lcms

LCMS (LIQUID CHROMATOGRAPHY MASS SPECTROSCOPY)

1. GURU JAMBHESHWAR UNIVERSITY OF SCIENCE AND TECHNOLOGY, HISAR LIQUID CHROMATOGRAPHY-MASS SPECTROSCOPY (LC-MS) SUBMITTED BY- Shikha Kamboj M Pharm 2 ND Sem Pharmaceutical Chemistry

2. CONTENTS CHROMATORAPHY MASS SPECTROSCOPY HYPHENATED TECHNIQUES ADVANTAGES OF HYPHENATION APPLICATIONS OF CHROMATOGRAPHY LC-MS Principle Parts of LC-MS 7. INSTRUMENTATION 8. WORKING 9. APPLICATIONS 10. ADVANTAGES 11.DISADVATAGES 12. RECENT ADVANCEMENT

3. CHROMATOGRAPHY It is defined as a separation technique in which an individual component is separated from the mixture by using stationary phase and a suitable mobile phase. The term chromatography is derived from the Greek words Chroma means “color” and graphein "to write“ The stationary phase remains fixed in place while the mobile phase carries the components of the mixture through the medium being used. The movement of the components in the mobile phase is controlled by the significance of their interactions with the mobile and/or stationary phases . Because of the differences in factors such as the solubility of certain components in the mobile phase and the strength of their affinities for the stationary phase, some components will move faster than others, thus facilitating the separation of the components within that mixture.

4. PRINCIPLE Chromatography is based on the principle where molecules in mixture applied onto the surface or into the solid, and fluid stationary phase (stable phase) is separating from each other while moving with the aid of a mobile phase . The factors effective on this separation process include molecular characteristics related to adsorption (liquid-solid), partition, and affinity. Because of these differences, some components of the mixture stay longer in the stationary phase, and they move slowly in the chromatography system, while others pass rapidly into the mobile phase, and leave the system faster .

5. Stronger interaction with the stationary phase will tend to move slower and having higher retention time, than others which have lower or no interactions with the stationary phase will tend to move faster. Generally maximum interactions between a solute and stationary phase takes place when both have similar characteristics i.e. polarity When their properties are so different, a solute will not tend to stay and will thus prefer to stay in the mobile phase.

6. MASS SPECTROSCOPY Mass spectroscopy is an instrumental technique in which sample is converted to rapidly moving positive ions by electrons bombardment and charged particles are separated according to their masses. Mass spectrum is a plot of relative abundance OR intensity against the ratio of mass/charge. PRINCIPLE :- Conversion of neutral molecule into a charged molecule to a positively charged molecule. Separation of positively charged fragments formed, based on their masses.

7. WHY WE COMBINE THESE TWO? Combining the two processes reduces the possibility of error, as it is extremely unlikely that two different molecules will behave in the same way in both a liquid chromatography and a mass spectrometer. While LC separates mixtures with multiple compounds and MS provides structural identification of the individuals. Therefore , when an identifying mass spectrum appears at a characteristic retention time in a LC-MS analysis, it typically tends to increased certainty that the analyte of interest is in the sample .

8. HYPHENATED TECHNIQUES A Hyphenated technique is combination or coupling of two different analytical techniques with the help of proper interface. Hirschfield (1980) introduced the term "hyphenation" to refer to the on-line combination of a separation technique and one or more spectroscopic detection techniques. Chromatography - Produces pure or nearly pure fractions of chemical components in a mixture. Spectroscopy – Produces selective information for identification using standards or library spectra. The hyphenated technique is developed from the coupling of a separation technique and an online spectroscopic detection technology. The number of existing techniques has been combined to expand the utility.

9. The direct conjugation of chromatographic technique with spectroscopic examination of separated fraction constitutes several powerful analytical techniques . The hyphenation does not always have to be between two techniques; the coupling of separation or detection techniques. Recently , more than two techniques coupled together to form a more powerful integrated system have revolutionized the trace element analysis industry. Also called double hybrid e.g. LC-PDA-MS; LC-MS-MS; LC-NMR-MS instruments have become available and have been applied to pharmaceutical problem solving. Online coupling with solid phase extraction (SPE), solid phase micro extraction or large volume injection can be incorporated to build in a more powerful integrated system e.g. SPE-LC-MS.

