Pharmaceutical Analysis

PHARMACEUTICAL ANALYSIS

Mass Spectrometry

Dr. Hinna Hamid Lecturer Dept. of Chemistry Faculty of Science Jamia Hamdard Hamdard Nagar New Delhi- 110062

(12.07.2007)

CONTENTS History Principle Applications Isotope patterns Nitrogen rule Ionization Techniques Q-TOF Mass Analyzer

Keywords Spectrometry, metastable Peaks, Mclafferty rearrangement, nitrogen rule, base peak Mass spectrometry is a powerful analytical technique that is used to determine the mass of a compound, identify unknown compounds, and to elucidate the structure and chemical properties of molecules. This technique uses the interaction of electric and/or magnetic fields with matter to determine weight or mass of the matter under study and unlike other spectroscopic techniques it does not measure the absorption or emission of electromagnetic radiations. Detection of compounds can be accomplished with very minute quantities (as little as 10-12g to 10-15 moles for a compound of mass 1000 Daltons).

History  Earliest mass spectrometer was built in 1918.  Francis Aston, a physicist working in Cambridge England, first put the concept into practice in 1919. In 1922, a Nobel Prize was awarded to him.  First mass spectrum was used by J.J. Thomson in 1911 to demonstrate the existence of Neon-22 in a sample of Neon -22.  The technique was designed to measure mass of elements (esp. isotopes) and now this technique has turned into one of the most powerful analytic tools in chemistry

Principle Different elements can be uniquely identified by their mass. The precise atomic masses of some stable isotopes, which might be commonly found in organic molecules, are ELEMENT MASS 1H 1.007825 12C 12.000000 13C 13.003355 14N 14.003074 16O 15.994915 32S 31.972070 Thus different compounds can be uniquely identified by their masses

N CH - - 2 OH COOH HO -CH CH-NH 2 2

HO HO Butorphanol L-dopa

NO C21H29 2 C9H11NO4 MW = 327.1 MW = 197.2 CH CH OH 3 2 Ethanol

MW = 46.1 2 Any two molecules of identical nominal (integral) mass and different elemental composition, such as HCOOH and CH3OCH3 will differ significantly; e.g. 46.0054 and 46.0340’. Any mass analyser, which can operate with a resolving power greater than 1600, will be able to distinguish between these two species. {Resolving Power required = 46/(46.0340-46.0054)= 1608}

It follows that, if a sufficiently precise and accurate measurement of the m/z of an ion can be obtained, the elemental composition(s) corresponding to this value can be deduced.

It can be done by finding a way to “charge” an atom or molecule (ionization), place the charged atom or molecule in a magnetic field or subject it to an electric field and measure its speed or radius of curvature relative to its mass-to-charge ratio (mass analyzer) and detect the ions using micro channel plate.

Sample + Ionizer -_ Mass Analyzer Detector

Advantages over other techniques:  Accurate mass measurements can be used to match empirical formulae.  Fragmentation fingerprints (specific to each compound) can be used to identify samples by comparison to fragment databases.  Controlled fragmentation (through MS/MS) can be used for structural elucidation of novel compounds.

 Relative isotope abundance's are used to get information regarding the elements making up a compound.

 Common peaks observed in a spectrum can give useful information regarding functional groups.  Complex mixtures can be analysed via 'hyphenated' techniques such as GC-MS and HPLC- MS, thus negating the need for time-consuming sample purification.  Data is easier to interpret than IR and/or NMR.

Applications:  Determination or confirmation of chemical structure of drugs and drug metabolites (MS-MS)  Detection/quantitation of impurities  Detection/quantitation of drugs and their metabolites in biofluids and tissues  High throughput drug screening  Analysis of liquid mixtures (LC-MS)

3  Clinical testing (detection of inborn errors of metabolism, cancer, diabetes, organic solvent poisoning, drugs of abuse, etc. etc.)  Fingerprinting nutraceuticals and herbal drugs  Fingerprinting or tracing source of natural products or drugs.

A Typical Spectrometer

Spectrum The output of the mass spectrometer shows a plot of relative intensity vs the mass-to-charge ratio (m/e). The most intense peak in the spectrum is termed the base peak and all others are reported relative to it's intensity.

The process of fragmentation follows simple and predictable pathways and the ions, which are formed, will reflect the most stable cations and radical cations, which that molecule can form.

The highest molecular weight peak observed in a spectrum will typically represent the parent molecule, minus an electron, and is termed the molecular ion (M+).

Mass Spectrum of Ethyl Benzene and Toluene

4 Highly branched substances undergo fragmentation very easily. Molecular ion stabilized by  e- systems, cyclic systems etc.

Benzene with side chain Benzyl cation Tropylium cation Rearrangement

+ R CH 2 R + R

The lifetimes of molecular ions vary according to the following generalized sequence.

