LC-MS

Liquid chromatography-mass spectrometry

1. Liquid Chromatography-mass Spectrometry(LC-MS) GPRCP S. Lakshmi narayana reddy M.PHARMACY Pharmaceutical analysis and quality assurance.

2. CONTENTS Hyphenated technique Introduction to LC-MS High performance liquid chromatography Mass spectrometry Instrumentation of LC-MS Sample preparation Ion sources Mass analyzers Applications 1

3. Hyphenated Technique The term “hyphenation” was first adapted by Hirschfeld in 1980. The technique developed from the coupling of a separation technique and an on-line spectroscopic detection technology is known as   hyphenated technique. What Is Hyphenated Technique ? Advantages of hyphenated techniques: 1. Fast and accurate analysis. 2. Higher degree of automation. 3. Higher sample throughput. 4. Better reproducibility. 5. Reduction of contamination due to its closed syste m. 6. Separation and quantification achieved at same time. List of Hyphenated Techniques : 1.GC-MS 2. LC-MS 3. LC-NMR 4. EC-MS 5. CE-MS 6. GC-IR 7. LC-MS-MS 8. GC-MS-MS 9. GC-GC-MS 10. GC-NMR 2

4. INTRODUCTION TO LC-MS Liquid Chromatography/Mass Spectrometry (LC/MS) is a powerful analytical technique that combines the resolving power of liquid chromatography with the detection specificity of mass spectrometry . Liquid chromatography (LC) separates the sample components and then introduces them to the mass spectrometer (MS). The MS creates and detects charged ions. The LC/MS data may be used to provide information about the molecular weight, structure, identity and quantity of specific sample components. 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. 3 GPRCP

5. High Pressure Liquid Chromatography (HPLC) Liquid chromatography generally utilizes very small particles packed and operating at relatively high pressure, and is referred to as  high performance liquid chromatography  (HPLC). Modern LC-MS methods use HPLC instrumentation, essentially exclusively, for sample introduction. In HPLC, the sample is forced by a liquid at high pressure (the  mobile phase ) through a column that is packed with a  stationary phase  generally composed of irregularly or spherically shaped particles chosen or derivatized to accomplish particular types of separations. RP-LC is most often used as the means to introduce samples into the MS, in LC-MS instrumentation. 4

6. Mass Spectrometry (MS) Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of charged particles. MS works by ionizing chemical compounds to generate charged molecules or molecule fragments and measuring their mass-to-charge ratios. In a typical MS procedure, a sample is loaded onto the MS instrument and undergoes vaporization. The components of the sample are ionized by one of a variety of methods (e.g., by impacting them with an electron beam), which results in the formation of charged particles (ions). The ions are separated according to their mass-to-charge ratio in an analyzer by electromagnetic fields. 5

7. Instrumentation of LC-MS 1)HPLC Constitutes The Lc Part: a) Solvent System(mobile Phase) b)Pumps c)Mixer d)Injector e)Column 2) Mass spectrometer A)Ion Sources i)Electrospray ionization. ii)Atmospheric Pressure Chemical Ionization. iii) Atmospheric pressure photoionization 6

8. B)Mass Analyzers 1) Quadrupole 2) Time-of-flight 3) Ion trap 4) Fourier transform-ion cyclotron resonance (FT-ICR) 7

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10. Sample preparation : Sample preparation generally consists of concentrating the analyte and removing compounds that can cause background ions or suppress ionization. Examples of sample preparation include: • On-column concentration to increase analyte concentration • Desalting to reduce the sodium and potassium adduct formation that commonly occurs in electrospray • Filtration to separate a low molecular-weight drug from proteins in plasma, milk, or tissue 9

11. Ion Sources Ion Sources : Electrospray ionization (ESI) Atmospheric pressure chemical ionization (APCI) Atmospheric pressure photoionization (APPI) Electrospray ionization (ESI) : Process API-ES is a process of ionization followed by evaporation. It occurs in three basic steps: nebulization and charging; desolvation and; ion evaporation. 10

12. Nebulization: The HPLC effluent is pumped through a nebulizing needle which is at ground potential. The spray goes through a semi-cylindrical electrode which is at a high potential. The potential difference between the needle and the electrode produces a strong electrical field. This field charges the surface of the liquid and forms a spray of charged droplets. There is a concentric flow of gas which assists in the nebulization process. Desolvation : The charged droplets are attracted toward the capillary sampling orifice. There is counterflow of heated nitrogen drying gas which shrinks the droplets and carries away the uncharged material. 11