10. Hyphenated techniques ranges from the combination of- 1. separation-separation, 2. separation-identification & 3. Identification-identification techniques.

11. ADVANTAGES OF HYPHENATION 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 System. 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 . LC-ESI-MALDI-TOF 9 . GC-MS-MS 10 . GC-NMR 11 . GC-AES 12 . ICP-MS 13 . ICP-AAS 14 . ICP-OES

12. APPLICATIONS OF CHROMATOGRAPHY Pharmaceutical sector To identify and analyze samples for the presence of trace elements or chemicals. Separation of compounds based on their molecular weight and element composition. Detects the unknown compounds and purity of mixture. In drug development. Food Industry In food spoilage and additive detection Determining the nutritional quality of food Forensic Science In forensic pathology and crime scene testing like analyzing blood and hair samples of crime place.

13. Molecular Biology Studies Various hyphenated techniques in chromatography such as EC-LC-MS are applied in the study of metabolomics and proteomics along with nucleic acid research. HPLC is used in Protein Separation like Insulin Purification, Plasma Fractionation, and Enzyme Purification and also in various departments like Fuel Industry, biotechnology, and biochemical processes. Chemical industry In testing water samples and also checks air quality. HPLC and GC are very much used for detecting various contaminants such as polychlorinated biphenyl (PCBs) in pesticides and oils. In various life sciences applications.

14. LIQUID CHROMATOGRAPHY-MASS SPECTROSCOPY What is LC-MS ? It is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry. LC-MS is a powerful technique used for many applications which has very high sensitivity and specificity. • 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 . •A LCMS system contains an interface that efficiently transfer the separated components from the LC column into the MS ion source. The interface is necessary because the

15. LC and MS devices are fundamentally incompatible. High sensitivity of mass spectroscopy provides the information for identification of compounds or structural elucidation of compounds. Combination of these two techniques leads the metabolites appear from the end of the column they enter the mass detector, where the solvent is removed and the metabolites are ionized. In the most of the cases the interface used in LC- MS are ionization source . It is application oriented towards the specific detection and potential identification of chemicals in the presence of other chemicals (in a complex mixture).

16. While the mobile phase in LC system is pressurized liquid, the MS analyzers commonly operates under high vacuum. Thus, it is not possible to directly pump eluate from the LC column into the MS source. T he interface is a mechanically simple part of the LC-MS system that transfers the maximum amount of analyte , removes a significant portion of the mobile phase used in LC and preserves the chemical identity of the chromatography products (chemically inert). As a requirement, the interface should not interfere with the ionizing efficiency and vacuum conditions of the MS system. Nowadays , most extensively applied LC-MS interfaces are based on atmospheric pressure ionization (API) strategies like electrospray ionization  (ESI), atmospheric-pressure chemical ionization (APCI), and atmospheric pressure photo-ionization (APPI).

17. PRINCIPLE OF LC-MS LC-MS is a hyphenated technique, combining the separation power of LC , with the detection power of mass spectrometry. Even with a very sophisticated MS instrument, HPLC is still useful to remove the interferences from the sample that would impact the ionization . In LC-MS we remove the detector from the column of LC and fit the column to interface of MS. The mass analyzer is then used to sort ions according to their mass to charge ratio and detector counts the ions emerging from the mass analyzer and may also amplify the signal generated from each ion.

18. As a result, mass spectrum (a plot of the ion signal as a function of the mass-to-charge ratio) is created, which is used to determine the elemental nature of a sample, the masses of particles and of molecules, and to elucidate the chemical structures of molecules .  In the most of the cases the interface used in LC-MS are ionization source.

19. INSTRUMENTATION OF LC-MS Mainly HPLC instrument contains mobile phase, pump, mixing unit (solvent degassing system), injector (manual/auto), guard column, analytical columns, detectors, recorder and integrators (Figure 1).