Aromatic compounds > conjugated alkenes > alicyclic compounds > organic sulides > unbranched hydrcarbons > mercaptans > ketones > amines > esters > ethers > carboxylic acids > branched hydrocarbons > alcohols.

Generally, small peaks are also observed above the calculated molecular weight due to the natural isotopic abundance of 13C, 2H, etc.

Isotopic Abundances Table 1: Relative natural abundances of isotopes

Element Isotopes Approx. ratio Carbon 12C & 13C 99:1 Chlorine 35Cl & 37Cl 75:25 Bromine 79Br & 81Br 50:50 Sulfur 32S, 33S & 34S 95:1:4 Silicon 28Si, 29Si & 30Si 92:5:3

Isotope patterns Mass spectrometers are capable of separating and detecting individual ions even those that only differ by a single atomic mass unit. As a result molecules containing different isotopes can be distinguished. "M+1" peaks are seen due the presence of 13C in the sample.

. 2-chloropropane

5 This is most apparent when atoms such as bromine or chlorine are present (79Br : 81Br, intensity 1:1 and 35Cl : 37Cl, intensity 3:1) where peaks at "M" and "M+2" are obtained. The intensity ratios in the isotope patterns are due to the natural abundance of the isotopes.

The isotope pattern at 78 and 80 in the figure above, represent the M and M+2 in a 3:1 ratio. Loss of 35Cl from 78 or 37Cl from 80 gives the base peak at m/z = 43, corresponding to the secondary propyl cation. The peaks at m/z = 63 and 65 still contain Cl and therefore also show the 3:1 isotope pattern.

Nitrogen rule A rule that can be used to verify the presence of the molecular ion is ‘Nitrogen Rule’. ‘It states that if a compound has an even number of nitrogen atoms (or no nitrogen atoms) its molecular ion will appear at an even mass value. On the other hand a molecule with an odd number of nitrogen atoms will form a molecular ion with an odd mass. It stems from the fact that nitrogen even if it has an even mass has an odd valency.’

Ethylamine C2H5NH2, has one nitrogen and has an odd mass number, i.e., 45. Ethylene diamine H2N-CH2 – CH2 – NH2 has two nitrogens and an even mass number, i.e., 60

How Does a Mass Spectrometer Work? Mass spectrometers - three fundamental parts:  Ionisation source, Analyser,  Detector

Ionization Techniques These techniques can be classified as hard and soft techniques depending on the impact on the analyte molecule. Hard techniques bring about extensive fragmentation of the ions formed.  Electron Impact (EI - Hard method): –It is used for small molecules of mol. wt. from 1-1000 Daltons.  Fast Atom Bombardment (FAB - Hard): – It is used for peptides, sugars, etc., of mol. wt. up to 6000 Daltons 

Electrospray Ionization (ESI - Soft): – Used for peptides, proteins, up to 200,000 Daltons mol. wt.

 Matrix Assisted Laser Desorption (Soft): Used for peptides, proteins, DNA, up to 500 kD mol. wt.

6 Electron Ionization (EI) Principle: Sample is introduced into instrument by heating it until it evaporates. Gas phase sample is bombarded with electrons. Electrons are produced by thermionic emission from a tungsten or rhenium filament. These electrons leave the filament surface and are accelerated towards the ion source chamber, which is held at a positive potential (equal to the accelerating voltage). The electrons acquire energy equal to the voltage between the filament and the source chamber - typically 70 electron volts (70 eV). The electron trap is held at a fixed positive potential with respect to the source chamber.

Molecule is “shattered” into fragments (70 eV >> 5 eV bonds) and the positive ions produced are accelerated through a charged array into an analyzing tube. The path of the charged molecules is bent by an applied magnetic field. A permanent magnet is positioned across the ion chamber to produce a magnetic flux in parallel to the electron beam. Ions having low mass (low momentum) will be deflected most by this field and will collide with the walls of the analyzer and, high momentum ions will not be deflected enough and will also collide with the analyzer wall. Ions having the proper mass-to-charge ratio, however, will follow the path of the analyzer, exit through the slit and collide with the Collector. This generates an electric current, which is then amplified and detected. By varying the strength of the magnetic field, the mass-to-charge ratio, which is analyzed, can be continuously varied.

Inside the spectrometer

7 Equation of mass spectrometry 1 2 Ion’s kinetic energy (E) is the function of accelerating voltage (V) and charge (z): mv= zV 2

Centrifugal force:

Applied magnetic field:

The forces balance as ion goes through flight tube: 2 2 Combine equations to obtain: m/ z= B R / 2 V

m = mass of ion B = magnetic field z = charge of ion R = radius of circle V = voltage

This is the fundamental equation of mass spectrometry

Thus one can scan B or V to sweep masses across a single detector.

Resolution of an instrument can be given by R = M/ ΔM Where M is the mass of an ion ΔM is the difference in mass of an ion of mass M and of the next higher mass ion that can be resolved by the instrument.

