 + m 2 ++ neutral fragment VIGNAN PHARMACY COLLEGE 16

17. If in the reaction m 1 + --------->m 2 ++ takes place in source then the daughter ion may be m 2 + . But m 1 + ----->m 2 ++ if occurs after the source and before arrival at collector at lower mass than m 2+ and is said to be metastable ion m*.The peak (m*) due to such fragmentation therefore occurs at lower mass than m 2+ and generally broad. The relation between the m* with that of m 1 + & m 2+ can be written as m*=(mz+)/m 1 VIGNAN PHARMACY COLLEGE 17

18. Relative abundance m/z values 110 108 81 79 29 –C 2 H 5 M +2 M + CH 3 CH 2 Br VIGNAN PHARMACY COLLEGE 18

19. Liquid chromatography–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 VIGNAN PHARMACY COLLEGE 19

20. It is the combination of liquid chromatography and the mass spectrometry. 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. LC-MS 20 VIGNAN PHARMACY COLLEGE

21. HPLC is a method for separating a complex mixture in to its individual components. High sensitivity of mass spectrometry provides the information for identification of compounds or structural elucidation of compounds. Combination of these two techniques is LC-MS VIGNAN PHARMACY COLLEGE 21

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23. DIRECT CHEMICAL IONIZATION: The simplest way to introduce HPLC effluent into mass spectrometer is to split the flow. Chemical ionization is most suitable in this technique because under CI pressure conditions, solvent rates as high as 10 micro lit / min can be tolerated. This permits10-20 micro lit/ min (1-2%) eluate from the HPLC to that of the ion source. VIGNAN PHARMACY COLLEGE 23

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25. MOVING WIRE OR BELT INTERFACE: The moving wire or belt interface consists of an auxiliary vacuum chamber through which a continuous train carries the column eluate, evaporates the solvent and subsequently vaporizes the solute. The moving interface was developed by Mc Fadden et al 84,85 which can transfer upto 30-40% of solute from HPLC to ion source. The residual solvent helps to maintain vaccum in the MS. The sample is finally conducted into the ion source, where it vaporizes. VIGNAN PHARMACY COLLEGE 25

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27. THERMOSPRAY: The eluent from the column is vapourised and a portion of vapour is transferred to the mass spectrometer and rest of the vapour is pumped to waste. As a result a supersonic jet vapour, containing a mist of particles and solvent droplet is created. There vaporization takes place in presence of an electrolyte the LC buffer, the droplets are charged. And finally they enter into the ionization chamber. VIGNAN PHARMACY COLLEGE 27

28. VIGNAN PHARMACY COLLEGE 28 IONIZATION SOURCES

29. Electron Spray Ionization: The LC eluent is sprayed into chambers in presence of strong electrostatic field and heated drying gas . Large molecules even acquires more than one charge this process mathematically called deconvolution. The heated gas causes solvent in the droplet evaporation. The repulsive force b/w ions with like charge exceeds cohesive force and ions are eject into gas phase and passed into the analyser. VIGNAN PHARMACY COLLEGE 29

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31. Atmospheric Pressure Chemical Ionization[APCI]: In APCI, the LC eluent is sprayed through a heated [250-400] vapourises at atmospheric pressure. The heat vapourises 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 analyser. VIGNAN PHARMACY COLLEGE 31

32. Atmospheric Pressure Photo Ionization[APPI]: Atmospheric pressure photo ionization is relatively never technique. Here a discharge UV lamp is placed which generates photons in a narrow range of ionization energies. It shows its ionization for highly non polar compounds and low flow rates[<100m/min] VIGNAN PHARMACY COLLEGE 32

33. VIGNAN PHARMACY COLLEGE 33 Mass Analyzers 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.

34. VIGNAN PHARMACY COLLEGE 34 Quadrupole: In a quadrupole mass analyser 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 .

35. VIGNAN PHARMACY COLLEGE 35 TIME OF FLIGHT: TOF mass analyser 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.

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37. The ion trap mass analyser 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. VIGNAN PHARMACY COLLEGE 37 ION TRAP MASS ANALYSER:

38. VIGNAN PHARMACY COLLEGE 38 ION TRAP MASS ANALYSER:

39. VIGNAN PHARMACY COLLEGE 39 FOURIER ION TRASNFORM ION CYCLOTRON RESONANCE: In this ions entering are trapped in circular well defined orbits for extended periods by electrical and magnetic fields. These are excited by radio frequency RF and generates the current. This current is converted to Fourier transform into orbital frequencies. The angular frequency of motion is called cyclotron frequency .