20. The two key components in this process are the ion source, which generates the ions, and the mass analyzer, which sorts the ions. Several different types of ion sources are commonly used for LC/MS . Source for the production of ions OR ionization source:- Ion spray , Most common ionization sources are Electrospray ionization (ESI), Atmospheric pressure chemical ionization (APCI) and Matrix-assisted laser desorption/ionization (MALDI). A part from this Electron impact (EI) and Chemical ionization (CI) or negative chemical ionization are also used as ionization source in MS.

21. Interface :- most important and critical. Ions are transferred from LC to MS region. MS analyzer:- Q uadrupole, time of flight, ion trap analyzer, magnetic sector mass analyzer, Detector:- Electron multipliers, Dynolyte photomultiplier and Micro channel Plate. MOBILE PHASE :-The mobile phase is the solvent that moves the solute through out column . General requirements:- (1)low cost , 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.

22. STATIONARY PHASE In the normal phase it contains the hydroxy group and in the reverse phase it contains ODS ( octa decyl silane ), octyl (C8), C4 (butyl), nitrile and amino column. COLUMN Guard column and analytical column are generally used . Column are generally made up of stainless steel, glass, polyethylene and ploy ether ether ketone (PEEK). Most commonly stainless steel columns are used which can withstand with high temperature. Most widely used columns for LCMS are:- ( 1) fast LC column. the use of short column. (15-50mm) ( 2) Micro LC column. the use of large column. ( 20-150mm)

23. PUMP- SOLVENT DELIVERY SYSTEM There are different types of pumps available such as mechanical pumps and pneumatic pumps. Mechanical pump operate with constant flow rate and pneumatic pump operate with high pressure. The solvents Or mobile phase used must be assed through the column at high pressure. INJECTOR Septum injectors, stop flow but rheodyne injectors are generally used.

24. 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.

25. 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 .

26. The interface between a liquid phase technique (HPLC) with a continuously flowing eluate, and a gas phase technique carried out in a vacuum was difficult for a long time. The advent of electrospray ionization changed this. Currently, the most common LC-MS interfaces are electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photo-ionization (APPI). These are newer MS ion sources that facilitate the transition from a high pressure environment (HPLC) to high vacuum conditions needed at the MS analyzer .  Although these interfaces are described individually, they can also be commercially available as dual ESI/APCI, ESI/APPI, or APCI/APPI ion sources.

27. IONIZATION GAS PHASE:- Electron ionization, chemical ionization DESORPTION:- Field desorption, fast atom bombardment (FAB), Matrix assisted laser desorption ionization (MALDI) EVAPORATIVE:- Thermospray , electrospray ionization. Ionization Hard ionization soft ionization High energy low energy Increased fragmentation decreased fragmentation

28. Electrospray ionization (ESI): Electrospray is produced by applying a strong electric field to a liquid passing through it. A solution containing the sample molecule is sprayed through the high voltage potential capillary by the help of nebulizer. Then the sprayed droplets are ionized due to high voltage potential at capillary. Heated disolvation gas will evaporate the solvent and it will produce the molecular ion. It large volume to increase the sensitivity of the detection. By using ESI high mass sample, non-volatile molecules, liquids can be ionized and disadvantage of this source of ionization is poor sensitivity, low fragmentation and source is instable.

29. Atmospheric pressure chemical ionization (APCI ) • APCI vaporizes solvent and sample molecules by spraying the sample solution into a heater (heated to about 400 C) using a gas, such as N2. • Solvent molecules are ionized by corona discharge to generate stable reaction ions . Principle of this technique involves nebulization of the mobile phase with nitrogen gas and vaporization by heating it to relatively high temperature (above 400°C). The resulting vapor is then subjected to a corona discharge electrode to create ions. APCI is most commonly used ionization source used in LC-MS. APCI are used for analysis of pharmaceutical, environmental, toxicological, clinical and chemical industrial/ laboratory samples.

30. Atmospheric pressure photoionization(APPI) • The LC eluent is vaporized using a heater at atmospheric pressure. The resulting gas is made to pass through a beam of photons generated by a discharge lamp (UV lamp) which ionizes the gas molecules.

