Analytical Chemistry

/ Instrumentation

Atomic Spectroscopy

Applications of Mass Spectrometry

Paper No. : 06 Atomic Spectroscopy

Module : 35 Applications of Mass Spectrometry

Principal Investigator: Dr. Nutan Kaushik, Senior Fellow The Energy and Resouurces Institute (TERI), New Delhi

Co-Principal Investigator: Dr. Mohammad Amir, Professor of Pharm. Chemistry, Jamia Hamdard University, New Delhi

Paper Coordinator: Dr. Mymoona Akhtar, Associate professor, Dept. of Pharm. Chemistry, Jamia Hamdard, New Delhi.

Content Writer: Dr. S.K.Raza, Former Director, Institute of Pesticide formulation Technology, Gurugram

Content Reviwer: Dr. Nutan Kaushik, Senior Fellow , The Energy and Resouurces Institute (TERI), New Delhi

Analytical Chemistry

/ Instrumentation

Atomic Spectroscopy

Applications of Mass Spectrometry

APPLICATIONS OF

MASS SPECTROMETRY

1. Aim of the Modules

To give an account of various applications of mass spectrometry in scientific

disciplines and areas.

2. Objectives of the Modules

At the end of this module one should be able to :

Understand the importance of mass spectrometric techniques vis-à-vis its application in

various scientific field and its role in solving complex problems.

3. Introduction

Mass spectrometry can be used to :

• Detect and identify the use of steroids in athletes

• Monitor the breath of patients by anesthesiologists during surgery

• Determine the composition of molecular species found in space

• Determine whether honey is adulterated with corn syrup

• Locate oil deposits by measuring petroleum precursors in rock

• Monitor fermentation processes for the biotechnology industry

• Detect dioxins in contaminated fish

• Determine gene damage from environmental causes

• Establish the elemental composition of semiconductor materials

• Identify structures of biomolecules, such as carbohydrates, nucleic acids and steroids

Description of Module

Subject Name Analytical Chemistry / Instrumentation

Paper Name Atomic Spectroscopy

Module Name/Title Applications of Mass Spectrometry

Module Id 35

Pre-requisites

Objectives

Keywords

Analytical Chemistry

/ Instrumentation

Atomic Spectroscopy

Applications of Mass Spectrometry

• Sequence biopolymers such as proteins and oligosaccharides

• Determine how drugs are used by the body

• Perform forensic analyses such as conformation and quantitation of drugs of abuse

• Analyze for environmental pollutants

• Determine the age and origins of specimens in geochemistry and archaeology

• Identify and quantitate compounds of complex organic mixtures

4. Important Applications of Mass Spectrometry

Mass spectrometry has both qualitative and quantitative uses. These include identifying

unknown compounds, determining the isotopic composition of elements in a molecule,

and determining the structure of a compound by observing its fragmentation. Other uses

include quantifying the amount of a compound in a sample or studying the fundamentals

of gas phase ion chemistry (the chemistry of ions and neutrals in a vacuum). MS is now in

very common use in analytical laboratories that study physical, chemical, or biological

properties of a great variety of compounds. Some of the important applications are

discussed below.

4.1 Identification of Small Molecules - “Known Unknowns”

The term known unknown was introduced to indicate compounds that are unknown to the

researcher, but actually described somewhere in the scientific literature and/or available in

compound databases. The “known unknowns” differ from the target compounds searched for

in targeted residue analysis, which can be considered as “known knowns.” The identification

of “known unknowns” is a highly challenging task. This task can generally not be performed

by using MS technologies alone, especially because MS often is not a very powerful tool in

clearing stereoisomerism issues. Thus, in this, MS analysis should be combined with other

techniques, especially Nuclear Magnetic Resonance (NMR) Spectroscopy. In practice, this is

not straightforward, if one keeps in mind that NMR needs ∼100–1000 times more (pure)

compound to get an interpretable spectrum. Besides, MS and MSn are readily performed

within the timescale of high-resolution chromatography, whereas NMR requires far longer

data acquisition times, typically 8–16 hrs. when only low concentrations are available. Thus,

either fraction collection or time-consuming stop-flow operations have to be performed, when

multiple unknowns within one LC run are to be identified by NMR. The general procedure

of the identification of known unknowns consists of the following steps.

i) One needs to collect as much information on the unknowns as possible.

