Pharmaceutical analysis

1. Mass Spectrometry
1

2. Introduction
 MS is an analytical technique that provides qualitative &
quantitative information, including the mass of molecules &
atoms in samples as well as the molecular structure of
organic and inorganic cpds
– It is an instrument that separates gas phase ionized
atoms, molecules, & fragments of molecules by the
difference in their mass-to-charge ratios.
 In MS, a substance is converted into fragment ions.
2

3. Introduction…
 The fragments (usually cations) are sorted on the basis of
mass-to-charge ratio, m/z.
 The bulk of the ions usually carry a unit positive charge, thus
m/z is equivalent to the MW of the fragment.
 The analysis of MS information involves the re-assembling of
fragments, working backwards to generate the original
molecule.
3

4.  A MS needs to perform three functions:
• Creation of ions – e.g. the sample molecules are
subjected to a high energy beam of electrons,
converting some of them to ions
• Separation of ions – as they are accelerated in an
electric field, the ions are separated according to mass-
to-charge ratio (m/z)
• Detection of ions – as each separated population of
ions is generated, the spectrometer needs to qualify
and quantify them
Mass Spectrometer
4

5. 5
Mass Spectrometer…

6. Inlet
Ion
source
Mass
Analyzer Detector
Data
System
High Vacuum System
Mass Spectrometer Block Diagram

7. 7
Inlet System:
To introduce a very small amount of sample (mol or less) into
the MS that converted to gaseous ions. ( a means for
volatilizing solid or liquid samples is presents).
Ion sources:
Convert the components of a sample into ions.
Mass analyzer
Analogous to grating in an optical spectrometer.
Dispersion is based upon the mass/charge ratios of the analyte
ions rather than upon the wavelength of photons.
Mass Spectrometer…

8. Detectors:
Convert the beam of ions into an electrical signal that can
then be processed, stored in the memory of a computer
and displayed or recorded in a variety ways.
Vacuum System:
To create low pressure (10-4 to 10-8 torr) in all the instrument
components except the signal processor and readout.
To prevent the ions of interest from colliding with
air molecules.
Mass Spectrometer…
8

9. 9
Ion sources
Starting point: Formation of gaseous analyte Ions.
Methods of ion formation: Two major categories:
1- Gas-phase sources
-The sample is first vaporized and then ionized.
-Restricted to thermally stable compounds of B.pt. < 500 0C.
-Limited to Compounds of MWt’s <103dalton .
2- Desorption sources
 The sample in a solid or liquid state is converted directly into
gaseous ions (not require volatilization of analyte molecules)
 Applicable to nonvolatile and thermally unstable samples
 Applicable to analytes having of 105 dalton or larger.
Ion sources

10. 10
Ionizing agent
Name
Basic Type
Energetic electrons
Electron Impact (E I)
Reagent gaseous ions
Chemical Ionization (CI)
High-potential electrode
Field ionization (FI)
Gas Phase
High-potential electrode
Field desorption (FD)
Desorption
High electric Field
Electrospray ionization (ESI)
Laser beam
Matrix-assisted desorption/ionization
(MALDI)
Fission fragments from
252Cf
Plasma desorption (PD)
Energetic atomic beam
Fast atom bombardment (FAB)
Energetic beam of ions
Secondary ion mass spectrometry (SIMS)
High temperature
Thermospray ionization (TI)
Ion sources

11. In a mass spectrometer, molecules in the gaseous state under low pressure are
bombarded with a beam of high energy electrons (about 70 electron-volts; eV)
This bombardment can first dislodge one of the electrons of the molecule and
produce a positively charged ion called “The molecular ion”
M + e’ M+• + 2 e’
Molecule high-energy electron molecular ion
The molecular ion contains also an odd number of electrons, thus it is a Free
radical, or generally “Radical Cation”
Electron Ionization
11

12. • This is done by adding a reagent gas into the ion
source.
• Electrons ionize the reagent gas (e.g. methane).
• These ions are highly reactive and react with the
analyte (XH).
• A molecular ion: or
Chemical Ionization
12

13. • At the end of this capillary, a fine aerosolis formed by nitrogen gas
flowing along the tip of the capillary.
• Because the mobile phase is volatile, the liquid droplets evaporate by
electrical potential & are flushed away by adrying gas.
• The analyte molecules remain charged & are extracted into the
vacuum area of MS.
• Used for Acidic or
basic analytes
Electrospray Ionization
13

14. Atmospheric Pressure Chemical Ionization
14
can then react with the analyte (MH):
&

15. hn
Laser
1. Sample is mixed with matrix (X)
and dried on plate.
2. Laser flash ionizes matrix
molecules.
3. Sample molecules (M) are
ionized by proton transfer:
XH+ + M  MH+ + X.
MH+
MALDI: Matrix Assisted Laser Desorption Ionization
+/- 20 kV Grid (0 V)
Sample plate

16. • ICP-MS
• The sample constituents are atomized into free
atoms, and some of the free atoms are also
ionized by losing an electron in hot plasma.
Inductively Coupled Plasma
16

17. 17
Mass Analyzers
Mass analyzers separate ions based on their mass-to-charge ratio (m/z)
– Several devices are available for separating ions with different m/z
ratios.
o magnetic-sector,
o quadrupole,
o time-of-flight and
o ion traps are most used.
Mass analyzer should be:
- Capable of distinguishing between minute mass differences.
- Allows passage of sufficient number of ions to yield measurable
currents.
- Key specifications are:
o resolution,
o mass measurement accuracy, and
o sensitivity.

