Chapter No. 1 INTRODUCTION

Transcription

1 Chapter No. 1 INTRODUCTION The Outlook The importance of bio-analysis is most appreciated in various fields of pharmaceutical sciences. During the development of new drugs, extensive studies are performed in the pre-clinical and clinical stages. At the pre-clinical stage, fluids from animals are analysed. In the clinical stage, human samples need to be examined. Virtually all aspects of a new drug will be investigated. The toxicological and therapeutic concentrations of the parent drug and its metabolites must be determined, in combination with the pharmacodynamic and pharmacokinetic properties of the potential drug. Finally, the formulation of the drug must be optimised, which also relies to a large extent on bio-analytical evaluation. The sooner a drug can be placed on the market, the better for all involved, thus rapid development is preferred. This means that many samples should be analysed in a rather short time. Once a drug is on the market, therapeutic drug monitoring can be a vital aspect. Other important applications of bioanalysis are, amongst others, the control of residues in food and food-producing animals, drug abuse testing, clinical and forensic toxicology and environmental control. Thus, in various application fields many biological samples need to be analysed. As a result, there is a strong demand for highly sensitive and selective methods that can be used in high-throughput analysis 42,43. Determination of drugs and their metabolites is difficult in biological matrix compared to in formulations. Biological matrix (e.g. blood, plasma, serum and urine) samples contain mostly water and other components like dissolved proteins, glucose, clotting factors, mineral ions, hormones and acids 44,45. K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 1 of 256

2 1.1 Bio analytical method development on LC-MS/MS The development of a LC-MS/MS method required three separate methodologies to be developed Sample Preparations Chromatography Mass spectrometry Sample Preparations The most critical step in the development of LC-MS methods is the sample preparation to obtain homogenous solutions suitable for injection onto column, as well as low ion suppression for reliable MS detection. It is necessary to clean the biological sample as much as possible to get matrix interferences free sample solution. An efficient extraction procedure need to develop that can give quantitative and reproducible recovery 46,47. The common sample preparation techniques used in bio-analytical are protein precipitation, Liquid-Liquid extraction (LLE), Solid-Phase extraction (SPE) techniques, etc Protein precipitation: It is a simplest procedure to remove proteins from biological matrix. The inorganic acid, organic acid or organic solvent such as perchloric acid (PCA), trichloroacetic acid (TCA), formic acid (FA), acetonitrile and methanol are used to precipitate proteins in biological matrix. The mixure is then centrifuged to remove denatured proteins. After centrifugation, clear supernatant is injected directly or after drying and reconstitution into LC-MS/MS. It is fast and cost effective extraction method but can give the sample with lots of matrix interferences that cause column clogging, ion suppression/enhancement and require frequent system clean-up 48. K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 2 of 256

3 Liquid-liquid extraction: It is a method used to separate compounds based on their relative solubility in two different immiscible liquids, usually water and organic solvent. During extraction, the compound should be in unionized form and so ph adjustment of sample is necessary. An essential characteristic of a solvent used for this purpose is its immiscibility with water. The most widely used solvents in increasing solvent strength are: ethyl acetate, methylene chloride, chloroform, methyl teritarybutylether (MTBE), chloroform, butyl chloride, hexane, petroleum ether and pentane. The actual order may vary depending upon the criteria used to determine solvent strength. Ideally the polarity of the solvent used should be sufficient to remove the drug from the aqueous phase without removing closely related endogenous compounds. In addition it is important to consider the volatility, density and toxicity of solvents. Sometimes it is required to back extract the compounds or multiple extractions to remove interferences from the sample. It is a cost effective method compared to solid phase extraction, but is tedious and time consuming as it requires drying followed by reconstitution. LLE is a simple and efficient method or the separation and concentration of relatively hydrophobic compounds. For some polar compounds, it is not possible to get matrix free clean sample using this extraction procedure 49, Solid-Phase extraction (SPE): SPE is a selective method for sample preparation where the analyte is bound onto a solid support, interferences are washed off and the analyte is selectively eluted. Due to many different choices of sorbents, SPE is a very powerful technique. SPE consists of four steps; conditioning the column, loading the sample, washing the column and finally eluting the analyte. First, the SPE column is conditioned by passing a solvent trough the sorbent to wet the packing material, activate functional groups and remove impurities as well as air present in the column. In reversed-phase SPE, common solvents are methanol and acetonitrile. The organic solvent is followed by a buffer for K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 3 of 256

4 compatibility with aqueous samples. The sample containing the analyte is then loaded on the column. In this step, the analyte is retained along with some matrix components, while other passes through. A wash step removes interferences while still retaining the analyte. Finally, the analyte is eluted from the sorbent by applying a solvent capable of disrupting analyte-sorbent interactions. Ideally, there will be no interferences present in the elute. Often evaporation and reconstitution is performed, in order to transfer the analyte into a solvent more compatible with the chromatographic settings, and also to concentrate the analyte. 1. Selection of Cartridge 2. Conditioning 3. Loading K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 4 of 256

