Mass spectroscopy Mass spectroscopy instrumentation

1. ANUBHAV GUPTA
B. PHARM 4TH YEAR
Institute of Pharmacy
VBS Purvanchal University

2. 1.INTRODUCTION
▪ Mass Spectrometry (MS) is a technique that helps to identify the amount and type of chemicals
present in a sample by measuring the mass-to-charge ratio and abundance of gas-phase ions.
▪ A mass spectrometer works by generating charged molecules or molecular fragments either in a
high vacuum or immediately prior to the sample entering the high-vacuum region.
▪ Mass spectrum is a plot of relative abundance against the ratio of mass/charge (m/e).
▪ These spectra are used to determine the elemental or isotopic signature of a sample, the masses of
particles and of molecules, and to elucidate the chemical structures of molecules and other chemical
compounds.
▪ Mass spectrometry provides a highly specific method for determining or confirming the identity or
structure of drugs and raw materials used in their manufacture.
▪ Mass spectrometry in conjunction with either gas chromatography (GC–MS) or liquid
chromatography (LC–MS) provides a method for characterising impurities in drugs and formulation
excipients.
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3. 2.PRINCIPLE
▪ The mass spectroscopy works on principle that when charged molecules or molecular fragments are
generated in a high-vacuum region, or immediately prior to a sample entering a high-vacuum
region, using a variety of methods for ion production.
▪ The ions are generated in the gas phase so that they can then be manipulated by the application of
either electric or magnetic fields to enable the determination of their molecular weights.
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4. 3.Working of Mass Spectrometry
▪ In a typical procedure, a sample, which may be solid, liquid, or gas, is ionized, for example by
bombarding it with electrons.
▪ This may cause some of the sample’s molecules to break into charged fragments. These ions are
then separated according to their mass-to-charge ratio, typically by accelerating them and
subjecting them to an electric or magnetic field:
▪ Ions of the same mass-to-charge ratio will undergo the same amount of deflection.
▪ The ions are detected by a mechanism capable of detecting charged particles, such as an electron
multiplier.
▪ Results are displayed as spectra of the relative abundance of detected ions as a function of the
mass-to-charge ratio.
▪ The atoms or molecules in the sample can be identified by correlating known masses (e.g. an
entire molecule) to the identified masses or through a characteristic fragmentation pattern.
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5. 4.FRAGMENTATION PATTERN
▪ Fragmentation is the dissociation of energetically unstable molecular ions formed from passing
the molecules in the ionization chamber of a mass spectrometer.
▪ Fragmentation of the molecular ions takes place in following forms:
1. Homolytic cleavage. This type of fragmentation is promoted by the presence of a hetero atom
such as oxygen, nitrogen or sulphur, and in molecules containing a hetero atom it often gives rise
to the most abundant ion in the mass spectrum
2. Heterolytic cleavage. Heterolytic cleavage occurs less often as a predominant fragmentation
mechanism in drug molecules since they usually contain a lot of heteroatoms to direct the
cleavage. In such cleavage, the positive charge is carried by the carbon atom not by the
heteroatom.
3. Retro Diels-Alders reaction. It occurs in compounds with ring systems. It involves the
cleavage of two bonds of a cyclic system which results in the formation of two stable
unsaturated fragment in which two new bonds are formed.
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6. 4.1.Fragmentation Pattern
ALOCOHOL(3-Pentanol) ALDEHYDE(3-Phenyl 2-Propenal)
ALKANE(Hexane) AMIDE(Methyl butyramide)
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7. 5.McLafferty Rearrangement
▪ The McLafferty rearrangement is a characteristic fragmentation of the molecular ion of a carbonyl
compound containing at least one gamma hydrogen, e.g., Migration of -hydrogen followed by
-bond cleavage and elimination of ethylene or substituted ethylene neutral molecule.
▪ The McLafferty rearrangement, is relatively uncommon
in drug molecules; it is most often encountered in fatty
acid esters.