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41. P hoto 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. VIGNAN PHARMACY COLLEGE 41

42. 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 VIGNAN PHARMACY COLLEGE 42

43. Electron multipliers: 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 2 n Where n= no of collisions with electron multiplier surface. VIGNAN PHARMACY COLLEGE 43

44. 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 upto 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 upto 90 min VIGNAN PHARMACY COLLEGE 44

45. APPLICATIONS MOLECULAR WEIGHT DETERMINATION DIFFERENTIATION OF SIMILAR OCTAPEPTIDES: The spectra of octa peptides whose m/z ratio differ only by 1m/z. The only difference in sequence is at C-terminus that is one having threonine and other having threonine amide. VIGNAN PHARMACY COLLEGE 45

46. DETERMINATION OF MOLECULAR WEIGHT OF GREEN FLORESCENT PROTEIN: GRF is 27000 Dalton protein with 238 amino acids. During electron spray ionization GFP acquires multiple charges. This allows to be analyzed by mass spectrometer by m/z range. Here the mass deconvolution is then used to determine the weight of the protein GFP. VIGNAN PHARMACY COLLEGE 46

47. STRUCTURAL DETERMINATION: LC/MS is also applied for the information about the molecular structure. this can be in addition of mol wt if the identity of the compound is already known. STRUCUTRAL DETERMINATION OF GINSENOSIDES USING LC/MS ANALYSIS: GINSENG root have dozens of biologically active saponins called gensinosides. In mass spectrometer, ion trap mass analyser permits multiplier stages of precursor ion isolation and fragmentation The most prominent feature is sodium adduct ion corresponding to cleavage of single bonded glycoside. Subsequent isolation and fragmentation at m/z 789.7 yields two products. VIGNAN PHARMACY COLLEGE 47

48. 1.A more abundant ion at m/z 365.1 losses oligosaccharide ion 2. Loss of less abundant ion at m/z 627.5 i.e. de-oxyhexose sugar VIGNAN PHARMACY COLLEGE 48

49. PHARMACEUTICAL APPLICATIONS RAPID CHROMATOGRAPH OF BENZODIAZEPINES: It allows compounds to be separated even they are chromatographically unresolved. A series of benzodiazepines are analyzed using both UV and MS detectors. In this chlorine cl - has a characteristic abundance of 2 most abundant isotopes. By which TRAIZOLAM spectrum shows a two cl - ions and DIAZEPAM shows only one cl - ion . VIGNAN PHARMACY COLLEGE 49

50. 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 . The second shows minor metabolite which is eluted at 9.41min. Third graph show product spectrum from the ion at m/z 407 which confirms identity . VIGNAN PHARMACY COLLEGE 50

51. Graph-1 Graph-2 Graph-3 VIGNAN PHARMACY COLLEGE 51

52. CLINICAL APPLICATIONS HIGH SENSITIVITY DETECTION OF TRIMIPRAMINE AND THIORIDAZINE: Triminpramine is a tricyclic anti depressant , Thioridazine is a tranquilizer when these compounds are detected by UV there is no extract level. For this single quadrupole of LCMS was been used for analysis by ion monitoring. VIGNAN PHARMACY COLLEGE 52

53. GENERAL CONSIDERATION: Immunoassays can be detected as it is more sensitive and very easily allocated. Similarly many drug and other small molecules assay are readily performed by immunoassay, LC/MS, GLC etc. Lc/ms is best suited for either problems i.e. those which have tedious specimen preparation protocols in which more than one compound must be measured simultaneously. BIOCHEMICAL GENETICS: One of the seminal applications of Lc/ms was multiple analyte screening for inborn errors of metabolism. Few of inherited metabolic disorders detected by newborn screening with Lc/ms. VIGNAN PHARMACY COLLEGE 53