5 4. Washing 5. Elution Figure 1- Steps involved in Extraction Procedure Typically, sorbents used in SPE consists of 40 μm diameter silica gel with approximately 60 Å pore diameters. To this silica gel, functional groups are chemically bonded, for different modes of action. The most commonly used format is a syringe barrel that contains a 20 μm frit at the bottom of the syringe with the sorbent material and another frit on top, referred to as packed columns. Extraction disks are also placed in syringe barrels. These disks consist of 8 12 μm particles of packing material imbedded into an inert matrix. Disks are conditioned and used in a similar way as packed columns. The major advantage of disks compared to packed columns is that higher flow rates can be applied. Packed columns and extraction disks are also available in 96-well plate format. Benefit of well plates is the possibility to process large numbers of samples, either manually via a special vacuum manifold, or via automated instruments. This format makes manual processing easier and quicker compared to working with individual cartridges. However, variation of vacuum between wells can be a problem 51. The sorbent functional groups used for SPE are similar to those used in LC i.e. normal phase, reversed phase and ion-exchange. Normal phase sorbents are polar and K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 5 of 256

6 used for samples where the analyte of interest is present in an organic solvent. Reversed phase is used for aqueous samples, where the analyte is hydrophobic and retained on a nonpolar sorbent. Ion exchange sorbents isolate analytes based on the ionic state of the analyte. Mixed sorbents are also available, combining these modes. Polymeric sorbents are made of divinylbenzene instead of silica, and are often comparable with reversed phase sorbents 51. Analytes can be classified into four categories; basic, acid, neutral and amphoteric compounds. Amphoteric analytes have both basic and acidic functional groups and can therefore function as cations, anions or zwitterions, depending on ph 52. Three issues are of high importance when developing a SPE method. First, one must choose the proper sorbent functional group to be able to retain and elute the analyte. Secondly, for all but neutral analytes, an efficient use of ph is needed to shift the analyte between ionic and uncharged form. Adjustment of ph is used to vary retention and improve selectivity. In some cases, functional groups in the column are affected by ph, and made ionic or uncharged. Thirdly, solubility of the analyte in all solvents used for the extraction method need consideration Chromatography: Chromatography basically involves the separation of mixtures due to differences in the distribution coefficient (equilibrium distribution) of sample components between two different phases. One of these phases is a mobile phase and the other is a stationary phase. Distribution Coefficient (Equilibrium Distribution) is defined as Concentration of component A in stationary phase Concentration of Component A in mobile phase Different affinity of these two components to stationary phase causes the separation. K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 6 of 256

7 Chromatographic techniques can be classified into the following basic categories; gas chromatography (GC), Liquid chromatography, supercritical fluid chromatography (SFC) and capillary electrophoresis (CE) Gas chromatography (GC) Since separation in GC occurs in the gas phase, liquid samples have to be vaporised. This represents the main constraint of the technique since the analytes have to be thermo stable and sufficiently volatile. Derivatisation can be used to convert the analytes to a more volatile form. Other disadvantages include the unsuitability of water or salt solutions and the small injection volumes 54. The strength of GC is its high efficiency and the high separation capability. GC can be divided into two categories: gas-solid chromatography (GSC) and gas-liquid chromatography (GLC). In GSC, the mobile phase is a gas and the stationary phase is a solid that retains the analytes by adsorption. GSC is most suitable for low molecular weight gaseous species like nitrogen oxides and carbon dioxide. GLC is based upon partition of the analytes between an immobilized liquid and a gas phase. Several different liquid phases exist for GLC with a wide range of applications. This technique is suitable for all kinds of volatile analytes, such as steroids, alcohols, amino acids, fatty acids and sugars Liquid chromatography (LC) Liquid chromatography (LC) was the first type of chromatography to be discovered and, in the form of liquid-solid chromatography (LSC) was originally used in the late 1890s by the Russian botanist, Tswett to separate and isolate various plant pigments. The colored bands he produced on the adsorbent bed evoked the term chromatography (color writing) for this type of separation. In the late 1930s and early 1940s Martin and Synge introduced a form of liquid-liquid chromatography by supporting the stationary phase, in this case water, on silica gel in the form of a packed bed and used it to separate some K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 7 of 256

8 acetyl amino acids. They published their work in 1941 and in their paper recommended the replacement of the liquid mobile phase with a suitable gas which would accelerate the transfer between the two phases and provide more efficient separations. Thus, the concept of gas chromatography was born. In the same paper in 1941, Martin and Synge suggested the use of small particles and high pressures in LC to improve the separation which proved to be the critical factors that initiated the development of high 55, 56 performance liquid chromatography. The basic liquid chromatograph consists of six fundamental units. They are the mobile phase supply system, the pump and programmer, the sample injection valve, the column, the detector and data processor. Figure 2- Block diagram of the basic liquid chromatograph Types of Liquid Chromatography Liquid chromatography can be classified into four different types based on mechanism of separation. K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 8 of 256

