The document discusses various applications of mass spectrometry including molecular mass determination, identification of elements through isotopic ratios, isotopic abundance determination, isotopic dilution methods, distinguishing between cis and trans isomers, evaluation of heat sublimation, environmental analysis, forensic analysis, clinical research applications like drug screening and disease biomarker detection, protein identification, protein sequencing, determination of ionization potential and bond dissociation energies, and impurity detection. Mass spectrometry is widely used across many fields due to its high sensitivity and selectivity.

1. Applications of mass
spectrometry
SUBJECT: MODERN PHARMACEUTICAL ANALYTICAL TECHNIQUES (MPA 101T)
PRESENTED BY: SHAIKH ASIF JAMIL
PRESENTED TO: DR OSAIR ALAM
DEPARTMENT: PHARMACEUTICAL CHEMISTRY
BRANCH: PHARMACEUTICAL ANALYSIS
1

2. Molecular mass determination
 When substance is bombarded with moving
electron and the mass spectrum is recoded
them the mass of the peak at the highest m/e
reveals the molecular mass accurately .
 Best method to determine molecular weight
accurately.
 This method is only accurate when no ions
heavier than parent ion is formed.
2

3.  Chlorine, bromine, and sulfur can all be identified by their isotopic ratios. The isotopic
ratios of commonly encountered elements are summarized in Table 1 below 3
Element
[M+] [M + 1] [M + 2]
Hydrogen 1H 100.0%
Carbon 12C 100.0% 13C 1.1%
Nitrogen 14N 100.0% 15N 0.4%
Oxygen 16O 100.0% 18O 0.2%
Sulphur 32S 100.0% 33S 0.8% 34S 4.4%
Chlorine 35CL 100.0% 37CL 32.5 %
Bromine 79BR 100.0% 81BR 98.0%
Iodine 127I 100.0%
Table1 Relative Isotopic Abundances of Common Elements

4.  The ratios have been normalized to the most abundant isotope to illustrate the expected peak
ratios in a mass spectrum.
4
Fig.01

5.  Compounds containing one bromine have a [M + 2] peak that is approximately the same
height as a [M+] peak (Figure02).
5
Fig 02

6. Determination Isotopic abundance
 The isotopic abundance of easily
vapourisable elements can be
determined using mass spectrometer .
 It can be done by converting the ratio of
peak heights into percentage abundance
of the isotopes of the element.
6

7. Isotopic dilution method
 Mass spectrometry is used to determine the amount of component of complicated
mixture from which it cannot be separated quantitatively.
7

8. Distinction between cis and trans-
isomers
 Mass spectrometry is successful in making distinction
between cis and trans-isomers.
 Both cis and trans-isomers yield similar mass spectra
but the molecular ion peak for trans is more intense
than cis-isomer.
 Another distinction is that the dehydration fragment
for cis-isomer is much stronger than trans-isomer.
8

9. Evaluation of heat sublimation
 If the sublimating solid is kept in thermostated reservoir at the given
temperature , the solid phase and vapour phase(of the solid that
sublimes)in the reservoir are in equilibrium.
 Suppose a small quantity of the vapour is diffused from reservoir into
ionisation chamber .
 If the mass spectrum is run , there occurs a change in peak intensity which
directly proportional to the vapour pressure of the sample in the ion
source.
 Similarly ,the vapour pressure of subliming substance are calculated from
the peak heights in the mass spectrum at different temperatures.
9

10. It is Used In Environmental Analysis
 Mass spectrometry coupled to gas chromatography
(GC) and liquid chromatography (LC) are ideal
techniques for the determination of organic
contaminants in wastewater due to the separation
capability of GC and LC coupled to the selectivity and
sensitivity of MS..
 In recent years, developments in chromatography such
as the introduction of ultra high-performance liquid
chromatography (UPLC or UHPLC), which utilizes the
same separation methodology as conventional HPLC,
but uses columns packed with smaller particles
(generally about 2 µm particle size or less) has further
enhanced the applicability of LC-MS in wastewater
analysis.
10

11. Used in forensic analysis
 Liquid chromatography-mass spectrometry (LC/MS) is a
forensics technique frequently used by toxicologists to
analyze substances for suspected illicit drugs and to
confirm the findings of presumptive drug tests.
 Analysts might test the substances themselves, or
human tissue or fluid samples to gauge whether an
individual has ingested the substance, whether
intentionally or not
11
.

12.  Here, a technique such as GC is used to to separate
samples before they enter an isotope ratio mass
spectrometer where specific elements and isotopes within
a sample can be analyzed to determine how old a sample is
using isotopic dating.
 The technique involves measuring the different isotopic
abundances of hydrogen, carbon, oxygen and nitrogen.
Such variations can arise when biological, physical or
chemical processes cause isotopic fractionation. The
lightest isotopes are the most affected by fractionation,
and the differences in isotope ratio values that occur as a
result of fractionation effects are often specific to particular
origins and production processes.
 In forensics, scientists can use these tiny differences to link
or distinguish between samples, even with only trace
amounts of samples.
12

13. Used In Clinical Research 13
RISE OF MS USE IN CLINICAL RESEARCH:
 A major impetus that moved MS from the research laboratory to the clinical laboratory was
the accident on the aircraft carrier Nimitz. On May 26, 1981, an aircraft crashed while landing
on the Nimitz, killing 14 and injuring 45 (5). Subsequent immunoassay tests demonstrated
that a large percentage of urine samples from servicemen were positive for marijuana
metabolites. This prompted President Reagan to develop a zero tolerance for drugs of abuse
in the military .
 Due to a large number of false-positive immunoassay results, antibody-based drug screens
began to be considered “presumptive” until confirmed by GC-MS . It was clear that the early
use of drug testing was effective at reducing the number of positive employee drug test
results, because positive rates dropped from 18% to 8% over a 10-year time span (8). Several
studies also demonstrated that urine drug testing was cost-effective (8, 9). The requirement
for GC-MS confirmation drove the development of MS in toxicology laboratories, where it also
began to be used for therapeutic drug monitoring.