18. =>
18
Magnetic-sector
•Amount of deflection depends on m/z.
•The detector signal is proportional to the number of ions hitting it.
•By varying the magnetic field, ions of all masses are collected and counted.

19. • Analyzer tube is surrounded by a magnet whose magnetic field deflects
the positively charged fragments in a curved path.
• At a given magnetic field strength, the degree to which the path is
curved depends on the mass-to-charge ratio (m/e) of the fragment:
• Path of a fragment with a smaller m/e value will bend more than that of
a heavier fragment. In this way, the particles with the same m/z values
can be separated from all the others.
• If a fragment’s path matches the curvature of analyzer tube, the
fragment will pass through the tube and out the ion exit slit (These ions
are said to be in register).
• A collector records the relative number of fragments with a particular
m/e passing through the slit. The more stable the fragment, the more
likely it will make it to the collector.
• Strength of magnetic field is gradually increased, so fragments with
progressively larger m/e values are guided through tube and out the exit
slit.
Mass Analyzers…
19

20. Quadrupole Mass Analyzer
Uses a combination of RF
and DC voltages to operate
as a mass filter.
• Has four parallel metal
rods.
• Lets one mass pass
through at a time.
• Can scan through all
masses or sit at one
fixed mass.

21. mass scanning mode
m1
m3
m4 m2
m3
m1
m4
m2
single mass transmission mode
m2 m2 m2 m2
m3
m1
m4
m2
Quadrupoles have variable ion transmission modes

22. 22
Time-of-flight (TOF) Mass Analyzer
•Ions are accelerated in pulses by means of an electric potential imposed
on a back plate right in the back of the ion source.
•The mass of each ion is thus determined based on its flight time to reach
the detector.
• Small ions reach the detector before large ones.

23. HYPHENATED TECHNIQUES
• Hyphenated Techniques combine
chromatographic and spectral methods to
exploit the advantages of both.
23

24. • GC-MS: the analytes are ionized under vacuum
conditions e.g. by EI, CI
• LC-MS: ionization is performed at atmospheric
pressure by ESI, APCI
• In both cases sample constituents are separated
by passage through a chromatographic column.
• Each cpd elutes into the mass spectrometer for mass
spectrometric analysis.
• MS - advanced chromatography detector.
Chromatography Coupled with MS
24

25. Fig. Total ion current chromatogram & mass spectrum for component 3 in a mixture
of four components
25

26. • Full scan and recording of spectra;
• Selected ion monitoring (SIM);
• Selected reaction monitoring (SRM).
26

27. The Mass Spectrum?
27
A mass spectrum is a presentation of the masses of the
positively charged ions (peaks) separated on the basis of
mass/charge (m/z) versus their relative concentrations.

28. Mass Spectrum (cont.)
28
Three types of peaks are observed in a MS Spectrum:
1. Base Peak
2. Molecular Ion Peak
3. Fragment Peaks
Base Peak
The most intense peak (most stable ion) in the mass spectrum
is called the Base Peak.
This Peak is assigned a value of 100%.
The intensities of the other peaks are reported as percentage
of the base peak.

29. Molecular Ion Peak
– In a Mass Spectrum Molecular Ion Peak is usually the peak
of highest mass number except for the isotope peak.
– It is important to note that there are possibilities and cases
where Base Peak and Molecular Ion Peak for a given
compound are same.
Mass Spectrum (cont.)
29

30. 30
Fragment Peaks
All other peaks obtained and seen in the Mass Spectrum are derived
from Molecular Ion Peak or Base Peak and are thus called Fragment
Peaks.
*Base peak can be also fragment peak
M+ peak
Base peak
F. peak
F. peak
Mass Spectrum (cont.)

31. Example: Pentane molecule:
Mass spectrum of pentane,
shown as a bar graph and in
tabular form.
Base peak represents fragment
that appears in greatest
abundance. Value of molecular
ion gives molecular mass of the
compound.
Note that:
The way by which the
molecular ion to be
fragmented depends
on the strength of its
bonds and stability of
the fragments.
31

32. Note that:
1- In some cases the base peak is the molecular ion peak, but in most cases it is
different from that of the molecular ion (according to the relative stability of ions
during fragmentation).
2- Peaks are commonly observed at m/e values one and two units less than the
m/e values of the carbocations because the carbocations can undergo further
fragmentation by losing one or two hydrogen atoms.
3- Small peak that occurs at m/e 73 (0.52%) is known as M+1 peak to indicate
that “It is one mass unit greater than the molecular ion(M+.),
The M+1 peak appear because most of elements have more than one naturally
occurring isotope.
32