▪ This rearrangement can occur in carbonyl compounds
e.g. amides, ketones and acids.
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8. 6.IONIZATION TECHNIQUES
▪ There are many types of ionization techniques that are used in mass spectrometry. Some of them
are:
1.Electron Impact ionization (EI)
2.Fast Atom Bombardment (FAB)
3.Electrospray ionization (ESI)
4.Atmospheric Pressure Chemical Ionization (APCI)
5.Matrix Assisted Laser Desorption Ionization (MALDI)
▪ APCI is considered best than EI because APCI forms a protonated molecule and is completely
compatible with Liquid Chromatography (LC), while EI is more likely to fragment the ion leading
to a possible more ambiguous identification of the molecule weight and is incompatible with LC.
▪ MALDI along with ESI allows for ionization and measurement of large molecular weight.
▪ ESI has an advantage in its easy compatibility with LC. While MALDI has advantages for imaging
mass spectrometry.
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6.1. Electron Impact Ionization(EI)
▪ EI is still used in conjunction with sample introduction either via a direct heated probe or via gas
chromatography (GC):
(i) The sample is introduced into the instrument source by heating it on the end of a probe until it
evaporates, assisted by the high vacuum within the instrument or via a capillary GC column.
(ii) Once in the vapour phase, the analyte is bombarded with the electrons produced by a rhenium or
tungsten filament, which are accelerated towards a positive target with an energy of 70 eV. The analyte is
introduced between the filament and the target, and the electrons cause
ionisation as follows: M þ e ! Mþ: þ 2 e.
(iii) Since the electrons used are of much higher energy than the
strength of the bonds within the analyte (4–7 eV), extensive
fragmentation of the analyte usually occurs.
(iv) The molecule and its fragments are pushed out of the source
by a repeller plate which has the same charge as the ions
generated.
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6.2. Fast Atom Bombardment(FAB)
▪ FAB is a technique that was popular in the 80's to early 90's because it was the first technique that
allowed ionization of non-volatile compounds that could be done simply.
▪ It was done by bombarding a sample in a vacuum with a beam of atoms, typically Ar or Xe,
accelerated to Kilovolt energies. The sample was typically mixed in a matrix.
▪ The two most common matrixes were glycerol and 3 Nitro-benzoic acid. The matrix allowed the
sample to refresh itself.
▪ The ions formed by FAB were adducts to the molecule, where the
adducts could be protons, sodium ions, potassium ions or
ammonium ions.
▪ A variation of FAB was replacement of the atom beam with a
beam of ions, typically cesium ions, which was called secondary
ion mass spectrometry (SIMS). SIMS spectra were typically
identical to FAB spectra and the terms became interchangeable.
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6.3. Electronspray Ionization(ESI)
▪ The electronspray is created by putting a high voltage on a flow of liquid at atmospheric pressure,
sometimes this is assisted by a concurrent flow of gas.
▪ The created spray is directed to an opening in the vacuum system of the mass spectrometer, where
the droplets are de-solvated by a combination of heat, vacuum and acceleration into gas by voltages.
▪ The ions are ejected in droplets and accelerated into
the mass analyzer by voltages. For larger molecules,
the ions may contain multiple charges, allowing the
detection of very large molecules on analyzers that
have limited mass to charge (m/Z)) ratio ranges.
Because of the natural use of a flowing liquid, it is
easily adapted to liquid chromatography (LC).
▪ There are many other techniques that are variations of
electrospray, for example, nanospray, picospray
desorption electrospray ionization (DESI).
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6.4. Atmospheric Pressure Chemical Ionization (APCI)
▪ Atmospheric pressure chemical ionisation (APCI) is closely related to ESI and instruments carrying
out ESI can be readily switched to operate in APCI mode.
▪ In APCI mode the eluent from the HPLC does not pass through a charged needle before entering
the mass spectrometer source but via a heated tube so that it forms an aerosol. Upon exiting the
heated tube an electric discharge is passed through the aerosol generating reactive species such as
H3Oþ and N2þ, which promote the ionisation of the
analytes.