54. AMINO ACIDOPATHIES Phenyl ketoxuria Tyrosinemia Non ketotic hyperglycemia Argininemia ORGANIC ACIDEMIAS Glutaric academia type I Propionic academia Methyl malonic academia DEFECTS OF FATTY ACID OXIDATION Short chain acyl-CoA dehydrogenase deficiency Ethylmalonic academia Carnitine transport defect VIGNAN PHARMACY COLLEGE 54

55. THERAPEUTIC DRUG MONITORING: A direct injection of HPLC/MS method has been developed for the rapid identification and quantitation of seven tricyclic antidepressants in human plasma can be done. A sensitive and specific method is determined for phenprocoumon , warfarin in human plasma by Lc/ms has been developed for monitoring of anticoagulant therapy. VIGNAN PHARMACY COLLEGE 55

56. FOOD APPLICATIONS INDENTIFICATION OF ALFATOXINS IN FOOD: Aflatoxins are fungi produced in food. By total in chromatogram we can detect 4 different aflatoxins. Even though they are structurally very similar to each other but can be uniquely identified by LC/ms. VIGNAN PHARMACY COLLEGE 56

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58. PHARMACOLOGY: Since most drugs have a core chemical structure, which is retained by their metabolites, the determination of drug metabolites in pharmacology is now dominated by Lc/ms. The core structure produces charged or neutral fragment. The charged can be detected using ion scanning strategy. Unchanged can be recognized by neutral loss scanning. VIGNAN PHARMACY COLLEGE 58

59. TOXICOLOGY An even simpler strategy solid phase extraction and direct flow injection with no HPLC has been shown to be viable using clinical specimens. Determination of designer drugs 3,4-methylene dioxymethamphetamine[MDMA], 3,4-methylenedioxyethyl amphetamine[MDEA] and 3,4-methylenedioxy amphetamine[MDA] by Lc/ms has been showing using fluorescent detection. The method was found suitable for determination for whole blood, serum, vitreous humor and urine. VIGNAN PHARMACY COLLEGE 59

60. IN DRUG DISCOVERY Drug discovery involves a number of phases, including target identification, lead identification, small molecule optimization and pre-clinical and clinical development. Target identification has been speeded up as result of genomics but the measurement of gene transcription through detection of RNA does not necessarily indicate proteins produced are, which are translated by glycosylation or phosphorycation. With advances in Lc/ms, identification of translated proteins is possible. VIGNAN PHARMACY COLLEGE 60

61. LC-MS has proved to be an extremely sensitive and specific technique for the analysis of pharmaceuticals. It plays important role in the studies of drug metabolism, discovery of new drug candidates and the analysis, identification and characterisation of impurities and degradants in drug substances and products. VIGNAN PHARMACY COLLEGE 61 Conclusion

62. VIGNAN PHARMACY COLLEGE 62 Pharmaceutical Analysis - A Text book for pharmacy students & chemical applications By- DAVID G. WATSON. Page no: 206-213. Instrumental analysis By- SKOOG, HOLLER,CROUCH . Page no- 607-631. Spectroscopy By- PAVIA, LAMPMAN,KRIZ, VYVYAM Page no- 401-417. REFERENCES

63. Elementary Organic Spectroscopy principles & chemical applications . By- Y.R.SHARMA Page No- 280-289 Instrumental Methods Of Chemical Analysis . By- B.K SHARMA Page No- C286-C291 Instrumental Methods Of Chemical Analysis By-GURDEEP R.CHATWAL,SHAM K.ANAND Page No 2.272-2.286 VIGNAN PHARMACY COLLEGE 63

64. VIGNAN PHARMACY COLLEGE 64 Organic Spectroscopy Principles & Applications. By-JAG MOHAN Page no 443-446 Vogel’s Text Book Of Quantitative Chemical Analysis. By- J.MENDHAN, R C DENNY, J.D. BARNES, M.THOMAS, B. SIVA SANKAR . Page no 720-741 AGILENT TECHNOLOGIES. Page no 6-30

65. ACKNOWLEDGEMENT I sincerely thank my guide Ch. DEVADASU sir for giving support. I thank our principal Dr. P. SRINIVASA BABU sir & seminar committee for giving me this opportunity. VIGNAN PHARMACY COLLEGE 65