31. ANALYZERS The analyzer is component of the mass spectrometer that takes ionized molecules and separates them based on charge to mass ratios and outputs them to the detector where they are detected and later converted to a digital output. Quadrupole and triple quadrupole are the most widely used analyzer because it is easy to operate and it will cover wide mass range (10 to 4000 A.M.U./atomic mass unit). Quadrupole gives good linearity for quantitative work and good resolution (up to 4000), quality of mass spectra, scanning speed (5000 A.M.U per second) and mass accuracy

32. A Quadrupole mass filter consists of four parallel metal rods with different charges • Two opposite rods have an applied + potential and the other two rods have a - potential • The applied voltages affect the trajectory of ions traveling down the flight path • For given DC and AC voltages, only ions of a certain mass-to-charge ratio pass through the quadrupole filter and all other ions are thrown out of their original path. Quadrupole is composed of two pairs of metallic rods. Each opposing rod pair is connected together electrically, and voltage is applied between one pair of rods and the other. A direct current voltage is then superimposed on the RF voltage. Ions travel down the quadrupole between the rods. Only ions of a certain mass-to-charge ratio (m/z) will reach the detector for a given ratio of voltages. Other ions have unstable trajectories and will collide with the rods. This permits selection of an ion with a particular m/z and allows the operator to scan for a range of m/z-values by continuously varying the applied voltage.

33. Ion trap analyzer: This analyzer is also known as the quadrupole ion trap analyzer ( QIT).Mostly it will be used on GC/MS rather than LC/MS. The principle of the trap is to store the ions in a device (ion trap) consisting of a ring electrode and two end cap electrodes. These ions are manipulated by using applied DC and RF fields. The amplitude of the applied voltages enables the analyzer to trap ions of specified mass to charge ratio within the analyzing device. Non selected ions are given a trajectory by the electrostatic field that causes them to exit the trap. By filling the trap with inert gas fragmentation of selected ions is possible. This is useful when structural information is required.

34. Detectors Three deferent kinds of detectors are used in Mass Spectrometry, i.e. Electron multipliers, Dynolyte photomultiplier and Micro channel plates. Electron multipliers dynode is used to convert either – ve , + ve ions into electrons, that will be amplified and detected. This will be widely used in quadrupole and ion trap instruments. The dynode of Dynolyte photomultipliers converts the charged ions into electrons. These electrons sticks to a phosphor and emits photons, and that photons are made to strike the photomultiplier to achieve multiplied signals for recording. Micro channel Plate (MCP) are commonly employed in ToF spectrometers. This will have very low time response and high degree of sensitivity.

35. Liquid chromatography is a method of physical separation in which the components of a liquid mixture are distributed between two immiscible phases, i.e., stationary and mobile. The practice of LC can be divided into five categories, i.e., adsorption chromatography, partition chromatography, ion-exchange chromatography, size-exclusion chromatography, and affinity chromatography. Among these, the most widely used variant is the reverse-phase (RP) mode of the partition chromatography technique, which makes use of a nonpolar (hydrophobic) stationary phase and a polar mobile phase. Hence the polar components get eluted first and non-polar component retained for the longer time. WORKING OF LC-MS

36. In common applications, the mobile phase is a mixture of water and other polar solvents (e.g., methanol, isopropanol, and acetonitrile), and the stationary matrix is prepared by attaching long-chain alkyl groups (e.g., n- octadecyl or C 18, C8, C4) to the surface of irregularly or spherically shaped 5 μm diameter silica particles . In HPLC, typically 20 μl of the sample of interest are injected into the mobile phase stream delivered by a high pressure pump. The mobile phase containing the analytes permeates through the stationary phase bed in a definite direction. The components of the mixture are separated depending on their chemical affinity with the mobile and stationary phases.

37. The separation occurs after repeated sorption and desorption steps occurring when the liquid interacts with the stationary bed. The liquid solvent (mobile phase) is delivered under high pressure into a packed column containing the stationary phase. The high pressure is necessary to achieve a constant flow rate for reproducible chromatography experiments. Depending on the partitioning between the mobile and stationary phases, the components of the sample will flow out of the column at different times . The column is the most important component of the LC system and is designed to withstand the high pressure of the liquid. Conventional LC columns are 100–300 mm long with outer diameter of 6.4 mm (1/4 inch) and internal diameter of 3.0 – 4.6 mm. For applications involving LC-MS, the length of chromatography columns can be shorter (30–50 mm) with 3–5 μm diameter packing particles. 

38. Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio ( m/z)  of charged particles (ions). Although there are many different kinds of mass spectrometers, all of them make use of electric or magnetic fields to manipulate the motion of ions produced from an analyte of interest and determine their  m/z. The basic components of a mass spectrometer are the ion source, the mass analyzer, the detector, and the data and vacuum systems. The ion source is where the components of a sample introduced in a MS system are ionized by means of  electron beams, photon beams (UV lights), laser  beams.

39.  In the case of electrospray ionization, the ion source moves ions that exist in liquid solution into the gas phase. The ion source converts and fragments the neutral sample molecules into gas-phase ions that are sent to the mass analyzer. While the mass analyzer applies the electric and magnetic fields to sort the ions by their masses, the detector measures and amplifies the ion current to calculate the abundances of each mass-resolved ion. In order to generate a mass spectrum that a human eye can easily recognize, the data system records, processes, stores, and displays data in a computer.

40. Therefore, the development of devices facilitating the transition from samples at higher pressure and in condensed phase (solid or liquid) into a vacuum system has been essential to develop MS as a potent tool for identification and quantification of organic compounds like peptides. MS is now in very common use in analytical laboratories that study physical, chemical, or biological properties of a great variety of compounds. Among the many different kinds of mass analyzers, the ones that find application in LC-MS systems are the quadrupole, time-of-flight (TOF ),  ion traps, and hybrid quadrupole-TOF (QTOF) analyzers.

41. APPLICATIONS OF LCMS LC-MS are most widely used in food industries, pharmaceutical and chemical industries for quantitative and qualitative analysis.34-36 Applications of LS-MSMS are as follows. Molecular weight determination: Able to determine the molecule weight of chemical substance, pharmaceutical substances, proteins, etc. Structural determination/elucidation : Tandem mass spectrometry used to determine structural information using mass spectral fragmentations.

42. Pharmaceutical applications: It’s used to determine the pharmacokinetic profile of the pharmaceuticals like drug, drug metabolites/degradation product, impurities and chiral impurities. The separation and detection of chiral impurities in pharmaceuticals are of great importance because the D-isomer of a drug can have different pharmacological, metabolic and toxicological activity from the L-isomer. • Clinical and biochemical applications : MALDI-TOF MS is used in SNP genotyping, quantification of DNA, gene expression analysis, DNA and RNA sequencing. • Food and Environmental applications: use to identify aflatoxins (toxic metabolic product in certain fungi), determine the vitamin D3 in poultry fed supplements, etc. • Capillary electrophoresis/MS applications : Used for analysis of peptides.

43. ADVANTAGES The major advantage of LC-MS is that it is the most robust analytical technique that provides higher sensitivity and selectivity required to detect an exact molecular weight of a wide range of samples . The combination of the two analytical techniques (MS and HPLC) reduces experimental error and improves accuracy. It can separate and identify solutes in low concentrations (which are in parts per million- PPM) in a complex mixture. LC-MS is widely applied as regulatory compliance in the pharmaceutical, bio-pharmaceutical, research, forensic, food, and environmental sectors. The LC-MS offers high selectivity, resolution, precise mass, and specificity as compared with other chromatography techniques. It is applied to a wide range of samples and able to identify the different types of impurities.

44. DISADVATAGES LC-MS is an expensive technique both in terms of capital and analysis costs. This is not a portable instrument; it requires special and more space. To operate and data analysis of the liquid chromatography and mass spectrometry (LC-MS) requires a skilled and trained person. The LC-MS has a high maintenance cost as compared with other analytical instruments. Phosphate buffer is not compatible with the LC-MS analysis, which is the most commonly used buffer in HPLC method development.

45. RECENT ADVANCEMENTS There has been a rising concern regarding the harmful impact of biotoxins , source of origin, and the determination of the specific type of toxin. With numerous reports on their extensive spread, biotoxins pose a critical challenge to figure out their parent groups, metabolites, and concentration. In that aspect, liquid chromatography-mass spectrometry (LC-MS) based analysis paves the way for its accurate identification and quantification . At the same time, the suspect screening and nontarget screening approach are facilitated by the HRMS platforms during the absence of reference standards.