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14. Ionization : As the droplets shrink, they approach a point where the electrostatic (coulombic) forces exceed the cohesive forces. This process continues until the analyte ions are ultimately desorbed into the gas phase. These gas-phase ions pass through the capillary sampling orifice into the low pressure region of the ion source and the mass analyzer. Atmospheric Pressure Chemical Ionization : Process : APCI, a process of evaporation followed by ionization, is complementary to API-ES. 13

15. Nebulization and Desolvation : APCI nebulization is similar to that in API-ES. However, APCI nebulization occurs in a hot (typically 250°C–400°C) vaporizer chamber. The heat rapidly evaporates the spray droplets, resulting in gas-phase HPLC solvent and analyte molecules. Ionization: The gas-phase solvent molecules are ionized by the discharge from a corona needle. In APCI there is a charge transfer from the ionized solvent reagent ions to the analyte molecules in a way that is similar to chemical ionization in GC/MS. These analyte ions then are transported through the ion optics to the filter and detector. 14

16. APCI - ION SOURCE 15

17. Atmospheric pressure photoionization : Atmospheric pressure photoionization (APPI) for LC/MS is a relatively new technique. As in APCI, a vaporizer converts the LC eluent to the gas phase. A discharge lamp generates photons in a narrow range of ionization energies. The range of energies is carefully chosen to ionize as many analyte molecules as possible while minimizing the ionization of solvent molecules. The resulting ions pass through a capillary sampling orifice into the mass analyzer. 16

18. APPI- ION SOURCE 17

19. Applications Of Ion Source 18

20. Mass Analyzers Mass Analyzers : Although in theory any type of mass analyzer could be used for LC/MS, four types: • Quadrupole • Time-of-flight • Ion trap • Fourier transform-ion cyclotron resonance (FT-ICR or FT-MS) 19

21. Quadrupole : A quadrupole mass analyzer consists of four parallel rods arranged in a square. The analyte ions are directed down the center of the square. Voltages applied to the rods generate electromagnetic fields. These fields determine which mass-to-charge ratio of ions can pass through the filter at a given time. Quadrupoles tend to be the simplest and least expensive mass analyzers. Quadrupole mass analyzers can operate in two modes: • Scanning (scan) mode. • Selected ion monitoring (SIM) mode. 20

22. In scan mode, the mass analyzer monitors a range of mass-to-charge ratios. In SIM mode, the mass analyzer monitors only a few mass-to-charge ratios. SIM mode is significantly more sensitive than scan mode but provides information about fewer ions. Scan mode is typically used for qualitative analyses or for quantitation when all analyte masses are not known in advance. SIM mode is used for quantitation and monitoring of target compounds. 21

23. 22 Quadrupole Mass Analyzer

24. 23 Time-of-flight : In a time-of-flight (TOF) mass analyzer, a uniform electromagnetic force is applied to all ions at the same time, causing them to accelerate down a flight tube. Lighter ions travel faster and arrive at the detector first, so the mass-to-charge ratios of the ions are determined by their arrival times. Time-of-flight mass analyzers have a wide mass range and can be very accurate in their mass measurements .

25. Time of flight mass analyzer 24

26. Ion trap : An ion trap mass analyzer consists of a circular ring electrode plus two end caps that together form a chamber. Ions entering the chamber are “trapped” there by electromagnetic fields. Another field can be applied to selectively eject ions from the trap. Ion traps have the advantage of being able to perform multiple stages of mass spectrometry without additional mass analyzers. 25

27. Ion trap mass analyzer 26

28. Fourier transform-ion cyclotron resonance (FT-ICR) : An FT-ICR mass analyzer (also called FT-MS) is another type of trapping analyzer. Ions entering a chamber are trapped in circular orbits by powerful electrical and magnetic fields. When excited by a radio-frequency (RF) electrical field, the ions generate a time dependent current. This current is converted by Fourier transform into orbital frequencies of the ions which correspond to their mass-to-charge ratios. 27

29. Collision-Induced Dissociation and Multiple-Stage MS : The atmospheric pressure ionization techniques discussed are all relatively “soft” techniques. They generate primarily: • Molecular ions M+ or M– • Protonated molecules [M + H]+ • Simple adduct ions [M + Na]+ • Ions representing simple losses such as the loss of a water [M + H – H2O]+ 28

30. The resulting molecular weight information is very valuable, but complementary structural information is often needed. To obtain structural information, analyte ions are fragmented by colliding them with neutral molecules in a process known as collision induced dissociation (CID) or collisionally activated dissociation (CAD). Voltages are applied to the analyte ions to add energy to the collisions and create more fragmentation. 29