9 a. Adsorption chromatography: This type of chromatography makes use of a solid stationary phase (silica gel or any other silica based packing) and a liquid or gaseous mobile phase. The solute gets adsorbed on the surface of the solid particles. Equilibration between the stationary phase and the mobile phase accounts for separation of different analytes. b. Partition chromatography: The separation of analytes is afforded by differential partitioning between a liquid stationary phase coated on the surface of a solid support. The solute equilibrates between the stationary liquid and the mobile phase. c. Ion-exchange chromatography: The stationary bed has an ionically charged surface of opposite charge to the sample ions. This technique is used almost exclusively with ionic or ionizable samples. Solute ions of the opposite charge are attracted to the stationary phase by electrostatic force. The mobile phase is an aqueous buffer, where both ph and ionic strength are used to control elution time. d. Size exclusion chromatography: Also called gel filtration or gel permeation chromatography, this technique separates molecules by size. The stationary phase is a porous gel with precisely controlled pore sizes through which the liquid mobile phase passes. The pores are small enough to exclude large solute molecules but not small ones. The sample is simply screened or filtered according to its solvated molecular size. Larger molecules are rapidly washed through the column; smaller molecules penetrate inside the porous of the packing particles and elute later. Unlike other forms of liquid chromatography, there is no attractive interaction between the stationary phase and the solute, only physical entrapment. Adsorption chromatography on bare silica is an example of normal-phase chromatography, in which a polar stationary phase and a less polar solvent is used. Reversed-phase chromatography is more commonly used in which the stationary phase is non-polar or weakly polar and the mobile phase is more polar. Reversed-phase chromatography eliminates peak tailing because the stationary phase has few sites that can strongly adsorb a solute to cause tailing. Thus, majority of chromatographic K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 9 of 256

10 applications are executed using reversed-phase chromatography. Liquid chromatography can be conducted either under isocratic or gradient elution conditions. Isocratic elution is performed with a single solvent or a constant solvent mixture. If one solvent does not provide sufficiently rapid elution of all components, gradient is the preferred choice. Under gradient elution there is a continuous change of solvent composition to increase eluent strength Selection LC Column Column is the heart of a chromatographic system, where compounds are retained and separated between the stationary phase and the mobile phase. Due to a wide variety of columns available, the challenge is to pick the right analytical column to analyze the sample correctly. The decision is based on several factors like column specifications, dimensions, particle and pore sizes, and chemistry of the bonded phase, all of which can affect the separation efficiency, inertness, durability, ph range, batch-to-batch reproducibility, resolution, solvent usage etc. There is also the complexity and quantity of the sample available and the desired cost and accuracy of analysis to be considered. Separation performance depends on both component retention and band broadening. Band broadening is, in general, a kinetic parameter, dependent on the adsorbent particle size, porosity, pore size, column size, shape, and packing performance. On the other hand, retention does not depend on the above mentioned parameters, but it reflects molecular surface interaction and depends on the total adsorbent surface. Consider a separation of a two component mixture dissolved in the eluent, where the component A has the same interaction with the adsorbent surface as an eluent, and component B has strong excessive interaction (Figure 3). Once injected into the column, these components will be forced through by eluent flow. Molecules of the component A will interact with the adsorbent surface and retard on it by the same way as an eluent molecules. K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 10 of 256

11 Figure 3. A typical chromatogram for separation of components A and B. Thus, as an average result, component A will move through the column with the same speed as an eluent. Molecules of the component B being adsorbed on the surface (due to their strong excessive interactions) will sit on it much longer. Thus, it will move through the column slower than the eluent flow Selection of LC Mobile Phases The choice of phase system can be very complex, particularly if multicomponent mixtures are to be separated. In the first instance the type of stationary phase needs to be chosen and this choice must be based on the interactive character of the solutes to be separated. If the solutes are predominantly dispersive then the stationary phase must also be dispersive (a reversed phase) to promote dispersive interaction with the solutes and provide adequate retention and selectivity. If the solutes are strongly polar then a polarizable stationary phase (one containing aromatic rings or cyano groups) would be appropriate to separate the solutes by polar and induced polar interactions. If the solutes are weakly polar then a strong polar stationary phase would be required (such as silica gel) to separate the solute by polar interactions. The mobile phase must be chosen to complement the stationary phase so that the selected interactions are concentrated in the stationary phase. Thus, a reversed phase having strong dispersive interactions would be used with a strongly polar mobile phase (e.g., mixtures of methanol and water, acetonitrile and water or tetrahydrofuran and water). In contrast, if the strongly polar silica gel is selected for the stationary phase then a strongly dispersive mobile phase would be appropriate (e.g., n-heptane, n- heptane/chloroform or n-heptane with a small quantity of n-propanol or ethanol). In K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 11 of 256