Parameters such as origin of the sample, solubility, thermal stability, and possibly

underlying chemistry may provide valuable pieces of information.

ii) One needs to establish whether the sample is actually amenable to MS analysis, by

GC–MS in EI mode, LC–MS in either positive-ion or negative-ion mode (or

preferably both), MALDI-TOF-MS, or by any of the other available MS

techniques.

iii) If the first MS data are acquired by HRMS, the calculation of the elemental

composition of the unknown is possible, especially when a soft-ionization

technique is applied.

iv) On the basis of the elemental composition and the general information on the

unknown, compound data bases may be searched for known structures, which will

be successful for the “known unknowns”.

v) Subsequently acquired MS–MS or MSn data allow filtering the known structures

from the database search by checking the observed fragmentation behavior against

Analytical Chemistry

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Atomic Spectroscopy

Applications of Mass Spectrometry

predicted fragmentation of the database-retrieved structures. In favorable cases,

this leads to a (number of) potential structure proposal(s) for the unknown.

vi) In the end, standards should be purchased or synthesized and analyzed to check

retention time, fragmentation behavior, and possibly other properties.

At this stage, as a result it may be reported that a structure proposal for the unknown is

available, for which the calculated elemental composition is in agreement with the measured

accurate mass of the precursor ion, the main fragments in the product ion spectra could be

assigned and seem to agree with the proposed structure, and chromatographic and MS

characteristics seem to be in agreement with that of a synthetic standard (or an “known

unknowns”). Further experiments may need to be performed, for example, preparative LC in

order to collect sufficient material for NMR analysis, to further confirm the structure and rule

out isomerism issues.

4.2 Identification of Drugs and their Metabolites

Mass Spectrometry has been very successfully employed for the identification of drugs and

their metabolites. The structure elucidation of related substances, be it synthetic by-products

or degradation products of active pharmaceutical ingredients drug metabolites, or natural

products within a particular compound class, can be performed by more or less similar

strategies. The acquisition of MS, MS–MS, and/or MSn spectra of the parent drug and the

thorough interpretation of these spectra is of utmost importance to the success of the study.

After the analysis of relevant samples mass spectrometry (GC-MS or LC-MS) and data

processing to search for potential related substances, MS–MS or MSn have to be acquired.

Nowadays, this is mostly done by DDA or data-independent MSE procedures, using

automatic switching between survey MS and (dependent) MS–MS or MSn experiments,

preferably using HRMS. Finally, interpretation of the data has to be performed, often

followed by additional LC–MSn experiments, isolation of particular compounds, synthesis of

standards, and NMR analysis.

4.2.1 Analysis of Haloperidol

Haloperidol is a dopamine inverse agonist of the typical antipsychotic class of medications.

Haloperidol is an older antipsychotic used in the treatment of schizophrenia and acute

psychotic states and delirium. A long-acting decanoate ester is used as an injection given

every four weeks to people with schizophrenia or related illnesses who have poor adherence

to medication regimens and suffer frequent relapses of illness, or to overcome the drawbacks

inherent to its orally administered counterpart that burst dosage increases risk or intensity of

side effects. In some countries, such as the United States of America, injections of

antipsychotics such as haloperidol can be ordered by a court at the request of a psychiatrist.

Analytical Chemistry

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Atomic Spectroscopy

Applications of Mass Spectrometry

4.2.2 Analysis of Ramelton

Ramelteon, marketed as Rozerem by Takeda Pharmaceuticals North America, is the first in a

new class of sleep agents that selectively binds to the MT1 and MT2 receptors in the

suprachiasmatic nucleus (SCN), instead of binding to GABA A receptors, such as with drugs

like zolpidem, eszopiclone, and zaleplon. Ramelteon is approved by the U.S. Food and Drug

Administration (FDA) for long-term use. Ramelteon does not show any appreciable binding

to GABAA receptors, which are associated with anxiolytic, myorelaxant, and amnesic effects.

4.2.3 Analysis of Nephazodone and its Metabolites

The experimental and data interpretation procedures in the identification of in-vitro drug

metabolites may be illustrated for the antidepressant drug nefazodone. The structure of

nefazodone, its MS–MS spectrum, the identity of a number of its fragments, and relevant

profile groups are given below.