Mechanisms of ion formation Consider the ionization of the analyte species AB: 1. AB + e-* -----> A+ + B- + e-

2. AB + e-* -----> A+ + B° + 2e-

3. AB + e-* -----> [AB+°*] + 2e- followed by [AB+°*] -----> AB+°

4. AB + e-* -----> [AB2+*]" + 3e-followed by [AB2+*]" -----> A+ + B+ - very low abundance

5. AH + e-* -----> AH* + e- followed by AH* + AH -----> [AH+H]+ + A- - 'self chemical ionization'  ° Radicals.  " Short lived intermediates which are not seen in the spectra.  1 and 2 - highest abundance  3 is fairly high abundance and is the process responsible for the molecular ion formation.

8  Radical intermediate [AB+°*] tends to undergo fragmentation (or rearrangement) as a stabilizing process; this is responsible for the lower mass fragment ions present in the spectra.  4 is a very low abundance process, but theoretically it can occur.  5 can occur at higher pressures (self Chemical Ionization), leading to the formation of the [M+H]+ pseudo-molecular ion.

EI Fragmentation of CH3OH CH OH+ CH OH 3 3 CH OH CH O=H+ + H 3 2 CH OH + CH 3 3 + OH CH O=H+ + 2 CHO=H + H Applications  The application of EI is restricted to thermally stable samples with low molecular masses (< ca. 2000 Da).  Since the ion source temperature and the bombarding electron's energy is 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. There is a 8000 EI mass spectra library available on-line. ( Nist Chemistry WebBook )

Advantages and Disadvantages  Can be used for GC/MS systems and direct inlet techniques.  Useful for positive compound identification and/or structure elucidation.  EI spectra are relatively easy to obtain.  Comparatively rugged and sensitive ionization technique.  Can be employed for analyzing air- and moisture-sensitive compounds.  Analytes have to be vaporized - problems with thermal degradation.

Chemical Ionisation (CI) Principle  This ion source is very similar to the EI source but the beam of electrons is used to create plasma of ionized reagent gas (e.g. isobutane, methane, ammonia) that is introduced into the ion source continuously.  Ionization is then achieved by interaction of the sample molecule with the reagent gas, not by direct interaction with the electron beam.

9  For methane electron collisions produce CH4+ and CH3+, which further react with methane to form CH5+ and C2H5+:  CH4+ + CH4 --> CH5+ + CH3

 CH3+ + CH4 --> C2H5+ + H2  This is a less energetic procedure than EI and the ions produced are generally either protonated ([M + H]+) or negative ions ([M-·] or [M - H]-).  These ions are often relatively stable, tending not to fragment as readily as ions produced by EI.

Electron Impact MS of CH3OH

Molecular ion

EI Breaks up Molecules in Predictable Ways

Isotopes can help in identifying compounds

Electron Impact MS of CH3Br

CI characteristics in summary

 Provides molecular weight information.  Quantification is almost impossible without internal standards.

10  CI can be used as ionization methods in GC/MS.

CI Reagent Gases Methane  Good for most organic compounds

 Usually produces [M+H]+, [M+CH3]+ adducts  Adducts are not always abundant  Extensive fragmentation Isobutane

 Usually produces [M+H]+, [M+C4H9]+ adducts and some fragmentation  Adducts are relatively more abundant than for methane CI  Not as universal as methane Ammonia  Fragmentation virtually absent

 Polar compounds produce [M+NH4]+ adducts  Basic compounds produce [M+H]+ adducts  Non-polar and non-basic compounds are not ionized

Comparison of EI and CI spectra of Ephedrine

Fast Atom Bombardment (FAB) and Liquid Secondary Ion Mass Spectrometry (LSIMS) The techniques of FAB and LSIMS involve the bombardment of a solid analyte and matrix mixture by a fast particle beam. The matrix commonly used is a small organic species (glycerol or 3-nitrobenzyl alcohol, 3-NBA).

In FAB, the particle beam is a neutral inert gas, typically Ar or Xe, at bombardment energies of 4-10 KeV. The high-energy beam of netural atoms strikes a solid sample causing desorprtion and ionization. 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. It is used for large biological molecules that are difficult to get into the gas phase.

11 In LSIMS, the particle beam is an ion, typically CS+, at bombardment energies of 2-30KeV. The particle beam is incident at the analyte surface, where it transfers much of its energy to the surroundings, setting up momentary collisions and disruptions. Some species are ejected off the surface as positive and negative ions by this process, and these 'sputtered' or secondary ions are then extracted from the source and analysed by the mass spectrometer. The polarity of the source extraction can be switched depending on what species are to be analysed.

Both FAB and LSIMS soft ionization techniques, and are thus well suited to the analysis of low volatility species, typically producing large peaks for the pseudo-molecular ion species [M+H]+ and [M-H]-, along with structurally significant fragment ions and some higher mass cluster ions and dimers.