12 general the mobile phase must be chosen so that the selected interactions strongly dominate in the stationary phase and are minimized in the mobile phase. In LC-MS/MS based assays, it is preferred to use simple volatile buffers like Formic acid solution, Acetic acid solution, etc of very mild concentrations. Other buffers generally used are weak solutions of Ammonium acetate or Ammonium formate. Strong buffers containing ionic salts and phosphates are not very well tolerated by the mass spectrometer and hence their use is very minimal to negligible ph of Mobile Phase Mobile phase ph is a primary tool for controlling this selectivity through the change of the analyte ionization state. A simplistic rule for the retention in reversed-phase HPLC is the more hydrophobic the component the more it is retained. By simply following this rule one can conclude that any organic ionizable component will have longer retention in its neutral form then in the ionized form. Ionization is ph dependent process; ionization of the analyte could be expressed as, AH A - + H + for acidic components B + H + BH + for basic components Equilibrium constants are usually written in one of the following forms: and using the definition for the ph, one can rewrite as (1) (2) K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 12 of 256

13 Similar expression could be written for bases. As mentioned above, compound in its ionic form is more hydrophilic so it not only tends to have less interaction with hydrophobic stationary phase; it also tends to be more solvated with water molecules. This also causes significant decrease of the retention of ionic components. Since the pk a is a characteristic constant of the specific analyte, from the above equation one can conclude that relative amounts of neutral and ionic forms of the analyte could be easily adjusted by varying the ph of mobile phase. Moreover, if the ph is about two units away from the component pk a more then 99% of the analyte will be in either ionic or neutral form, depending upon the direction of the ph shift. Chromatographic resolution between two or more peaks depends upon three factors column efficiency, selectivity and retention. With ionizable analytes (bases and acids), all of these factors change dramatically with ph. For example retention can be improved by changing the separation ph, so that analytes are separated in their non-ionized form. A change in mobile phase ph also improves column efficiency because the ionization of the analyte and the residual silanols can both be altered. This minimizes secondary interactions between analytes and the silica surface that cause poor peak shape. Achieving optimum resolution requires changing the mobile phase ph. The following method development strategy explains how this is done with superior column lifetime. K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 13 of 256

14 Figure 4. Three ph regions for HPLC separation of basic compounds. The inflection point of the curve corresponds to the component pk a. Low, mid and high phs are the three general regions for chromatographic separations as defined in Figure 4. This figure highlights the benefits of performing separations of ionizable analytes in each ph region. Method development proceeds by investigating chromatographic separations at low ph and then higher ph until optimum results are achieved Ultra Performance Liquid Chromatography (UPLC) UPLC can be regarded as new invention for liquid chromatography. UPLC refers to Ultra Performance Liquid Chromatography. UPLC brings dramatic improvements in sensitivity, resolution and speed of analysis.it has instrumentation that operates at high pressure than that used in HPLC & in this system uses fine particles(less than 2.5µm) & mobile phases at high linear velocities decreases the length of column, reduces solvent consumption & saves time. The UPLC is based on the principal of use of stationary phase consisting of particles less than 2 μm (while HPLC columns are typically filled with particles of 3 to 5 μm). The underlying principles of this evolution are governed by the van Deemter equation, which is an empirical formula that describes the relationship between linear velocity (flow rate) and plate height (HETP or column efficiency) 57. The Van Deemter curve, governed by an equation with three components shows that the usable flow range for a good efficiency with a small diameter particles is much greater than for larger diameters 58, 59. H=A+B/v+Cv Where A, B and C are constants and v is the linear velocity, the carrier gas flow rate. The A term is independent of velocity and represents "eddy" mixing. It is smallest when the packed column particles are small and uniform. The B term represents axial diffusion or the natural diffusion tendency of molecules. This effect is diminished at high flow rates K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 14 of 256

15 and so this term is divided by v. The C term is due to kinetic resistance to equilibrium in the separation process. The kinetic resistance is the time lag involved in moving from the gas phase to the packing stationary phase and back again. The greater the flow of gas, the more a molecule on the packing tends to lag behind molecules in the mobile phase. Thus this term is proportional to v. Therefore it is possible to increase throughput, and thus the speed of analysis without affecting the chromatographic performance. The advent of UPLC has demanded the development of a new instrumental system for liquid chromatography, which can take advantage of the separation performance (by reducing dead volumes) and consistent with the pressures (about 8000 to 15,000 PSI, compared with 2500 to 5000 PSI in HPLC). Efficiency is proportional to column length and inversely proportional to the particle size 60.Therefore, the column can be shortened by the same factor as the particle size without loss of resolution. The application of UPLC resulted in the detection of additional drug metabolites, superior separation and improved spectral quality 61, Supercritical fluid chromatography SFC is a hybrid technique between LC and GC. A supercritical fluid is formed when a liquid is heated above its critical temperature. Above the critical temperature a substance cannot be converted back to a liquid state by application of pressure. The supercritical fluid has properties of both a liquid and a gas. Carbon dioxide is the most common mobile phase but ethane and nitrous oxide are used as well 63.They all have critical temperatures around 30 C. The stationary phase are immobilised in columns similar to those used in LC. The columns can be much longer than for ordinary LC due to the low viscosity of a supercritical fluid. The retention mechanism in SFC is explained by dissolution-precipitation and is dependent of the salvation power of the mobile phase.the solvation power is a function of the density, the latter regulated by the pressure over the column. The technique is most suitable for the analysis of thermally unstable, large non-volatile compounds like polycyclic aromatic hydrocarbons and n- alkanes 64. K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 15 of 256

