LCMS

Liquid chromatography Mass spectroscopy
By: Afshan Gohar & Group
Biology department
Preston University Islamabad

1. LIQUID CHROMATOGRAPHY MASS SPECTROMETRY PRESENTED BY : Afshan Gohar Marvi Altaf Shumaila Raza Amna Liaqat Mehwish Khalid Namal Khan

2. CONTENTS : INTRODUCTION BASIC PRINCIPLE OF LC/MS INSTRUMENTATION COMPONENTS OF MASS SPECTROMETER IONIZATION METHODS MASS ANALYZER DETECTORS TANDEM MASS SPECTROMETRY APPLICATIONS OF LCMS FUTURE PROSPECTS OF LCMS

3. LIQUID CHROMATOGRAPHY MASS SPECTROMETRY INTRODUCTION: Liquid chromatography-mass spectrometry (LC/MS) is a technique that uses liquid chromatography (or HPLC) with the mass spectrometry. It is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry.

4. Basic Principle of LC/MS: LC/MS combines the separating power of High Performance Liquid Chromatography (HPLC ), with the detection power of Mass Spectrometry. High Performance Liquid Chromatography (HPLC): Liquid chromatography involves two main phases: Mobile phase: Liquid (solvents i.e. ethanol, Acetonitrile). Stationary phase: Column packed with very small particles 2 . Mass Spectrometry : Analytical technique that measures the mass-to-charge ratio of charged particles.

5. Working Principle: MS works by ionizing chemical compounds to generate charged molecules or molecule fragments and measuring their mass-to-charge ratios. The components of the sample are ionized by one of a variety of methods. The ions are separated according to their mass-to-charge ratio in an analyzer by electromagnetic field. The ions are detected, usually by a quantitative method. The ion signal is processed into mass spectra.

6. INSTRUMENTATION: Instrumentation of HPLC

7. Instrumentation of MS

8. Instrumentation of LCMS

9. Components of Mass Spectrometer : Ion source , which can convert gas phase sample molecules into ions. Analyzer , where ions are separated according to their mass-to-charge ratio by applying electromagnetic fields. Detector , where ions are counted thus calculating the abundances of each ion present.

10. Ionization Methods : Following are the most common ionization methods : Electrospray Ionization Atmospheric Pressure Chemical Ionization Atmospheric Pressure Photo- ionisation Electrospray Ionization : Method of molecule ionization used in the analysis of multicomponent mixtures in an LC-MS system. It belongs to a group of methods whereby “soft” ionization is carried out under atmospheric pressure. Electrospray ionization is a highly efficient technique that does not cause the dissociation of the molecules under study

11. . Under this method, the eluate stream leaving the chromatographic column is introduced into the ionization source through the capillary. At the outlet of the capillary, the sample dissolved in the solvent is exposed to a strong nebulizing gas (typically, nitrogen) and a very strong electric field, which results in the atomization of the sample into charged microdroplets .

12. Electrospray Ionization

13. Atmospheric Pressure Chemical Ionization: Atmospheric pressure chemical ionization (APCI) is a very similar technique to ESI. The manner of sample ionization is the fundamental difference between the two methods. In APCI, the eluate leaving the chromatographic column is heated and sprayed from the capillary and then captured, in gaseous form, by a stream of gas and carried to the electrode ions are formed.

14. Atmospheric Pressure Chemical Ionization :

15. Atmospheric Pressure Photo-ionization : Atmospheric pressure photo- ionisation (APPI) uses photons to excite and ionise molecules after nebulisation . The energy of the photons is chosen to minimise concurrent ionisation of solvents and ion source gases. The technique also gives predominantly singly-charged ions and has been used for the analysis of neutral compounds such as steroids and has been reviewed.

16. Mass Analyzer : 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

17. There are following kinds of mass analyzers that can be used in LC/MS : 1. Quadrupole Analyzer: The quadrupole analyzer (Q) consists of four metal electrodes in the form of symmetrically arranged rods. It works like a mass filter that, with the specific parameters of the electromagnetic field, allows the passage of ions with the selected mass-to-charge ratio values, while other ions are dispersed and do not reach the detector.