Plot Help /

Nefazo

done

12

0

Plot Help / Software

Nefazodon

e

120

100

Analytical Chemistry

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EI Mass Spectrum of Nephazodone

Detection of Nefazodone Metabolites by HR-MS

20

50 100 150 200 250

m/

300 350 400

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HR-MS Analysis of GSH-trapped Reactive Metabolites of Ticlopidine in rat LMs using

various data mining methods

4.3 Analysis of Lipids by Mass Spectrometry

Lipids are composed of eight categories; around 1.68 million species. The large amount of

categories and the extremely complex structures of lipids lead to a formidable challenge to

fully analyze all lipids. Nowadays, there are two strategies to analyze lipids: targeted lipids

analysis and non-targeted lipid analysis. The targeted lipids analysis focuses on known lipids,

and develops a specific method with a high sensitivity for the quantitative analysis of these

specific lipids. Non-targeted lipids analysis aims to detect every lipid species simultaneously.

In order to successfully realize the qualitative and quantitative analysis of lipids, many

analytical methods have been developed, including thin-layer chromatography (TLC), gas

chromatography (GC), liquid chromatography (LC), enzyme-linked immunosorbent assays

(ELISA), nuclear magnetic resonance (NMR) and mass spectrometry (MS). Among them, the

MS-based method is the best in terms of high sensitivity and specificity, high throughput and

high accuracy. In particular, the extensive use of electrospray ionization for lipid analysis and

the improvement of mass analyzers in mass spectrometer, including the combination of

different mass analyzers and the development of a high-resolution mass analyzer, has greatly

increased the performance of MS in lipid analysis and revived lipid studies. In addition, the

biological system is extremely complex, and it is required to extract the lipids from the

biological system for further analysis. Furthermore, the studies in lipidomics have generated

overwhelming amounts of data, which need bioinformatics technology to aid in data

processing for acquiring meaningful biology information. Taken together, lipid analysis

needs a serial of methods and technologies, including lipid extraction methods, MS-based

analytical technologies and bioinformatics tools. A flowchart of the study of lipidomics is

shown in the following Figure.

Analytical Chemistry

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A Flowchart of the Study of Lipidomics

The following figure shows the total ion chromatograms (TICs) in positive and negative

ionization mode of a yeast WT lipid extract with highlighted regions where

lysoglycerophospholipids (LGPs), glycerophospholipids (GPs), diacylglycerols (DGs),

sphingolipids (SPLs) and triacylglycerols (TGs) elute. (A) Corresponding chromatogram of

yeast WT grown in YPD in positive ESI mode (B) Chromatogram of the same WT sample

under identical chromatographic conditions in negative ion mode.

4.4 Analysis of Proteins and Peptides by Mass Spectrometry

Proteins and peptides are linear polymers made up of combinations of the 20 amino acids

linked by peptide bonds. Proteins undergo several post translational modifications, extending

the range of their function via such modifications. The term Proteomics refers to the analysis

of complete protein content in a living system, including co- and post-translationally

modified proteins and alternatively spliced variants. Mass Spectrometry has now become a

Analytical Chemistry

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crucial technique for almost all proteomics experiments. It allows precise determination of

the molecular mass of peptides as well as their sequences. This information can very well be

used for protein identification, de novo sequencing, and identification of post-translational

modifications. The basic lab experimental steps for protein analysis by MS are given below.

i) Proteins digested with an enzyme to produce peptides

ii) Peptides charged (ionized) and separated according to their different m/z ratios

iii) Each peptide fragmented into ions and m/z values of fragment ions are measured

iv) Steps 2 and 3 performed within a tandem mass spectrometer.

v) Proteins consist of 20 different types of amino acids with different masses (except

for one pair Leu and i-Leu)

vi) Different peptides produce different spectra; The spectrum of a peptide is used to

determine its sequence.

Matrix Assisted Laser Desorption Ionization (MALDI) and Electrospray Ionization (ESI) are

the most commonly used techniques for mass spectrometric analysis of proteins and peptides.

MALDI is however, limited to solid state while ESI is best suited for liquids. ESI is better

for the analysis of complex mixture as it is directly interfaced to a separation technique (i.e.

