Mass Spectrometry Applications and spectral interpretation: Basics

Mass Spectrometry Mass Spectrometry applications in applications in

Medicinal ChemistryMedicinal Chemistry

Shreekant Deshpande

Mass spectrometry is a very powerful and versatile analytical tool.

Extensively used in every fields to detect one or the other things.

Here I try to give some outlines about HOW TO extract a INFORMATION FROM MASS SPECTRA.

What is mass spectrometry?

Where are mass spectrometers used?

How does a mass spectrometer work?

What information can it provide?

Interpretation of spectra. and

Applications in medicinal chemistry.

Overview

What is Mass Spectrometry?

Analytical tool measuring molecular weight (MW) of sample.Only picomolar concentrations required.With an high accuracy and within 5 ppm of std. Error for small organic molecules.For a mass of 40 kDa, there is a 4 Da error.This means it can detect amino acid substitutions / post-translational modifications in biological systems.

Where are they used?

Biotechnology: Analysis of proteins, peptides, oligonucleotides

Pharmaceutical: Drugs discovery, combinatorial chemistry, pharmokinetics, Drug metabolism

Clinical:Neonatal screening, haemoglobin analysis, drug testing

Environmental: Water, food, air quality

Geological:Oil composition

Detect and identify the use of steroids in athletes(Doping)

Ex. Sunita Rani’s case!!!

Monitor the breath of patients by anesthesiologists during surgery.

Determine the composition of molecular species found in space

Adulteration in food stuff

Ex. Presence of pesticide in …..

Mass Spectrometry is also used to...

How does a Mass Spectrometer work?

3 fundamental parts: the ionisation source, the analyser, the detectorSamples easier to manipulate if ionisedSeparation in analyser according to mass-to-charge ratios (m/z)Detection of separated ions with their relative abundanceSignals sent to data system and formatted in a m/z spectrum

Either directly or via chromatography for component separation (HPLC, GC, capillary electrophoresis)

Ionisation can be positively charged (for proteins; Org. molecules) or negatively charged (for saccharides and oligonucleotides)

Electron Impact (EI)

Chemical Ionisation (CI)

Atmospheric Pressure Chemical Ionisation (APCI)

Fast Atom Bombardment (FAB)

Field Desorption / Field Ionisation (FD/FI)

Electrospray Ionisation (ESI)

Matrix Assisted Laser Desorption Ionisation (MALDI)

• Magnetic Sector

• Time of flight (tof)

• Quadrupole

• Quadrupole ion trap

• Fourier Transform Ion Cyclotron Resonance

The resolving power of analysers

The respective resolving powers correspond to the resolution typically obtained for time-of-flight, quadrupole, and Fourier-transform mass analyzers. Notice that the individual isotope peaks can be distinguished at the higher resolution.

• Photo MultiplierPhoto Multiplier

• Faraday CupFaraday Cup

Detector monitors ion current, amplifies it and then transmits signal to data system

Mass spectrometry is a very powerful method to analyse the structure of organic compounds, but suffers from 3 major limitations:

1 It is a destructive method

2 Compounds cannot be characterised without clean samples

3 The dynamic properties of molecules can not be detected.

Most of the times chem. Strs. of the products are known are presumed.Sometime unexpected peaks will come then we’ll scratch our head.And we try to see +23/ +39 peaks(Na/ K adducts)If MI is observed what else we can see for further confirmations.

Isotope peaksmass no- odd/even

Spectrum speaks

What Molecular ion peak tells !!

m/z spectrum example

Natural abundance of isotopes

Elements Relative abundance

Carbon 12C 100 13C 1.08

Nitrogen 14N 100 15N 0.38

Oxygen 16O 100 17O 0.04 18O 0.20

Sulfur 32S 100 33S 0.78 34S 3.35

Chlorine 35Cl 100 3:1 37Cl 32.5

Bromine 79Br 100 1:1 79Br 98.0

The Rule of Thirteen – Molecular Formulas from Molecular Mass

When a molecular mass, M+, is known, a base formula can be generated from the following equation:

M = n + r

13 13

the base formula being: CnHn + r

For this formula, the HDI can be calculated from the following formula:

HDI = ( n – r + 2 )

2

Remember and Review!

Interpretation

How to interpret our spectra? • Check the quality of the data; does the total ion spectrum look

ok?

• Does the mass spectrum show significant peaks above the baseline?

• Identify any major peaks.

• Look for telltale isotope patterns.

• Remember the Nitrogen Rule. (The Nitrogen Rule: A neutral compound containing an odd number of nitrogen atoms will always have an odd molecular mass.)

• Do the masses fit with the proposed structure?

• Have you used the appropriate masses in your calculations?

Interpretation

How do I interpret my spectra?

Identify the MIIdentify the MI

Check isotope pattern - halogens, sulphur etc.

Odd/Even Mass - Nitrogen Rule

If FAB check intensity of MI - aromatic or aliphatic

Accurate mass - calculate r + db

See neutral losses out of your mass. Amine protecting groups CxHyNzOn

r + db = x - 0.5y + 0.5z + 1

In Med. Chemistry Accurate molecular weight measurements:Unknown sample, Purity of sample, About other side products.

Reaction monitoring:enzyme activity, chemical modification, protein digestion.

Structural elucidations:Natural products, unknown products through fragmentations.

Identification of Mechanism of actions:Both covalent n noncovalent interactions.

Active site Identifications:

Quantifying the amount of a compound in a sample using carefully designed methods.

Impurity profiling for the bulk drugs.

-lends to the analysis of samples without any preparation and provides data with accurate mass measurement. A complete analysis can be done within minutes by simply holding a sample in the open gas stream.

Its an atmospheric pressure source capable of ionizing liquid, solid, and gas samples in real time. Principle: on an atmospheric pressure interaction of a sample with long-lived electronic excited-state atoms or molecules and atmospheric gases. The system uses helium or nitrogen to produce metastable atoms or molecules that interact with the analytes.

AccuTOF-DART Technology

The DART can even analyze samples directly on surfaces such as concrete, currency, food, pills and clothing.

The revolutionary DART ion source gives an entirely new meaning to the phrase "open access".

MS is a highly applicable analytical tool in Medicinal Chemistry.

The full structural information can get out of single mass spectrum with min. qty. of sample.

Elemental analysis can b carried by using HRMS.

Conclusion

“I feel sure that there are many problems in chemistry which could be solved with far greater ease by this than by any other method. The method is surprisingly sensitive,……..requires an infinitesimal amount of material and does not require this to be specially purified.”

In his book,

“Rays of Positive Electricity” (1913),

J.J.Thomson remarked:

Thank you very much