Laser ionization (LIMS) The study of polar compounds has always been a problem for mass spectrometry. The extension of Laser Desorption to the analysis of non-volatile polar biological and organic macromolecules and polymers was a groundbreaking step in the development of LD. of LD. Laser Molecular “plume” ProbeProbeProbe Tip TipwithTip sample+matrix withwith sample+matrixsample+matrix

A laser pulse ionizes some of the sample constituents and makes them available for ionization. There are a number of LIMS techniques e.g. RIMS or MALDI.

Resonance ionization (RIMS) One or more laser beams are tuned in resonance to transitions of a gas-phase atom or molecule to promote it in a stepwise fashion above its ionization potential to create an ion. Solid samples must be vaporized by heating, sputtering, or laser ablation.

12 Matrix assisted laser desorption method (MALDI) MALDI is a LIMS method of vaporizing and ionizing large biological molecules such as proteins or DNA fragments. MALDI allows to determine the molecular weight of molecules up to 500 kDa, routinely 5 to 100 kDa (polymers, biomolecules, complexes, enzymes). 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. The mechanism of MALDI is not totally understood, but it is believed to work along the following lines.

The Formation of a 'Solid Solution'. The analyte molecules are distributed throughout the matrix so that they are completely isolated from one other. This is necessary if the matrix is to form a homogenous 'solid solution'

Matrix Excitation. Some of the laser energy incident on the solid solution is absorbed by the matrix, causing rapid vibrational excitation, bringing about localized disintegration of the solid solution, forming clusters made up of a single analyte molecule surrounded by neutral and excited matrix molecules. The matrix molecules evaporate away from these clusters to leave the excited analyte molecule.

Analyte Ionization: The analyte molecules an become ionized by simple protonation by the photo-excited matrix, leading to the formation of the typical [M+X]+ type species (where X= H, Li, Na, K, etc.). Some multiply charged species, dimers and trimers can also be formed. Negative ions are formed from reactions involving deprotonation of the analyte by the matrix to form [M- H]- and from interactions with photoelectrons to form the [M]-° radical molecular ions.

13 Matrix Properties  Needs to be involatile (most are solids at room temperature)  Needs to absorb the laser wavelength that you are using. (Most cases 337 nm)  Preferably dissolves in same solvent as the sample  Typically, the matrices are acidic.  Needs to have a proton available to donate during ionization.  Should have a proton affinity that is below that of analyte.  Typically is a crystalline solid.

Typical Matrixes Hot and Cold Matrices: DHB is a cold matrix, i.e., the samples are not as likely to be fragmented and it may not ionize some molecules. Alpha-cyano dihydroxybenzoic acid is considered a hot matrix, as it is more likely to fragment the molecules. It can also produce multiply charged proteins.

MALDI characteristics in summary  Soft ionization method, it provides molecular weight information.  Suitable for analyzing very large bio- or synthetic polymers.  Sensitivity depends strongly upon the analyte.  Suitable for analyzing polar and even ionic compounds (e.g. metal complexes).  Less fragmentation.

OH OH OH COOH OH3C COOH OH

Ferulic acid Dithranol 5-Dihydroxy benzoic acid (DHB)

COOH OH3C COOH

CN OH OH

4-Hydroxy-a-cyanocinnamic acid (4HCCA) Sinapinic acid

Atmospheric pressure ionization (API) I n this technique the ions are formed at atmospheric pressure. It is a very soft ionization method and molecular ion is seen. No fragmentation of the molecular ion is observed.

There are two common types of atmospheric pressure ionization: ESI and APCI.

14 Electro spray Ionization (ESI) : Large charged droplets are produced by 'pneumatic nebulization'; i.e. the forcing of the analyte solution through a needle at the end of which is applied a potential. The potential used is sufficiently high to disperse the emerging solution into a very fine spray of charged droplets all at the same polarity. The solvent evaporates away, shrinking the droplet size and increasing the charge concentration at the droplet's surface. Eventually coulombic repulsion overcomes the droplet's surface tension and the droplet explodes. This 'Coulombic explosion' forms a series of smaller, lower charged droplets. The process of shrinking followed by explosion is repeated until individually charged 'naked' analyte ions are formed. Increasing the rate of solvent evaporation, by introducing a drying gas flow counter current to the sprayed ions increases the extent of multiple-charging. Decreasing the capillary diameter and lowering the analyte solution flow rate i.e. in nanospray ionization, will create ions with higher m/z ratios (i.e. it is a softer ionization technique).

ESI characteristics in summary  Soft ionization method, it provides molecular weight information.  Suitable for analyzing large bio- or synthetic polymers.  Sensitivity depends strongly upon the analyte.  Suitable for analyzing polar and even ionic compounds (e.g. metal complexes).  Less fragmentation.  Enables LC / MS coupling.

