Atomic Spectroscopy
Transcript of Atomic Spectroscopy
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
/ Instrumentation
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
/ Instrumentation
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
/ Instrumentation
Atomic Spectroscopy
Applications of Mass Spectrometry
EI Mass Spectrum of Nephazodone
Detection of Nefazodone Metabolites by HR-MS
20
50 100 150 200 250
m/
300 350 400
Analytical Chemistry
/ Instrumentation
Atomic Spectroscopy
Applications of Mass Spectrometry
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
/ Instrumentation
Atomic Spectroscopy
Applications of Mass Spectrometry
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
/ Instrumentation
Atomic Spectroscopy
Applications of Mass Spectrometry
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
/ Instrumentation
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
/ Instrumentation
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
/ Instrumentation
Atomic Spectroscopy
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
/ Instrumentation
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
/ Instrumentation
Atomic Spectroscopy
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
/ Instrumentation
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
/ Instrumentation
Atomic Spectroscopy
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
/ Instrumentation
Atomic Spectroscopy
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.