31. Mass spectrum of sulfamethazine acquired without collision-induced dissociation exhibits little fragmentation Mass spectrum of sulfamethazine acquired with collision-induced dissociation exhibits more fragmentation and thus more structural information 30

32. Detectors : once the ions have passed the mass analyser they have to be detected and transformed into a usable signal. The detector is an important element generating secondary electrons, which are further amplified, or by inducing a current (generated by moving charges). Ion detector fall into two main classes: Point detectors: Ions are not spatially resolved and sequentially impinge upon a detector situated at a single point within the spectrometer geometry. Array detectors: Ions are spatially resolved and all ions arrive simultaneously (or near simultaneously) and are recorded along a plane using a bank of detectors. 31

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34. Applications Molecular Weight Determination : One fundamental application of LC/MS is the determination of molecular weights. This information is key to determining identity. Differentiation of similar octapeptides : Figure 1 shows the spectra of two peptides whose mass-to-charge ratios differ by only 1 m/z. The only difference in the sequence is at the C-terminus where one peptide has threonine and the other has threonine amide. The smaller fragments are identical in the two spectra, indicating that large portions of the two peptides are very similar. The larger fragments contain the differentiating peptides. 33

35. Mass spectra differentiating two very similar octapeptides 34

36. Determining the molecular weight of green fluorescent protein : Green fluorescent protein (GFP) is a 27,000- Dalton protein with 238 amino acids. It emits a green light when excited by ultraviolet light. During electrospray ionization, GFP acquires multiple charges. This allows it to be analyzed by a mass spectrometer with a relatively limited mass (mass-to-charge) range. Mass deconvolution is then used to determine the molecular weight of the protein . Molecular weight determination of green fluorescent protein by electrospray LC/MS 35

37. Pharmaceutical Applications Rapid chromatography of benzodiazepines The information available in a mass spectrum allows some compounds to be separated even though they are chromatographically unresolved. In this example, a series of benzodiazepines was analyzed using both UV and MS detectors. The UV trace could not be used for quantitation, but the extracted ion chromatograms from the MS could be used. MS identification and quantification of individual benzodiazepines from an incompletely resolved mixture 36

38. Clinical Applications High Sensitivity Detection of Trimipramine and Thioridazine : For some compounds, MS provides more sensitive detection. Trimipramine is a tricyclic antidepressant with sedative properties. Thioridazine is a tranquilizer. Figure 2 shows these compounds in a urine extract at a level that could not be detected by UV. To get the maximum sensitivity, the analysis was done by selected ion monitoring. Figure 2 shows trimipramine and thioridazine in urine extract 37

39. Food Applications : Aflatoxins are toxic metabolites produced by certain fungi in foods. Figure 3 shows the total ion chromatogram for four aflatoxins ; each could be uniquely identified by their mass spectra. Figure 3 shows Aflatoxins by API-ES 38

40. Environmental Applications Carbamates by APCI-LC/MS Carbamates are a class of pesticides usually analyzed by post-column derivativation and fluorescence detection. The chromatogram in Figure 4 is that of a tomato extract that has been spiked with 11 carbamates. Using APCI, these compounds are detected without derivitization. The extracted ion chromatogram displays the peaks for the individual compounds. Detection of Phenylurea Herbicides Many of the phenylurea herbicides are very similar and difficult to distinguish with a UV detector (Figure 5).Monuron and diuron have one ring and differ by a single chlorine. Chloroxuron has two chlorines and a second benzene ring attached to the first by an oxygen. 39

41. The UV-Vis spectra are similar for diuron and monuron, but different for chloroxuron. Each of these compounds has a distinct mass spectrum. The standards were run with an API-electrospray LC/MS. Contd ”” Carbamates by APCI(Fig. 4) Phenylurea herbicides by API-ES (Fig. 5) 40

42. REFERENCES : https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry . http://www.agilent.com/cs/library/support/documents/a05296.pdf. http://www.ecs.umass.edu/eve/background/methods/chemical/Openlit/Chromacademy%20LCMS%20Intro.pdf. http://ccc.chem.pitt.edu/wipf/Agilent%20LC-MS%20primer.pdf. http://www.shimadzu.com/about/magazine/i7rr0a0000008xh6-att/28_2.pdf.

43.   W. Paul & H. Steinwedel; Zeitschrift für Naturforschung, 8A; 1953, p448. SPECTROMETRIC IDENTIFICATION OF ORGANIC COMPOUNDS BY ROBERT M. SILVERSTEIN AND FRANCIS X. WEBSTER page no.2-6

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