Generates ions directly from solution

15 Atmospheric pressure chemical ionization (APCI): The ion source is similar to the ESI ion source. In addition to the electro hydrodynamic spraying process, a corona-discharge needle at the end of the metal capillary creates plasma. In this plasma, proton transfer reactions and to a small amount fragmentation can occur. Depending on the solvents, only quasi-molecular ions like [M+H]+, [M+Na]+ and M+. (in the case of aromatics), and/or fragments can be produced. Multiply charged molecules [M+nH]n+, as in ESI, are not observed.

APCI characteristics in summary  Provides molecular weight information.  Sensitivity depends strongly upon the analyte.  Suitable for analyzing less polar compounds compared to ESI.  Increased fragmentation compared to ESI.  Enables coupling MS and LC with flow rate up to 1 ml/min.

Plasma-desorption ionization (PD) Decay of 252Cf is used to produce two fission fragments that travel in opposite directions. One fragment strikes the sample knocking out 1-10 analyte ions. The other fragment strikes a detector and triggers the start of data acquisition. This ionization method is especially useful for large biological molecules.

Secondary ionization (SIMS) For SIMS a n ion beam; such as 3He+,16O+, or 40Ar+; is focused onto the surface of a sample and sputters material into the gas phase. Approximately 1% of the sputtered material comes off as ions. SIMS is nearly identical to FAB except the primary particle beam. Ions can also be focused and accelerated to higher kinetic energies than are possible for neutral beams, and sensitivity is improved for higher masses. The use of SIMS for moderate-size (3000-13,000 Da) proteins and peptides has largely been supplanted by electrospray ionization.

Analyzer

16 Immediately following ionization, gas phase ions enter a region of the mass spectrometer, known as the mass analyzer.

The mass analyzer is used to separate ions within a selected range of mass-to-charge (m/z) ratios. Ions are typically separated by magnetic fields, electric fields, or by measuring the time it takes an ion to travel a fixed distance.

Types of Detectors

Magnetic Sector Analyzer

Ions leaving the ion source are accelerated to a high velocity. The ions then pass through a magnetic sector in which the magnetic field is applied in a direction perpendicular to the direction of ion motion. We know that when acceleration is applied perpendicular to the direction of motion of an object, the object's velocity remains constant, but the object travels in a circular path. Therefore, the magnetic sector follows an arc. A magnetic sector alone will separate ions according to their mass-to-charge ratio. To achieve better resolution, it is necessary to add an electric sector that focuses ions according to their kinetic energy. The electric sector applies a force perpendicular to the direction of ion motion, and therefore has the form of an arc. The simplest mode of operation of a magnetic sector mass spectrometer keeps the accelerating potential and the electric sector at a constant potential and varies the magnetic field. Ions that have a constant kinetic energy, but different mass-to-charge ratio are brought into focus at the detector slit (called the 'collector slit") at different magnetic field strengths.

17 Double focusing magnetic sector mass analyzers are the "classical" model against which other mass analyzers are compared.

Benefits

 Classical mass spectra  Very high reproducibility  Best quantitative performance of all mass spectrometer analyzers  High resolution  High sensitivity  High dynamic range  Linked scan MS/MS does not require another analyzer  High-energy CID MS/MS spectra are very reproducible

Limitations  Not well-suited for pulsed ionization methods (e.g. MALDI)  Usually larger and higher cost than other mass analyzers  Linked scan MS/MS gives either limited precursor selectivity with unit product-ion resolution, or unit precursor selection with poor product-ion resolution

Applications  All organic MS analysis methods  Accurate mass measurements  Quantitation  Isotope ratio measurements

Quadrupole Quadrupoles are four precisely parallel rods with a direct current (DC) voltage and a superimposed radio-frequency (RF) potential. Combined DC and RF potentials on the quadrupole rods can be set to pass only a selected mass- to-charge ratio and by scanning a pre-selected radio-frequency field one effectively scans a mass range. All other ions do not have a stable trajectory through the quadrupole mass analyzer and will collide with the quadrupole rods, never reaching the detector.

Benefits  Classical mass spectra  Good reproducibility  Relatively small and low-cost systems  Low-energy collision-induced dissociation (CID) MS/MS spectra in triple quadrupole and hybrid mass spectrometers have efficient conversion of precursor to product

Limitations  Limited resolution  Peak heights variable as a function of mass (mass discrimination). Peak height vs. mass response must be 'tuned'.

18  Not well suited for pulsed ionization methods  Low-energy collision-induced dissociation (CID) MS/MS spectra in triple quadrupole and hybrid mass spectrometers depend strongly on energy, collision gas, pressure, and other factors.