14. 14
 major impacts of MS include confirmation of immunoassay-positive drug
screens
 (1) identification of inborn errors of metabolism
 (2) analysis of steroid hormones
 (3.More recently, MS mostly used in for microbial identifications

15. Determination of ionisation potential
 The ionisation potential of a molecule or
atom may be defined as the energy
required to remove an electron from the
molecule.
 AB AB + e
 Minimum energy that bombarding
electron must posses in order to produce
a molecular ion from the neutral molecule
is referred as ionisation potential .
15
+ -

16. Used in Proteomics and genomics.
 Evidence of gene expression at the protein level using mass spectrometry-
based proteomics is increasingly used in refinement of raw genome
sequencing data. In a typical "proteogenomic" experiment, the whole
proteome of an organism is extracted, digested into peptides and
measured by a mass spectrometer.
16

17. Determination of bond dissociation
energies
 When a molecule AB is bombarded with electron positive molecular ion is
produced
 AB + e- AB + 2 e
17
+ -

18. Peptide isolation ,identification 18

19. Protein Identification 19
The process of protein identification through mass spectrometry is done in two main ways:
1.Peptide Mass Fingerprinting
 Peptide mass fingerprinting typically
uses the masses of peptides derived
from a spectrum as to check against a
database of predicted peptide masses.
These predicted masses are recorded
from digestion of a list of well
documented proteins.
 Matrix Assisted Laser Desorption
Ionization - Time of Flight(MALDI-TOF)
mass spectrometers are the
instrumentation that is commonly used
for this type of peptide analysis.

20.  This technique utilizes collision-induced dissociation. This process breaks proteins within
the peptide backbone and because of this fragmentation, comparisons between the
observed fragment sizes and the database of predicted masses is possible.
20
2.Tandem MS

21. Protein sequencing
 Tandem MS can be used in conjunction with fragmentation methods such as
trypsin digestion. This allows a sample to generate overlapping peptides of varying
length.
 This method works in a similar fashion to the approach taken by genome
sequencers, when they generate overlapping DNA sequence fragments and
assemble into the genome.
 On the mass spectra, the mass difference between two overlapping peaks can be
used to determine the difference in sequence based on the well documented data
on the weights of the amino acids.
21

22.  Sequences are usually determined using one or more search algorithms linked
to an appropriate database
22

23. Disease Biomarker detection 23
 Research has shown that tumors can introduce proteins into various bodily fluids which
might not normally exist at those concentrations in those locations.
 This has led to the interest in the study of mass spectrometry in the use of clinical early
detection of cancer through analysis of these fluids.
 This method was used in a study, A Density-Based Proteomics Sample Fractionation
Technology: Folate Deficiency–Induced Oxidative Stress Response in Liver and Brain, on
biomarkers for oxidative stress, glutathione peroxidase 1 (GPx1) and glucose-regulated
protein 75 (GRP75), as a result of folate deficiency.

24. 24
These disease-associated molecules are known as biomarkers.
Edge Fractionation Technology provides a powerful density based separation and enrichment
method for rapid screening of potential Biomarkers

25. Determination of latent
heat of vaporisation of
liquid
 Mass spectrometry is can be used for the
determination of latent heat of vaporisation of
liquids by using Clausius -Clapeyron equation.
 logp= - H/RT
25

26. Studies involving reaction mechanism
26
Tenamfetamine
Medomafetamine

27. Impurity detection
 Mass spectrometry is one of the best method for detection of impurity in
parts per million only if their structure differ considerably from those of
the major component.
 If the molecular weights of the impurity are much larger than major
components their detection is more easy because of the higher mass
peaks are free from contributions by those of the major components .(MW
of impurity should be higher than major component /analyte)
 On the other hand molecular weights of the impurities are much lower
than the major component it is hard to detect impurity .
27

28. Quantitative analysis of mixture
 In general experience interpretation of spectrum of pure compound is a doubtful
process hence, identification of mixture is even more difficult.
 An experienced analyst can however interpret characteristics in the mass spectrum
provided there are unique peaks for each compound present .
 If the identities of compound in the sample mixtures are known , the quantitative
analysis is feasible.
28

29. Characterisation of polymer
 As polymers are pyrolyzed (decomposed by heating ) and then the
pyrolyzed product is fed into the inlet of mass spectrometer and identified.
 From the results structure of the polymer can be anticipated.
 For example, the mass spectrometry will be able to distinguish chlorinated
polymer to know weather the chlorine occurs randomly or occurs in the
block in the structure of polymers.
29

30. Reference
 Instrumental method of chemical analysis by Gurdeep r chatwal ,Sham K Anand
Himalaya publication page no. 2.296-3.302
 Mass Spectrometry as a Tool in Forensic Science (news-medical.net)
 https://academic.oup.com/clinchem/article/62/1/92/5611766
 (PDF) Mass spectrometry at the interface of proteomics and genomics (researchgate.net)
 Pharmastuff4u: Applications Of Mass Spectrometry
 protein sequencing mass spectrometry - Bing images
 (PDF) Mass Spectrometry for Biomarker Development (researchgate.net)
 https://images.app.goo.gl/EYY8sUrkfjbnDB2s8
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31.  https://www.google.com/search?client=ms-android-xiaomi-
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