18. Time-of-Flight Analyzer 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 charge .

19. Ion Trap Analyzer: 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.

20. 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; Electron Multipliers: used to convert either – ve , + ve ions into electrons, that will be amplified and detected.

21. Dynolyte Photomultiplier: The dynode of Dynolyte photomultipliers converts the charged ions into electrons. These electrons stick to a phosphor and emit photons, and that photons are made to strike the photomultiplier to achieve multiplied signals for recording.

22. Micro Channel Plates: Microchannel Plate (MCP) is commonly employed in ToF spectrometers. This will have very low time response and high degree of sensitivity.

23. Interface : The interface is most often an electrospray ion source; however atmospheric pressure chemical ionization interface is also used. Various techniques of deposition and drying have also been used such as using moving belts; however the most common of these is off-line MALDI deposition. LC/ MS for Detailed Structure-Dependent Analysis: It is hard in HPLC to be certain about purity of a particular peak, and if it contains only a single chemical. Adding a Mass Spectrometry to this will tell you the masses of all the chemicals present in the peak, which can be used for identifying them, and an excellent method to check for the purity.

24. Tandem Mass Spectrometry Tandem mass spectrometry (MS/MS) is a system of two combined analyzers of the same type or different types, characterized by high separation efficiency. The ions produced by the source are separated in the first analyzer (MS1). Ions with the selected  m / z  value reach the collision cell where, depending on the analysis conditions, they undergo dissociation or remain unchanged. In comparison with analysis using a single analyzer, tandem analysis shows a considerable improvement in selectivity and considerably increased sensitivity.

25. APPLICATIONS OF LCMS : Molecular Weight Determination Structural Elucidation Pharmaceutical Applications Food and Environmental Applications Characterization and Identification of Compounds Carotenoids Proteomics Glycopeptides Characterization Peptide Mapping Bioanalysis of various Biological Samples

26. Analysis of Complex Lipid Mixtures : It is a LC-MS-based methodology for the investigation of lipid mixtures where it has described, and its application to the analysis of human lipoprotein-associated lipids is demonstrated. Phytoconstituents : LC–MS provides a tool for differentiating this immense plant biodiversity due to this technique’s capability of analyzing a broad range of metabolites and highly polar and / or higher molecular weight molecules (oligosaccharides and lipids).

27. FUTURE PROSPECTS OF LCMS Metabolomics : The next 5–10 years will inevitably witness increased inter-laboratory cooperation in order to collate as much LC-MS-based metabolite data as possible. The integration of NMR to LC-MS-based metabolic profiling and metabolomic studies will likely increase, either through the offline analysis of collected LC fractions or through hybrid LC-NMR-MS instrumentation. Proteomics: performance of proteomic experiments. Cleanness of samples in relation to non-protein contaminants . affects the protein identification rate.

28. Pharmacovigilance : Referred to as Drug Safety Relates to the collection, detection, assessment, monitoring, and also prevention of adverse side effects with pharmaceutical products. The detection and monitoring can be done by LC-MS based disease modifying technique which provides detailed profiles.

29. Organic/Inorganic Hybrid Nanoflowers Helps in the development of drug delivery systems, biosensors, biocatalysts, and bio - related devices is anticipated to take multiple directions. The application of nanoflowers in bio-catalysis and the design of highly sensitive bio-sensing kits, as well as industrial bio-related devices with advanced functions should receive increasing attention.

30. References: Kumar, P. R., & Rini , R. (2016). LCMS-A REVIEW AND A RECENT UPDATE. Stachniuk , A., & Fornal , E. (2016). Liquid Chromatography-Mass Spectrometry in the Analysis of Pesticide Residues in Food. Food Analytical Methods, 9(6), 1654-1665. Parasuraman , S., Anish , R., Balamurugan , S., Muralidharan , S., Kumar, K. J., & Vijayan , V. (2014). An Overview of Liquid Chromatography-Mass Spectroscopy Instrumentation. Pharmaceutical Methods, 5(2), 47.  

31. THANKS 