Applications  Majority of benchtop GC/MS and LC/MS systems  Triple quadrupole MS/MS systems  Sector / quadrupole hybrid MS/MS systems

Quadrupole: Changes DC and RF Voltages to isolate a given m/z ion. PRO: cheap, fast, easy

Time-of-Flight Mass Analyzers Time of flight mass spectrometer measures the mass-dependent time it takes ions of different masses to move from the ion source to the detector. This requires that the starting, ions are either formed by a pulsed time (the time at which the ions leave the ion source) is well defined. Therefore ionization method (usually matrix-assisted laser desorption ionization, or MALDI), or various kinds of rapid electric field switching are used as a 'gate' to release the ions from the ion source in a very short time. Kinetic energy

The ion velocity, v, is the length of the flight path, L, divided by the flight time, t:

Substituting this expression for v into the kinetic energy relation, we can derive the working equation for the time-of-flight mass spectrometer:

Reflectron The ions leaving the ion source of a time-of-flight mass spectrometer have neither exactly the same starting times nor exactly the same kinetic energies. Various time-of-flight mass spectrometer designs have been developed to compensate for these differences.

A reflectron is an ion optic device in which ions in a time-of-flight mass spectrometer pass through a "mirror" or "reflectron" and their flight is reversed. A linear-field reflectron allows ions with greater kinetic energies to penetrate deeper into the reflectron than ions with smaller

19 kinetic energies. The ions that penetrate deeper will take longer to return to the detector. If a packet of ions of a given mass-to-charge ratio contains ions with varying kinetic energies, then the reflectron will decrease the spread in the ion flight times, and therefore improve the resolution of the time-of-flight mass spectrometer.

Benefits  Fastest MS analyzer  Well suited for pulsed ionization methods (method of choice for majority of MALDI mass spectrometer systems)  High ion transmission  MS/MS information from post-source decay  Highest practical mass range of all MS analyzers Limitations  Requires pulsed ionization method or ion beam switching (duty cycle is a factor)  Fast digitizers used in TOF can have limited dynamic range  Limited precursor-ion selectivity for most MS/MS experiments Applications  Almost all MALDI systems  Very fast GC/MS systems

Q-TOF Mass Analyzer Trapped-Ion Mass Analyzers There are two principal trapped-ion mass analyzers:  Three-dimensional quadrupole ion traps ("dynamic" traps)  Ion cyclotron resonance mass spectrometers ("static" traps) Both operate by storing ions in the trap and manipulating the ions by using DC and RF electric fields in a series of carefully timed events.

Principal of Operation Ions move in a circular path in a magnetic field. The cyclotron frequency of the ion's circular motion is mass dependent. By measuring the cyclotron frequency, one can determine an ion's mass.

We know that

Solving for the angular frequency (omega), which is equal to v/r:

20 A group of ions of the same mass-to-charge ratio will have the same cyclotron frequency, but they will be moving independently and out-of-phase at roughly thermal energies.

If an excitation pulse is applied at the cyclotron frequency, the "resonant" ions will absorb energy and be brought into phase with the excitation pulse. The packet of ions passes close to the receiver plates in the ICR cell and induces image currents that can be amplified and digitized. The signal induced in the receiver plates depends on the number of ions and their distance from the receiver plates. Since several different masses are present, one must apply an excitation pulse that contains components at all of the cyclotron frequencies. This is done by using a rapid frequency sweep ("chirp"), an "impulse" excitation, or a tailored waveform. The image currents induced in the receiver plates will contain frequency components from all of the mass-to-charge ratios. The various frequencies and their relative abundances can be extracted mathematically by using a Fourier transform, which converts a time-domain signal (the image currents) to a frequency-domain spectrum (the mass spectrum).

A small potential is applied to the trapping plates to keep the ions contained within the ICR cell because the magnetic field does not constrain the ion motion along the direction of the applied magnetic field. Beside the cubic cell, many other ICR cell designs have been evaluated, and each has its own special characteristics. Excitation events can be used to increase the kinetic energy of ions, or to eject ions of a given mass-to-charge ratio from the cell by increasing the orbital radius until ions are lost by collisions with the cell plates.

The background pressure of an FTICR should be very low to minimize ion-molecule reactions and ion-neutral collisions that damp the coherent ion motion.

FT-Ion Cyclotron Analzyer

Benefits  The highest recorded mass resolution of all mass spectrometers  Powerful capabilities for ion chemistry and MS/MS experiments  Well-suited for use with pulsed ionization methods such as MALDI  Non-destructive ion detection; ion remeasurement  Stable mass calibration in superconducting magnet FTICR systems

21 Limitations  Limited dynamic range  Strict low-pressure requirements mandate an external source for most analytical applications  Subject to space charge effects and ion molecule reactions  Artifacts such as harmonics and sidebands are present in the mass spectra  Many parameters (excitation, trapping, detection conditions) comprise the experiment sequence that defines the quality of the mass spectrum  Generally low-energy CID, spectrum depends on collision energy, collision gas, and other parameters Applications  Ion chemistry  High-resolution MALDI and electrospray experiments for high-mass analytes  Laser desorption for materials and surface characterization

Quadrupole Ion Traps Principal of Operation Ions are dynamically stored in a three-dimensional quadrupole ion storage device. The RF and DC potentials can be scanned to eject successive mass-to-charge ratios from the trap into the detector (mass-selective ejection).