16 Capillary electrophoresis CE is based on the migration of charged analytes in a fused silica capillary under the effect of an electric field. The capillary is filled with a aqueous buffer and connected to two reservoirs at the ends containing the same aqueous buffer. The movement of a compound depends on the electrophoretic mobility of the ion and the electrosmotic flow. The electrophoretic mobility of an ion is a function of the ion nature (cation or anion), ion size, form and physicochemical properties of the mobile phase. Fused silica capillaries naturally have a negative surface due to silanol groups that are mainly protonated above ph 2. Cations in the mobile phase form an ion-pair layer to neutralise the charges. This layer starts to move towards the cathode when the electric field is applied and creates the electro osmotic flow 64. Capillary electro chromatography (CEC) is a type of CE where the fused silica capillary is replaced by a capillary column filled with stationary phase. The stationary phase is comparable to the stationary phase for ordinary LC but with smaller particles. CE has better resolving power than ordinary LC but is less robust. Analytes that can be analysed by LC can normally also be analysed with the CE technique. CE can also be used for the analysis of macromolecules (e.g. peptides and proteins) where LC fails. Disadvantages with the technique include the need for more optimization than ordinary LC and that it is less robust and requires high voltage Mass Spectrometry Mass Spectrometry is a powerful technique for identifying unknowns, studying molecular structure, and probing the fundamental principles of chemistry. Applications of mass spectrometry include identifying and quantitating pesticides in water samples, it identifying steroids in athletes, determining metals at ppq (Parts Per Quadrillion) levels in water samples, carbon-14 dating the Shroud of Turin using only 40 mg of sample (1), looking for life on Mars, determining the mass of an 28Si atom with an accuracy of 70 ppt 65, and studying the effect of molecular collision angle on reaction mechanisms. K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 16 of 256

17 Mass spectrometry is essentially a technique for "weighing" molecules. Obviously, this is not done with a conventional balance or scale. Instead, mass spectrometry is based upon the motion of a charged particle, called an ion, in an electric or magnetic field. The mass to charge ratio (m/z) of the ion affects this motion. Since the charge of an electron is known, the mass to charge ratio a measurement of an ion's mass. Formation of gas phase samples ions is an essential prerequisite to the mass sorting and detection processes that occur in a mass spectrometer. Early mass spectrometers required a sample to be a gas, but due to modern developments, the applicability of mass spectrometry has been extended to include samples in liquid solutions or embedded in a solid matrix. The sample, which may be a solid, liquid, or vapor, enters the vacuum chamber through an inlet. Depending on the type of inlet and ionization techniques used, the sample may already exist as ions in solution, or it may be ionized in conjunction with its volatilization or by other methods in the ion source. K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 17 of 256

18 Figure 5- Block diagram of Mass spectrometer The above block diagram shows the basic parts of a mass spectrometer. 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. All mass spectrometers also have a vacuum system to maintain the low pressure, which is also called high vacuum, required for operation. High vacuum minimizes ion-molecule reactions, scattering, and neutralization of the ions Ionization in Mass spectrometer 66 Ionization is the physical process of converting an atom or molecule into an ion by adding or removing charged particles such as electrons or other ions. K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 18 of 256

19 This process works slightly differently depending on whether an ion with a positive or a negative electric charge is being produced. A positively charged ion is produced when an electron bonded to an atom (or molecule) absorbs enough energy to escape from the electric potential barrier that originally confined it, thus breaking the bond and freeing it to move. The amount of energy required is called the ionization potential. A negatively charged ion is produced when a free electron collides with an atom and is subsequently caught inside the electric potential barrier, releasing any excess energy. Ionization can generally be broken down into two types: sequential ionization and non-sequential ionization Sequential ionization Sequential ionization is basically a description of how the ionization of an atom or molecule takes place. More specifically, it means that an ion with a +2 charge can only be created from an ion with a +1 charge or a +3 charge. That is, the numerical charge of an atom or molecule must change sequentially, always moving from one number to an adjacent or sequential number Non-sequential ionization An electron that tunnels out from an atom or molecule may be sent right back in by the alternating field, at which point it can either recombine with the atom or molecule and release any excess energy, or it also has the chance to further ionize the atom or molecule through high energy collisions. This additional ionization is referred to as non-sequential ionization for two reasons: one, there is no order to how the second electron is removed, and two, an atom or molecule with a +2 charge can be created straight from an atom or molecule with a neutral charge, so the integer charges are not sequential. K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 19 of 256