▪ The technique has never achieved the level of
popularity of ESI since most drug molecules can be
ionised under ESI conditions; however, APCI can be
employed for the analysis of drug molecules of low
polarity that do not ionise efficiently under ESI
conditions.
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6.5. Matrix Assisted Laser Desorption Ionization (MALDI)
▪ MALDI uses a nitrogen laser to promote ionisation of molecules prior to ion separation in a mass
spectrometer. It is usually combined with time of flight (TOF) separation of the ions generated.
▪ In order for the sample to be ionised it has to be dissolved in a matrix that absorbs UV radiation at
around the wavelength (337 nm) produced by the laser. A simple example of a matrix is
dihydroxybenzoic acid and there are a number of similar aromatic compounds which are used to
promote ionisation of different classes of molecules.
▪ The sample solution is mixed with matrix solution on a metal plate and allowed to dry prior to being
introduced into the instrument. The laser is then directed at the target plate to promote ionisation.
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7. INSTRUMENTATION
Mass spectrometer has following components:
A. Sample Inlet, through HPLC, GC, Syringe etc.
B. Ionization, can be achieved by :
1.Electron Impact ionization (EI)
2.Fast Atom Bombardment (FAB)
3.Electrospray ionization (ESI)
4.Atmospheric Pressure Chemical Ionization (APCI)
5.Matrix Assisted Laser Desorption Ionization (MALDI)
C. Acceleration & Deflection
D. Analyser, can be achieved by analysers like :
1.Magnetic sector mass analyser
2.Double focussing analyser
3.Quadrupole mass analyser
4.Time of Flight analyser (TOF)
5.Ion trap analyser
6.Ion cyclotron analyser
E. Detector
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7.1. Sample Inlet
▪ Sample stored in large reservoir from which molecules reaches ionization chamber at low
pressure in steady stream by a pinhole called “Molecular leak”.
▪ Molecular leak–It is pin-hole restriction (0.01 to 0.05mm diameter) and made up of gold foil. It
is used for metering the sample to ionization chamber.
7.2. Ionization
▪ Atoms are ionized by knocking one or more electrons off to give positive ions by bombardment
with a stream of electrons. Most of the positive ions formed will carry charge of +1.
7.3.Acceleration & Deflection
▪ Ions are accelerated so that they all have same kinetic energy.
▪ Positive ions pass through 3 slits with voltage in decreasing order.
▪ Middle slit carries intermediate and finals at zero volts.
▪ Ions are deflected by a magnetic field due to difference in their masses.
▪ The lighter the mass, more they are deflected.
▪ It also depends upon the no. of +ve charge an ion is carrying; the more +ve charge, more it will be
deflected.
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7.4. Analyser
▪ A mass analyser is a device that can separate atoms and molecules according to their mass.
▪ The five main characteristics for measuring the performance of a mass analyser are
1) The mass range limit or dynamic range
2) The analysis speed [u (m)S-1]
3) The transmission = No. of ion reaching the ions/No. of ions entering mass analyzer
4) The mass accuracy
5) The resolution.
▪ Mass analysers used are:
1.Magnetic sector mass analyser
2.Double focussing analyser
3.Quadrupole mass analyser
4.Time of Flight analyser (TOF)
5.Ion trap analyser
6.Ion cyclotron analyser
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7.4.1. Quadrupole Mass Analyzer
▪ A cheaper and more sensitive mass spectrometer than a magnetic sector instrument is based on the
quadrupole analyser.
▪ It uses two electric fields applied at right angles to each other, rather than a magnetic field, to
separate ions according to their m/z ratios. One of the fields used is DC and the other oscillates at
radiofrequency.
Disadvantage
▪ Limited resolution
▪ Peak heights variable as a function of mass (mass
discrimination).
▪ Peak height vs. mass response must be 'tuned'.
▪ 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.
