LC-MS

1. ADICHUNCHANAGIRI UNIVERSITY Sri Adichunchanagiri college of Pharmacy LC-MS Presented By, Niranjan Kumar C V M Pharm 2 nd sem Presented To, Dr. Yunus Pasha Professor and Head Department of Pharmaceutical Chemistry

2. INTRODUCTION Why Liquid Chromatography? Analysis of labile analytes Analysis of more polar compounds without derivatization. Analysis of significantly higher masses. Separation of the mixed component before to the MS detector.

3. Mass spectroscopy It measures the masses of individual molecules, fragments of molecules and atoms. It provides ultrahigh detection sensitivity The mass spectrum of each compound is unique and can be used as chemical “fingerprint” together with its retention time to characterize the compounds.

4. LIQUID CHROMATOGRAPHY COUPLED WITH MASS SPECTROSCOPY LC with detectors like Refractive index, electrochemical, fluorescence, and ultraviolet-visible (UV-Vis) detectors generate two dimensional data; that is, data representing signal strength as a function of time When coupled with MS In addition to signal strength, they generate mass spectral data that can provide valuable information about the molecular weight, structure, identity, quantity, and purity of a sample.

5. Historical Perspective Goldstein – 1886 – Existence of positively charged particles Wein – 1898 – Positively charged ions can be deflected in electrical and magnetic fields • J.J. Thomson – 1913 – Demonstrated isotopes of Neon – “Father of mass spectrometry” • First GC-MS interface - 1960’s

6. First LC-MS interface developed - 1969 – 1uL/min flow into an EI source • Transport devices applied to LC/MS - 1970’s – Loss of volatile components – Thermally-reactive compound losses • Thermospray (TSI) gains popularity - 1983 – Mobile phases consist primarily of an aqueous buffer

7. Pr inciple LC/MS is a hyphenated technique combining the separation power of HPLC, with the detection power of mass spectrometry. It uses an interface that will eliminate the solvent and generate gas phase ions, transferred to the optics of the mass spectrometry(or). It is an analytical chemical technique that combines the physical separation capability of liquid chromatography (HPLC) with the mass analysis capabilities of mass spectrometry. LC MS is a powerful technique used for many application which has very high sensitivity and specificities.

8. It is application is oriented towards the specific detection and potential identification of chemical in the presence of other chemicals(in a complex mixture). • 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 LCMS are ionization source.

9. Instrumentation Detector/ Liquid Chromatography Ionization Mass Analyzer Detector/ Data Collection

10. Mass Spectrometer Liquid Chromatograph Rough pump UV Detector Sampler Injection port Column Solvents Ion source Pumps Interface Computer

11. Mobile phase The mobile phase is the solvent that moves the solute through out column. General requirements: (1) Low cost, UV transparency, high purity. (2) Low viscosity, low toxicity, non flammability. (3) Non corrosive to LC system component. Solvent strength and selectivity: It is the ability of solvent to elute solutes from a column.

12. COLUMN The use of di-functional or tri-functional silanes to create bonded groups with two or three attachment points leading to phases with higher stability in low or higher pH and lower bleed for LCMS Most widely used columns for LC-MS are: (1) fast LC column. the use of short column. (15-50mm) (2) Micro LC column. the use of large column. ( 20-150mm).

13. Sample preparation • Sample preparation generally consists of concentrating the analyte and removing compounds that can cause background ion or suppress ionization. • Example of sample preparation include: 1. On Column concentration -to increase analyte concentration. 2. Desalting - to reduce the sodium and potassium adduct formation that commonly occurs in electro spray. 3. Filtration- to separate a low molecular-weight drug from proteins in plasma, milk, or tissue.

14. INTERFACES LC-MS systems include a device for introducing samples (such as an HPLC )an interface for connecting such device, an ion source that ionizes samples, an electrostatic lens that efficiently introduces the generated ions, a mass analyzer unit that separates ions based on their mass-to-charge (m/z) ratio, and a detector unit that detects the separated ions. In an LC-MS system, however, if the LC unit is simply connected directly to the MS unit, the liquid mobile phase would vaporize, resulting in large amounts of gas being introduced into the MS unit. This would decrease the vacuum level and prevent the target ions from reaching the detector. So interfaces are to be used

15. Electro Spray Ionization (ESI) ESI draws sample solutions to the tip of a capillary tube, where it applies a high voltage of about 3 to 5 kV. • A nebulizer gas flows from outside the capillary to spray the sample. This creates a fine mist of charged droplets with the same polarity as the applied voltage. • As these charged particles move, the solvent continues to evaporate, thereby increasing the electric field on the droplet surface. When the mutual repulsive force of the charges exceeds the liquid surface tension, then fission occurs.