20 Ionization mechanism There are two major competing theories about the final production of lone ions, the charged residue model (CRM) and the ion evaporation model (IEM) 67. Electro spray droplets start off highly charged, and as they shrink through evaporation the Coulomb repulsion forces approach the force of surface tension that holds droplet together. The droplet then becomes unstable and disintegrates into several droplets of smaller radius. The Charged Residue Model suggests that electrospray droplets undergo evaporation and disintegration cycles, with each initial droplet leading to a multitude of much smaller "daughter" droplets. Each final "daughter" droplet contains on average one or less molecule of analyte. When the last solvent molecules evaporate from such droplet the analyte molecule is left with the charges that the droplet carried. The Ion Evaporation (Desorption) Model suggests that as the droplet reaches a certain radius the field strength at the surface of the droplet becomes great enough to push or desorb ions directly out of the droplet. Characteristically, the fission event corresponds to an almost negligible loss in droplet mass, but a significant drop in charge. It has been suggested that both models probably occur for different analytes/solvents and in the limit of both models they have a tendency to converge. That is to say that the distinction between a droplet containing an analyte molecule and an analyte molecule surrounded by a layer of solvent eventually disappears and coulombic fission looks a lot like ion evaporation. The real question is scale and timing and the techniques to definitively determine this are not yet available. The use of the word "ionization" in "electrospray ionization" is criticized by some because many of the ions observed are thought to be preformed in solution before the electrospray process or created by simple changes in concentrations as the aerosolized droplets shrink. It is argued that electrospray is simply an interface for transferring ions from the solution phase to the gas phase. K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 20 of 256

21 Figure 6- Ionization Mechanism Ionization Techniques A variety of ionization techniques are used for mass spectrometry. Most ionization techniques excite the neutral analyte molecule which then ejects an electron to form a radical cation (M +- ). Other ionization techniques involve ion molecule reactions that produce adduct ions (MH + ).The most important considerations are the physical state of the analyte and the ionization energy. Electron ionization and chemical ionization are only suitable for gas phase ionization. Fast atom bombardment, secondary ion mass spectrometry, electrospray, and matrix assisted laser desorption are used to ionize condensed phase samples. The ionization energy is significant because it controls the amount of fragmentation observed in the mass spectrum. Although this fragmentation complicates the mass spectrum, it provides structural information for the identification of unknown compounds. Some ionization techniques are very soft and only produce molecular ions, other techniques are very energetic and cause ions to undergo extensive fragmentation. Although this fragmentation complicates the mass spectrum, it provides structural information for the identification of unknown compounds. K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 21 of 256

22 Electron Impact Electron impact ionization is the classical ionization technique in mass spectrometry. In the ion source, the gaseous sample is bombarded with 70 ev electrons usually generated from a tungsten filament. Because the pressure is kept that low, ionmolecule reactions do not occur, e. g. a [M+H] + signal due to proton transfer is not observed. The application of EI is restricted to thermally stable samples with low molecular masses (< 2000 Da). Since the ion source temperature and the bombarding electron's energy are kept constant, the number and amount of fragments is constant for (almost) every mass spectrometer, too. Therefore, the number and amount of ionic fragments ('daughter ions') and the amount of the M+ is characteristic for each substance. Therefore most mass spectra libraries are only available for EI - ionization. Figure 7- Electron Ionization source Chemical ionization Chemical Ionization (CI) is especially useful technique when no molecular ion is observed in EI mass spectrum, and also in the case of confirming the mass to charge ratio of the molecular ion. Chemical ionization technique uses virtually the same ion source device as in electron impact, except, CI uses tight ion source, and reagent gas. Reagent gas (e.g. ammonia) is first subjected to electron impact. Sample ions are K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 22 of 256

23 formed by the interaction of reagent gas ions and sample molecules. This phenomenon is called ion-molecule reactions. Reagent gas molecules are present in the ratio of about 100:1 with respect to sample molecules. Positive ions and negative ions are formed in the CI process. Depending on the setup of the instrument (source voltages, detector, etc...) only positive ions or only negative ions are recorded Fast-atom bombardment (FAB) In FAB a high-energy beam of netural atoms, typically Xe or Ar, strikes a solid sample causing desoprtion and ionization. It is used for large biological molecules that are difficult to get into the gas phase. The atomic beam is produced by accelerating ions from an ion source though a charge-exchange cell. The ions pick up an electron in collisions with neutral atoms to form a beam of high energy atoms. The FAB spectrum contains often only a few fragments and a signal for the pseudo molecular ion, e. g. [M+H] +, [M+Na] +, adducts. This makes FAB useful for molecular weight determination. However, the low m/z region is crowded with signals resulting from the matrix. These matrix signals are not very reproducible. Therefore, spectra correction and interpretation is not easily accomplished. Matrices commonly used with peptides are glycerol and 3-nitrobenzyl alcohol, which are nonvolatile and do not overload the vacuum pumps. The sample is dissolved in the matrix and inserted into the ionization camera over a metallic plate. When a highenergy beam impacts the sample, a fraction of its energy is transferred, mainly to the solvent molecules. Some analyte and matrix molecules are desorbed to the gas phase, and if they are not already charged, they can be charged in the gas phase by reaction with the surrounding gas-phase ions. Charged molecules are propelled electrostatically to the mass analyzer and, eventually, reach the detector 68. Fast atom bombardment was the first soft ionization method, and was introduced in One year later, molecules as large as human insulin were analyzed, and became the benchmark for resolution and sensitivity 69. K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 23 of 256