HPLC or CE). MALDI is more “flexible” (MW from 200 to 400,000 Da). The whole

strategy is based on the breaking of protein molecules into peptides by using enzymes

(proteases) such as trypsin, MS/MS then breaks the peptide molecules into fragment ions and

measures the masses of each peace by giving the m/z ration of each ion. Following figure

shows the fragmentation of a peptide under MS/MS.

Analytical Chemistry

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Atomic Spectroscopy

Applications of Mass Spectrometry

The following figure shows Large-scale Analysis of in Vivo Phosphorylated Membrane

Proteins by Immobilized Metal Ion Affinity Chromatography and Mass Spectrometry,

Analytical Chemistry

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Atomic Spectroscopy

Applications of Mass Spectrometry

4.5 Analysis of Oligonucleotides by Mass Spectrometry

Oligonucleotides (DNA or RNA), are linear polymers of nucleotides. These are composed of

a nitrogenous base, a ribose sugar and a phosphate group. Oligonucleotides may undergo

several natural covalent modifications which are commonly present in tRNA and rRNA, or

unnatural ones resulting from reactions with exogenous compounds. Mass spectrometry

plays an important role in identifying these modifications and determining their structure as

well as their position in the oligonucleotide. It not only allows determination of the

molecular weight of oligonucleotides, but also in a direct or indirect manner, the

determination of their sequences.

Analytical Chemistry

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Applications of Mass Spectrometry

(A) The MALDI linear TOF mass spectrum of a DNA 40-mer in negative

ion mode. (B) The MALDI reflection TOF mass spectrum of an

RNA 195-mer in the positive ion mode. In both cases, the spectra

were obtained using 3-HPA as matrix and a laser at 337 nm.

4.6 Mass Spectrometry in Plant Biotechnology

Plants produce more than 200,000 metabolites, many of which play specific roles in allowing

adaptation to specific ecological niches. Therefore, the main problems encountered when

characterizing the plant metabolome have to do with the fact that in comparison to the

proteome or transcriptome, the metabolome is highly complex in nature, due to the enormous

chemical diversity of the compounds. In addition, there is a wide range of metabolite

concentrations, which can vary over nine orders of magnitude (pM to mM). These large

variations in the nature and the concentration of analytes to be studied provide challenges to

all the analytical technologies employed in metabolomic strategies. GC-MS is one of the

most widely used analytical techniques in plant metabolomics. Qualitative and Quantitative

analysis of wide range of volatile and/or derivatized nonvolatile metabolites with high

thermal stability can be performed by using mass spectrometric techniques. After separation,

the eluted metabolites are identified by mass spectrophotometers. Direct injection MS

analysis may also be applied for the phenotyping of plants, that is, Fourier transformed-MS

(FT-MS) provides ultimate limit of detection and mass measurement precision to enable

metabolomic analyses.

DNA 40-mer

(M−2H)2−

(M−H)−

A

B RNA 195-mer

(M+2H)2+

(M+H)+

(2M+H)+

Analytical Chemistry

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Atomic Spectroscopy

Applications of Mass Spectrometry

GC-MS Chromatogram of a Metabolic Mixture

LC/TOF-MS Ion Chromatograms of detected flavonoids acquired at ESI+

(a) and ESI- (b) from the ethanol extract of R. rosea aerial parts.

4.7 Analysis of Anabolic Steroids by Mass Spectrometry

Anabolic steroids, also known more properly as anabolic–androgenic steroids (AAS) are

steroidal androgens having large medicinal values. Enhancement of athletic performance

Analytical Chemistry

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Applications of Mass Spectrometry

through anabolic steroids is forbidden in human sports. Global fight against doping in sports

is supervised by the World Anti-Doping Agency (WADA). In practice, drug abuse is

controlled by way of testing of athletes. Urine or blood samples are collected from athletes,

either prior to or during contests; Test samples are analyzed for banned substances in

analytical laboratories accredited by WADA. Detection of Anabolic Steroids is demanding

due to the presence of numerous different steroids, their extensive metabolism and their low

concentration in urine. A capillary gas chromatograph coupled to a benchtop quadrupole

mass spectrometer (GC/MS) has been the backbone of testing of anabolic steroids. Although

GC/MS allows fairly successful large scale screening, more efficient instrumental techniques

such as high resolution mass spectrometry (HRMS), tandem mass spectrometry (MS/MS) and

liquid chromatography/mass spectrometry (LC/MS) are also needed to enhance selectivity

and sensitivity of the measurements.