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7.4.2. Time of flight (TOF) analyser
▪ The basis of the separation is that smaller ions move more quickly than larger ions and thus reach the
detector first.
▪ The technique relies on gating the signal from the ion source, so that one packet of ions is allowed
time to reach the detector before the next set is ejected from the ion source, so that there is no
overlap between ion packets. The ions leaving the ion source have different kinetic energies and this
compromises the mass resolution, indeed early instruments gave very wide peaks for a single mass.
▪ The problem of mass resolution was resolved by
using a device called a reflectron, which opposes
the direction that the ions are moving by sending
them back in the opposite direction.
▪ The faster moving ions penetrate deeper into the
reflectron and thus a lag effect is produced for
faster moving ions so that they are focused with
the slower moving ions which do not penetrate as
far into the reflectron. Pushing the ions back in the
opposite direction also increases the length of the
flight tube, increasing instrument resolution.
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7.4.3. Magnetic Sector Mass Analyzer
▪ In a magnetic sector instrument the ions generated are pushed out of the source by a repeller
potential of same charge as the ion itself (most often positive).
▪ They are then accelerated in an electric field of ca 3–8 kV and travel through an electrostatic field
region so that they are forced to fall into a narrow range of kinetic energies prior to entering the
field of a circular magnet.
▪ They then adopt a flight path through the magnetic field depending on their charge to mass (m/z)
ratio; the large ions are deflected less by the magnetic field:
𝒎
𝒛
=
𝑯𝟐𝒓𝟐
𝟐𝑽
where H is the magnetic field strength, r is the
radius of the circular path in which the ion travels,
and V is the accelerating voltage.
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Comparison of various analyzers
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7.5. Detectors
▪ The ion collection system measures the relative abundance of ion fragments of each mass.
▪ Several types of detectors are available for mass spectrometers. The detector used for most routine
experiments is the electron multiplier.
▪ Another type of detector is photographic plates coated with a silver bromide emulsion, it is sensitive
to energetic ions. A photographic plate can give a higher resolution than an electrical detector.
Faraday Cup:
▪ It consists of a hollow conducting electrode
connected to ground via a high resistance.
▪ The ions hitting the collector cause a flow of
electrons from ground to the resistor.
▪ The resulting potential drop across the resistor
is amplified. A plate held at about -80 V in
front of the collector, prevents any ejected
secondary electrons from escaping and
causing an anomalous reading.
Photographic Plates:
▪ This detector system is most sensitive than any
other detector because the photoplate
integrates the ion signal over a period of time.
▪ The photoplate are processed by the usual
photographic techniques and read with the aid
of densitometer.
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Electron Multipliers:
▪ It is most common means of detecting ions. It is made up of a series (12 to 24) of aluminum oxide
dynodes maintained at ever increasing potentials.
▪ Ions strike the first dynode surface causing an emission of electrons. These electrons are then
attracted to the next dynode held at a higher potential and therefore more secondary electrons are
generated.
▪ Ultimately, as numerous dynodes are involved, a cascade of electrons is formed that reult in an
overall current gain on the order of one million or higher.
▪ The high energy dynode(HED) uses an electrostatic field to increase the velocity of the ions and
serves to increase signal intensity and therefore sensitivity.
Faraday Cup Photoplate Electron Multiplier
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8.APPLICATIONS
▪ Mass spectrometry provides a highly specific method for determining or confirming the identity or
structure of drugs and raw materials used in their manufacture.
▪ Mass spectrometry in conjunction with either gas chromatography (GC–MS) or liquid
chromatography (LC–MS) provides a method for characterising impurities in drugs and
formulation excipients.
▪ Pharmaceutical Analysis
Bioavailability studies
Drug metabolism studies
Characterization of potential drugs
Drug degradation product analysis
Identifying drug targets
▪ Biomolecular characterization
Characterization of Proteins an Peptides
Oligonucleotides.
▪ Environmental Analysis
▪ Forensic Analysis
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