33. Principal stable isotops of common elements:
From the table :
•For C, H, N and Si the principal heavier isotope is M+1.
•For O, Si, S, Cl and Br , the principal heavier isotope is the M+2
•For F and I, there is no heavier isotopes (not affect M+1 or M+2)
33

34. Some guides to determine molecular formula of
organic compounds:
 Nitrogen rule:
– If molecular ion (M+) is even number, compound must contain an
even number of N atoms (zero is considered as even number)
 Number of Carbon atoms:
– Relative abundance of M+1 peak can be used for determination of
number of carbons assuming that silicon and large number of
nitrogens (more than 3 N) are not present.
No of Carbons = Relative abundance of M+1/ 1.10
 Relative abundance of M+2 peak indicates the presence (or
absence) of Oxygen (0.2), Silicon (3.35),Sulfer (4.4), Chlorine
(32.5) & Bromine (98.0)
34

35. Some guides to determine molecular formula of
organic compounds…
 Molecular formula after this can be established by adding
the suitable number of hydrogens and oxygens if necessary.
 The index of hydrogen deficiency (Due to double and triple
bonds and cyclization) can be calculated for the compounds
containing C, H, N, O, S & halogens from the formula:
Index = Carbons - Hydrogens/2 – Halogens/2 + Nitrogens/2 +1
– Divalent atoms like O and S are not counted in formula. The index
mainly used to indicate the number of double bonds.
35

36. High Resolution Mass Spectroscopy:
• Resolution: A measure of how well a mass
spectrometer separates ions of different mass.
– Low resolution: Refers to instruments capable of
separating only ions that differ in nominal mass;
that is ions that differ by at least 1 or more atomic
mass units (amu).
– High resolution: Refers to instruments capable of
separating ions that differ in mass by as little as
0.0001 amu.
36

37. High Resolution Mass Spectroscopy…
• Example:
– A molecule with mass of 44 could be C3H8, C2H4O, CO2, or
CN2H4.
– Thus, high resolution mass spectrometer is capable of
measuring mass with a high accuracy so can easily
distinguish among these 4 molecules.
C3H8 C2H4O CO2 CN2H4
44.06260 44.02620 43.98983 44.03740
37

38. Fragmentation rules in MS
1. Intensity of M.+ is Larger for linear chain than for branched compound
2. Intensity of M.+ decrease with Increasing M.W.
3. Cleavage is favored at branching
 reflecting the Increased stability of the carbocations
 This is a consequence of the increased stability of a 30 carbocation over a 20 ,
which in turn is more stable than a 10 one.
R
R”
CH
R’
Loss of Largest Subst. Is most favored
38

39. C
H3
CH3
C
H3
C
H3
CH3
C
H3
C
H3
MW=170
M.+ is absent with heavy branching
Fragmentation occur at branching: largest fragment loss
Branched alkanes
39

40. Fragmentation rules in MS…
4. Aromatic Rings, Double bond, Cyclic
structures stabilize M.+
5. Double bond favor Allylic Cleavage
 Resonance – Stabilized Cation
C
H2
+
CH CH2 R
- R
.
C
H2
+
CH CH2
C
H2 CH CH2
+
40

41. 41

42. Fragmentation rules in MS….
6. a) Saturated Rings lose a Alkyl Chain (case
of branching)
b) Unsaturated Rings  Retro-Diels-Alder rxn
(Two Bond Cleavage)
CH2
CH2
CH2
CH2
+
+ . + .
R
+ .
+
-R
.
42

43. Fragmentation rules in MS….
7. In alkyl-substituted aromatic compounds,
cleavage is very probable at the bond β to the
ring.
– Giving the resonance-stabilized benzyl ion or more
likely, the tropylium ion:
Tropylium ion 43

44. 8. C-C Next to Heteroatom cleave leaving
the charge on the Heteroatom
Fragmentation rules in MS….
44

45. 9- Cleavage associated with elimination of small, stable, neutral molecules
such as carbon monoxide, olefines, water, ammonia, ketones or alcohols,
often occur with some sort of rearrangement.
 One important example is the so-called McLafferty rearrangement (Two
step fragmentation or two bond cleavage)
 To undergo a McLafferty rearrangement, a molecule must possess an
 Appropriately located heteroatom
 A П -system (usually a double bond), and
 An abstractable hydrogen atom g to the C = O system
45
Fragmentation rules in MS….

46. Pharmaceutical analysis
Bioavailability studies
Drug metabolism studies, pharmacokinetics
Characterization of potential drugs
Drug degradation product analysis
Screening of drug candidates
Identifying drug targets
Biomolecule characterization
Proteins and peptides
Oligonucleotides
Environmental analysis
Pesticides on foods
Soil and groundwater contamination
Forensic analysis/clinical
Applications of Mass Spectrometry

47. MS/MS
MS/MS
+
+
+ +
+
1 peptide
selected for
MS/MS
The masses of all
the pieces give an
MS/MS spectrum
Peptide
mixture
Have only masses
to start

48. Interpretation of an MS/MS spectrum to derive
structural information is analogous to solving
a puzzle
+
+
+ +
+
Use the fragment ion masses as specific pieces of
the puzzle to help piece the intact molecule back
together