Ions are formed within the ion trap or injected into an ion trap from an external source. The ions are dynamically trapped by the applied RF potentials. The trapped ions can be manipulated by RF events analogous to the events in FTICR to perform ion ejection, ion excitation, and mass- selective ejection. This provides MS/MS and MS/MS/MS... experiments analogous to those performed in FTICR.

Benefits  High sensitivity  Multi-stage mass spectrometry (analogous to FTICR experiments)  Compact mass analyzer

Limitations  Poor quantitation  Very poor dynamic range (can sometimes be compensated for by using automatic gain control)  Subject to space charge effects and ion molecule reactions  Collision energy not well-defined in CID MS/MS  Many parameters (excitation, trapping, detection conditions) comprise the experiment sequence that defines the quality of the mass spectrum

22 Applications  Benchtop GC/MS, LC/MS and MS/MS systems  Target compound screening  Ion chemistry

Detectors

Once the ion passes through the mass analyzer it is then detected by the ion detector, which is the final element of the mass spectrometer. Early detectors used photographic film. Today’s detectors produce electronic signals when struck by an ion. Timing mechanisms integrate these signals with scanning voltages to allow the instrument to report which m/z has struck the detector. The detector allows a mass spectrometer to generate a signal current from incident ions by generating secondary electrons, which are further amplified.

23 Faraday Cup A Faraday cup operates on the basic principle that a change in charge on a metal plate results in a flow of electrons and therefore creates a current. One ion striking the surface of the Faraday cup induces several secondary electrons to be ejected and temporarily displaced. This temporary emission of electrons induces a current in the cup and provides for a small amplification of signal when an ion strikes the cup. This detector is relatively insensitive, yet robust and simple in design.

Electron multiplier

Whereas a Faraday cup uses one surface, an electron multiplier is made up of a series of surfaces maintained at ever increasing potentials. Ions strike the surface, resulting in the emission of electrons. These secondary electrons are then attracted to the next surface where more secondary electrons are generated, ultimately resulting in a cascade of electrons. Typical amplification or current gain of an electron multiplier is one million.

Photomultiplier dynode With the photomultiplier conversion detector, electrons strike a phosphorus screen. The phosphorus screen, releases photons once an electron strikes. These photons are then detected by a photomultiplier, which operates with a cascading action much like an electron multiplier. The primary advantage of the conversion dynode setup is that the photomultiplier tube is sealed in a vacuum unexposed to the internal environment of the mass spectrometer.

Microchannel plates A microchannel plate consists of an array of glass capillaries (10-25 um inner diameter) that are coated on the inside with a electron-emissive material. The capillaries are biased at a high voltage and like the channeltron, an ion that strikes the inside wall one of the capillaries creates an avalanche of secondary electrons. This cascading effect creates a gain of 103 to 104 and produces a current pulse at the output.

Different Types of MS  ESI-QTOF

24 – Electrospray ionization source + quadrupole mass filter + time-of-flight mass analyzer  MALDI-QTOF – Matrix-assisted laser desorption ionization + quadrupole + time-of-flight mass analyzer  GC-MS - Gas Chromatography + MS – Separates volatile compounds in gas column and ID’s by mass  LC-MS - Liquid Chromatography + MS – Separates delicate compounds in HPLC column and ID’s by mass  MS-MS - Tandem Mass Spectrometry – Separates compound fragments by magnetic field and ID’s by mass

MS of Different classes of compounds

The process of fragmentation follows simple and predictable chemical pathways and the ions, which are formed, will reflect the most stable cations and radical cations, which that molecule can form.

Fragmentation

 Cleavage of weak bonds is often observed.  Gives more stable carbocations.  Loss of small neutral molecules, like: water, ethene, carbon dioxide, HCN etc.  Precise location of charge is difficult.  If oxygen, nitrogen or sulphur present: charge is often located on it.

Alkanes In alkanes, the C-C bonds are weaker than the C-H bonds. Ionization of the molecule results in greatly reduced bond strengths. Simple alkanes tend to undergo fragmentation by the initial loss of a methyl group to form a (m- 15) species. This carbocation can then undergo stepwise cleavage down the alkyl chain,

25 expelling neutral two-carbon units (ethene). The mass spectra of unbranched alkanes show groups of ions separated by 14Da corresponding to a difference of CH2 groups.

All the electrons are in  - orbitals and the molecular ion is usually strongly energized and fragments easily. The molecular ion is therefore usually weak or non-existent

The composition of the fragment ions is CnH2n+1, together with a series of less intense peaks at CnH2n-1, due to elimination of H2 from the higher fragment ions.

Typical fragments lost from straight chain Alkanes.