24 Figure 8- Fast Atom bombardment source Matrix-Assisted Laser Desorption/Ionization [MALDI] The term matrix-assisted laser desorption ionization (MALDI) was coined in 1985 by Franz Hillenkamp, Michael Karas and their colleagues 70. MALDI is a method of vaporizing and ionizing large biological molecules such as proteins or DNA fragments. The biological molecules are dispersed in a solid matrix such as nicotinic acid. A UV laser pulse ablates the matrix which carries some of the large molecules into the gas phase in an ionized form so they can be extracted into a mass spectrometer. MALDI allows to determine the molecular weight of molecules up to 500 kda, routinely 5 to 100 kda (polymers, biomolecules, complexes, enzymes), depending on the analyzer. In this ionization method, the analyte is combined with a matrix compound, in a molar ratio of 1:1000, and evaporated onto a metallic plate. The matrix compound is chosen to absorb strongly at the laser wavelength used. Absorption causes a rapid increase of temperature, allowing vaporization of the sample without extensive fragmentation,. Short pulses of laser radiation are used, typically at 337 nm, (nitrogen laser) but UV or infrared (IR) lasers can also be employed, depending on the matrix K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 24 of 256

25 compound selected. Common matrix compounds are 2,5-dihydroxybenzoic acid, nicotinic acid, sinapinic acid, and a-cyanocarboxylic acid. Figure 9- MALDI ionization Electrospray Ionization ESI is one of the most recent ionization techniques and the mode of gas phase ion formation is quite different to Electron Impact, Chemical Ionization and Fast Atom Bombardment. The major difference is that this technique involves spraying a solution of the sample through a needle which is kept at a potential (typically 3.5 kv). The voltage on the needle causes the spray to be charged as it is nebulized. The resultant droplets evaporate in a region maintained at a vacuum of several torr, until the solvent is essentially completely stripped off, leaving a charged ion. For samples which either exist as multiply charged ions in solution, or can accomodate several charges, multiply charged gas phase ions can be observed (this is in stark contrast to EI, CI or FAB, where multiply charged ions are very rarely observed). Indeed, the most striking feature of an ESI spectrum is that the resultant ions often carry multiple charges, which reduces their mass-to-charge ratio compared to a singly charged species. An important consequence K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 25 of 256

26 is that this allows mass spectra to be obtained for large molecules. ESI is a very sensitive technique, which is ideally suited for the analysis of small amounts of large and/or labile molecules such as peptides, proteins, organometallics, and polymers. Electrospray relies in part on chemistry to generate analyte ions in solution before the analyte reaches the mass spectrometer. The LC eluent is sprayed (nebulized) into a chamber at atmospheric pressure in the presence of a strong electrostatic field and heated drying gas. The electrostatic field causes further dissociation of the analyte molecules. The heated drying gas causes the solvent in the droplets to evaporate. As the droplets shrink, the charge concentration in the droplets increases. Eventually, the repulsive force between ions with like charges exceeds the cohesive forces and ions are ejected (desorbed) into the gas phase. These ions are attracted to and pass through a capillary sampling orifice into the mass analyzer. Some gas-phase reactions, mostly proton transfer and charge exchange, can also occur between the time ions are ejected from the droplets and the time they reach the mass analyzer. Electrospray is especially useful for analyzing large biomolecules such as proteins, peptides, and oligonucleotides, but can also analyze smaller molecules like benzodiazepines and sulfated conjugates. Large molecules often acquire more than one charge. Thanks to this multiple charging, electrospray can be used to analyze molecules as large as 150,000 u even though the mass range (or more accurately mass-to-charge range) for a typical LC/MS instruments is around 3000 m/z. For example: 100,000 u / 10 z = 1,000 m/z When a large molecule acquires many charges, a mathematical process called de-convolution is often used to determine the actual molecular weight of the analyte. K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 26 of 256

27 Figure 10- Electrospray ion source Figure 11- Desorption of ions from solution Atmospheric pressure chemical ionization In APCI, the LC eluent is sprayed through a heated (typically 250 C 400 C) vaporizer at atmospheric pressure. The heat vaporizes the liquid. The resulting gasphase 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. K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 27 of 256