4.8 Mass Spectrometry in Food Safety

The use of powerful mass spectrometric detectors in combination with Gas Chromatography

(GC) and Liquid Chromatography (LC) has played a vital role to solve many problems

related to food safety. These techniques are especially well suited for, but not restricted to

the analysis of food contaminants within the food safety area. There are basic legislation in

different parts of the world for the control of these contaminants. The latest innovations in

mass spectrometry have influenced the best control of food allowing an increase in the food

safety and quality standards. The major contaminants in food are :

• Pesticides

• Drugs – Antibiotics

• Heavy Metals

• Aflatoxins

• Environmental Contaminants such as :

• Poly Aromatic Hydrocarbons (PAHs)

• Poly Chlorinated Biphenyls (PCBs)

• Dioxins

• Furans

Mass Spectrometry can be used for all the above chemicals present in traces in various food

matrices. Mass spectrometry coupled a gas chromatograph has been successfully employed

for the analysis of both organochlorine (OC) and organophosphorus (OP) pesticides in

various food matrices. The following figures for example depict the analysis of these

pesticides in sugarcane.

Analytical Chemistry

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Atomic Spectroscopy

Applications of Mass Spectrometry

More than 280 pesticide residues, including difficult polar species, show excellent peak shape

and retention on a C18 LC/MS as shown below.

RT Pesticides 20.126 α-HCH 20.795 β-HCH

21.012 γ-HCH

21.757 δ-HCH

22.690 Alachlor

22.887 Heptachlor

23.293 Fenitrothian

23.783 Aldrin

24.480 Pendimethalin

25.357 O.P- DDE

25.451 Butachlor

25.630 α-Endosulfan

26.129 P,P-DDE

26.290 O, P-DDD

27.034 β-Endo

27.128 PP-DDD

27.185 OP-DDT

27.920 Endosulfan sulfate

28.005 PP-DDT

29.089 Bifenthrin

29.324 Fenpropathrin

30.304 λ-Cyhalothrin

32.350 Cypermethrin

34.348 Fenvalerate

34.602 Fluvalinate

36.129 Deltamethrin

RT Pesticides 19.815 Monocrotophos 20.012 Phorate 20.470 Dimethorate 20.798 Atrazine 22.503 Chlorpyriphosmethyl 23.668 Methyl parathion 23.520 Malathion 23.684 Chlorpyriphos 24.079 4-4‘- Dichlorobenzophenone 24.904 Qninalphos 26.032 Prefenophos 27.118 Ethian 27.469 Triazophos

OC Pesticides

OP Pesticides

Analytical Chemistry

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Applications of Mass Spectrometry

Liquid chromatography coupled to mass spectrometry and tandem mass spectrometry has

been used for the detection of aflatoxins in various food matrices at ng/ml levels as shown in

the following figure.

5. Conclusions

• Mass Spectrometry due to its high sensitivity and selectivity can found application in

a variety of fields.

• Recent innovations in mass spectrometric techniques has made it a highly sought after

technique in almost all areas of scientific research.

• Only a few of these applications have been discussed in this limited presentation.

6. Bibliography

• Edmond E. Hoffmann and Vincent Stroobant, Mass spectrometry-Principles and

Applications, 3rd Edition, John Wiley & Sons (2007).

• H. Steen and M. Mann. “The ABC’s (and XYZ’s) of Peptide Sequencing” Molecular

Cell Biology, Nature Reviews. 5, 699, (2004)

Analytical Chemistry

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Applications of Mass Spectrometry

• T. S. Nuhse, A. Stensballe, O. Jensen, and S. Peck. “Large-scale Analysis of in

Vivo Phosphorylated Membrane Proteins by Immobilized Metal Ion Affinity

Chromatography and Mass Spectrometry” Molecular & Cellular Proteomics,

2.11, 1234 (2003).

• R. Aebersold and D. Goodlett. “Mass Spectrometry in Proteomics” Chem. Rev.,

101, 269 (2001).

• Malik AK , Blasco C, Picó Y., Liquid chromatography-mass spectrometry in food

safety, J Chromatogr A, 1217(25):4018-40, 2010.