26 Nonane: C9H20 = 128 + + + + CH CH CH CH 2 2 2 2 + + CH CH CH CH CH M 128 3 + 2 + 2 + 2 3

15 29 43 57 71 85 99 + CH CH CH CH 2 2 2 2  CH3 CH2 CH2 CH2 113 CH3

Fragmentation43 of Butane

29 [Insert Fig. 37 71about here]85 15 99 113 128

Fragmentation of Butane

27 Fragmentation - Branched Alkanes Branched alkanes exhibit lower molecular ion abundances than in straight chain alkanes and fragment preferentially at the points of branching. When branched alkanes fragment, stable secondary and tertiary carbocations are formed. For this reason the molecular ion peak is much less intense. Mass spectrometry may thus be used to determine branching points in alkan

Mechanism of fragmentation for isobutane.

Aromatic Hydrocarbons

28 Molecular ion peaks are strong due to the stable structure. The fragmentation of the aromatic nucleus is generates a series of peaks having m/e = 77, 65, 63, etc. If the molecule contains a benzyl unit, to generate the benzyl carbocation, which rearranges to form the tropylium ion, will be the major cleavage. The tropylium ion, expels acetylene (ethyne) to generate a characteristic m/e = 65 peak.

MS of Naphthalene

Aldehydes and Ketones Cleavage of bonds next to the carboxyl group is common which results in the loss of hydrogen (molecular ion less 1) or the loss of CHO (molecular ion less 29). In aldehydes and ketones there is loss of one of the side-chains to generate the substituted oxonium ion. This is the predominant cleavage and the ion often represents the base peak in the spectrum.

One more important fragmentation patterns observed in carbonyl compounds (and in nitriles, etc.) is the expulsion of neutral ethene via McLafferty rearrangement. The McLafferty Rarrangement is a β Cleavage with the associated transfer of a γ hydrogen atom in a six- membered transition state in mono-unsaturated systems, irrespective of whether the rearrangement is formulated by a radical or an ionic mechanism, and irrespective of the position of the charge.

Esters, Acids and Amides The major cleavage observed for these compounds is expulsion of the "X" group to form the substituted oxonium ion and hydrogen rearrangements

29 Primary amides often show a base peak due to the McLafferty rearrangement and in short chain acids, peaks due to the loss of OH (molecular ion less 17) and COOH (molecular ion less 45) are important peaks due to cleavage of bonds next to C=O.

Alcohols In alcohols molecular ion is small or non-existent. A loss of H2O may occur. Cleavage of the C- C bond next to the oxygen is common. For primary alcohols, this generates a peak at m/e = 31; secondary alcohols generate peaks with m/e = 45, 59, 73, etc., according to substitution.

Ethers Fragmentation often occurs alpha to the oxygen atom, i.e., C-C bond next to the oxygen atom may break to form a substituted oxonium ion.

Halides Organic halides fragment with simple expulsion of the halogen. The presence of chlorine or bromine atoms is usually recognizable from isotopic peaks.

30 Metastable peaks Ions with lifetimes on the order of 10-6 sec are accelerated in the ionization chamber before they have an opportunity to disintegrate. These ions may disintegrate into fragment ions while they are passing into the analyzer region of the mass spectrometer. These ions have lower energy and thus follow an abnormal path on its way to detector. The ion appears at m/e ratio that depends on its mass as well as the mass of the original ion. Such ion gives rise to a metastable ion peak in the spectrum. It is broad and appears at non-integer values.

The equation that relates the position of the metastable ion peak in the mass spectrum to the mass of the original ion.

m1+ m2+ + fragment

(m2)2 m* = m 1 m* is the apparent mass of the metastable ion in the mass spectrum, m1 is the mass of the original ion from which the fragment formed and m2 is the mass of the new fragment ion.

Metastable ions can be used to prove a proposed fragmentation pattern or to aid in the solution of structure proof problems. For example a metastable peak at m/z 92.1 is observed in the spectrum of acetophenone (m/z 120) corresponding to the fragmentation scheme

+. + . C6H5COCH3 C6H5CO + CH3

Predicted mass

(105)2 m* = = 91.88 120

Interpretation of mass spectral data

31 Suggested Readings: . Introduction to spectroscopy: Pavia; Lampman, Kriz, Books/cole. . Spectrometric identification of organic compounds, R. M. Silverstein, John Wiley and Sons publication. . Spectroscopic methods in organic chemistry; H. Williams; I. Fleminig, Tata Mc Grawhills . Organic spectroscopy, W. Kemp, Palgrave publications.

N CH - -OH 2 COOH HO -CH CH-NH HO 2 2 HO Butorphanol L-dopa CH CH OH 3 2 NO C21H29 2 Ethanol C9H11NO4 MW = 327.1 MW = 197.2

MW = 46.1

CH OH CH OH+ 3 3 CH OH CH OH+ 3 3

CH O=H+ CH OH 2 + + H CH3OH CH O=H + H 3 2

+ CH OH + CH CH3OH CH 3 ++ OHOH 3 3 32

CH O=H+ CHO=HCHO=H++ ++ HH 2