28 APCI is applicable to a wide range of polar and non-polar molecules. It rarely results in multiple charging so it is typically used for molecules less than 1,500 u. Due to this, and because it involves high temperatures, APCI is less well-suited than electrospray for analysis of large bio-molecules that may be thermally unstable. APCI is used with normal-phase chromatography more often than electrospray is because the analytes are usually non-polar. Figure 12- APCI ion source 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 K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 28 of 256

29 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. APPI is applicable to many of the same compounds that are typically analyzed by APCI. It shows particular promise in two applications, highly nonpolar compounds and low flow rates (<100 μl/min), where APCI sensitivity is sometimes reduced. In all cases, the nature of the analyte(s) and the separation conditions has a strong influence on which ionization technique: electrospray, APCI, or APPI, will generate the best results. The most effective technique is not always easy to predict. Figure 13- APPI ion source MASS ANALYZERS: After ions are formed in the source region they are accelerated into the mass analyzer by an electric field. The mass analyzer separates these ions according to their m/z value. All mass spectrometers are based on dynamics of charged particles in electric and magnetic fields in vacuum. There are many types of mass analyzers, using either K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 29 of 256

30 static or dynamic fields, or magnetic or electric fields. Each analyzer type has its strengths and weaknesses. Many mass spectrometers use two or more mass analyzers for tandem mass spectrometry (MS/MS) Time-of-flight It uses an electric field to accelerate the ions through the same potential, and then measures the time they take to reach the detector. If the particles all have the same charge, the kinetic energies will be identical, and their velocities will depend only on their masses. Lighter ions will reach the detector first 71. Time-of-flight mass spectrometry (TOFMS) is a method of mass spectrometry in which ions are accelerated by an electric field of known strength 72. This acceleration results in an ion having the same kinetic energy as any other ion that has the same charge. The velocity of the ion depends on the mass-to-charge ratio. The time that it subsequently takes for the particle to reach a detector at a known distance is measured. This time will depend on the mass-to-charge ratio of the particle (heavier particles reach lower speeds). From this time and the known experimental parameters one can find the mass-to-charge ratio of the ion. K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 30 of 256

31 Figure 14- Time-of-flight mass analyzer K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 31 of 256

32 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. Figure 15- Ion trap mass analyzer K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 32 of 256

33 Quadrupole mass analyzers Quadrupole mass analyzers use oscillating electrical fields to selectively stabilize or destabilize ions passing through a radio frequency (RF) quadrupole field. A quadrupole mass analyzer acts as a mass selective filter and is closely related to the Quadrupole ion trap, particularly the linear quadrupole ion trap except that it operates without trapping the ions and is for that reason referred to as a transmission quadrupole. A common variation of the quadrupole is the triple quadrupole. The quadrupole mass analyzer is one type of mass analyzer used in mass spectrometry. In a quadrupole mass spectrometer the quadrupole mass analyzer is the component of the instrument responsible for filtering sample ions, based on their massto-charge ratio (m/z). A quadrupole mass analyzer is essentially a mass filter that is capable of transmitting only the ion of choice. A mass spectrum is obtained by scanning through the mass range of interest over time. The quadrupole consists of four parallel metal rods. Each opposing rod pair is connected together electrically and a radio frequency voltage is applied between one pair of rods, and the other. A direct current voltage is then superimposed on the R.F. voltage. Ions travel down the quadrupole in between the rods. Only ions of a certain m/z will reach the detector for a given ratio of voltages: other ions have unstable trajectories and will collide with the rods. This allows selection of a particular ion, or scanning by varying the voltages. K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 33 of 256

34 Figure 16 - Quadrupole Mass Analyzer Triple Quadrupole The arrangement of three quadrupoles was first developed by Jim Morrizon of LaTrobe University, Australia for the purpose of studying the photodissociation of gasphase ions. Yet, the first triple-quadrupole for mass analysis was developed at Michigan State University by Dr. Christie Enke and graduate student Richard Yost in the late 1970's A linear series of three quadrupoles can be used; known as a triple quadrupole mass spectrometer. The first (Q1) and third (Q3) quadrupoles act as mass filters, and the middle (Q2) quadrupole is employed as a collision cell. This collision cell is an RF only quadrupole (non-mass filtering) using He or N gas (~10-3 Torr, ~30 ev) to induce collisional dissociation of selected parent ion(s) from Q1. Subsequent fragments are passed through to Q3 where they may be filtered or scanned fully. This process allows for the study of fragments (daughter ions) which are crucial in structural elucidation. For example, the Q1 may be set to "filter" for a drug ion of a known mass, which is fragmented in Q2. The third quadrupole (Q3) can then be set to K.B.I.P.E.R. Kadi Sarva Vishwavidyalaya Page 34 of 256