16. As this evaporation and fission cycle is repeated, the droplets eventually become small enough that the sample ions are liberated into the gas phase. ESI provides the softest ionization method available, which means it can be used for highly polar, least volatile, or thermally unstable compounds. The main advantage of the use of ESI for quantitative LC-MS is the formation of protonated or de-protonated molecules with little fragmentation, ideal for selection of precursor ions and for maximising sensitivity.

17. Atmospheric pressure chemical ionization (APCI)

18. In APCI, the LC eluent is sprayed through a heated vaporizer at atmospheric pressure. The resulting gas-phase solvent molecules are ionized by electrons discharged from a corona needle. The solvent ions then transfer charge to the analyte molecules through chemical reactions (chemical ionization). The analyte ions pass through a capillary sampling orifice into the mass analyzer.

19. APCI is applicable to a wide range of polar and nonpolar molecules. typically used for molecules less than 1,500 u for analysis of large biomolecules that may be thermally unstable. APCI is used with normal-phase chromatography more often than electrospray is because the analytes are usually nonpolar

20. 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 mass analyzer.

21. Mass Analyzers Quadrupole Time-of-flight Ion trap Magnetic sector mass analyzer Electrostatic sector mass analyzer

22. The analyte ions are directed down the center of four parallel rods arranged in a 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.

23. two modes: Scanning (scan) mode & Selected ion monitoring (SIM) mode 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 more sensitive than scan mode but provides information about fewer ions. Scan mode is typically used for all analyte masses are not known in advance. SIM mode is used for quantitation and monitoring of target compounds

24. Time of flight

25. TOF Analyzers separate ions by time without the use of an electric or magnetic field. • In a crude sense, TOF is similar to chromatography, except there is no stationary/ mobile phase, instead the separation is based on the kinetic energy and velocity of the ions. 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.

26. Ion trap analyzer

27. It 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.

28. DETECTORS 3 different types of detector are used with the analyzer's Electron multipliers, dynolyte photomultiplier, microchannel plates.

29. A typical MCP consists of ~ 10 microns diameter and has thickness of ~1 mm. Channels are parallel and often enter the plate at a small angle to the surface (~8° from normal). 12.5 um diameter channels Due to the angle, an ion that enters one of the channels is guaranteed to hit the wall of the channel. The impact frees several electrons, which are accelerated along the channel until they in turn strike the channel surface, giving rise to more electrons. Eventually this cascade process yields a cloud of several thousand electrons which emerge from the rear of plate. The large planar detection area of MCPs results in a large acceptance volume. And, only a few MCP channels out of millions are affected by the detection of a single ion, thus it is possible to detect many ions at the same times.

30. Electron multipliers are probably the most common means of detecting ions, especially when positive and negative ions need to be detected on the same instrument. The  ions pass the conversion dynode (depending on their charge) and strike the initial amplification dynode surface producing an emission of secondary electrons which are then attracted either to the second dynode, or into the continuous dynode where more secondary electrons are generated in a repeating process ultimately resulting in a cascade of electrons. 

31. Application of LC MS Biomedical Applications: LC-MS technique is useful for the detection of steroid drugs in body fluids and in profiling endogenous steroids. Steroid sulphates have been detected with high sensitivity using this method. Plasma spray has been used to test saliva for steroid hormones in patients suffering from congenital adrenal hyperplasia. Amino acids were one of the first compounds analysed using LC-MS coupled with laser desorption and thermospray . Nucleosides, nucleotides, saccharides, peptides, and proteins were all analysed and their molecular weights were determined using LC-MS coupled with electrospray. Bile acids have also been determined using LC-MS and thermospray .

32. Environmental Applications: LC-MS is used in the analysis of diverse samples such as soil, drinking water or waste water, and sludge. Several pesticides and herbicides including triazine derivatives, chlorophenols, phenoxyalkanoic acids, and sulfonylurea herbicides can be analysed using LC-MS. Separation of polycyclic aromatic hydrocarbons and organometallic compounds is also possible using the technique.

33. Detection of phenyl urea herbicide

34. Food Applications Identification of aflatoxins in food

35. Pharmaceuticals: LC-MS is widely used in the determination of pharmaceutical compounds and especially in the separation of optically active drugs. Antibiotics and potential antimalarial have been studied using thermospray . The use of LC-MS in the identification of bromazepam and similar drugs in case of intoxication has been successfully demonstrated. Detection, isolation, and purification of drug metabolites is another major application of LC-MS, as they are chemically or thermally labile, and need liquid chromatography.  Separation and characterization of components in a crude mixture of natural products such as complex lipids, alkaloids, and hydroxylated or unsaturated fatty acids has been achieved using LC-MS.

36. Clinical Applications High-sensitivity detection of trimipramine and thioridazine In urine sample

37. Thank you