Mass Spectrometry (MS)

Presented by: Naraino Majie NabiilahDate: 2nd March 2015

Table of contentsIntroduction

Principle of MS

Components of MS

Manipulation of MS

Components of MS

Tandem MS

Interpretation of MS

Some applications of MS in Pharmacognosy

Conclusion

References

Introduction

• Mass spectrometry is a physical measuring technique whose foundations were developedin the early 20th century. Since the 1970s, and especially in the past few years, ongoingtechnological developments have contributed substantially to progress in biochemistry,molecular biology, and medicine.

• Mass spectrometry is a powerful analytical technique used to

• quantify known materials,

• to identify unknown compounds within a sample, and

• to elucidate the structure and chemical properties of different molecules.

• The complete process involves the conversion of the sample into gaseous ions, with orwithout fragmentation, which are then characterized by their mass to charge ratios (m/z)and relative abundances.

• This technique basically studies the effect of ionizing energy on molecules.

Basic Principles of Mass Spectrometry

MASS SPECTRUM

The formed ions are separated by deflection in Magnetic field according to their mass and charge

Further break up onto smaller ions

(Fragment ions or Daughter ions)

Converted into Highly energetic positively charged ions

(Molecular ions or Parent ions)

Organic molecules are bombarded with electron

Components of Mass Spectrometry

The instrument consists of three major components:

1. Ion Source: For producing gaseous ions from the substance being studied.

2. Analyzer: For resolving the ions into their characteristics mass components according

to their mass-to-charge ratio.

3. Detector System: For detecting the ions and recording the relative abundance of each of

the resolved ionic species.

Manipulation of Mass Spectrometry

Manipulation in MS comprise of the following:

1. Changing the different components of mass spectrometry apparatus

•Sample Introduction (Coupling)

•Ionisation Techniques

•Mass Analysers

•Detectors

2. Tandem Mass Spectrometry (MS/MS)

Mass Spectrometry

Sample introduction

Ionisation

Mass Analysers

Detectors

1. Changing the different components of mass spectrometry

apparatus

Sample Introduction

• Direct Vapour Inlet

• Gas Chromatography

• Liquid Chromatography

• Direct Insertion Probe

• Direct Ionization of Sample

Sample Introduction

DVI•Simplest Method•Introduced by

needle•Works for solid,

liquid & gas of High Vapour

pressure only

GC•Most common

technique•Complex mixtures

can be separated•Quantification

possible•Pressure should be

maintained

LC•Thermally labile

compounds•Temperature

sensitive compds; ionisation from

condensed phase

DIP•Low vapour

pressure liquids & solids sample

•Higher temperatures•Sample under

vacuum•Wider range of

sample

DIoS•Compds that decompose &

have no sign. VP•Introduced by

direct ionisation from condensed

form

Types of ionisation techniques

• Volatile samples

• Electron Ionisation

• Chemical Ionisation

• Non-volatile samples

• Fast Atom Bombardment

• Thermospray

• Matrix Assisted Laser Desorption Ionisation

• Electrospray Ionisation

• Atmospheric Pressure Chemical Ionisation

Ionisation Techniques

Volatile samples Non-Volatile samples

EI•Heated filament emits é; accelerated by PD of

70 eV•Ionisation: removal of é

from molecule•Produces +ve charged ions with 1 unpaired é

CIProduces M+H+ ions or

M–H- ionsIonisation: gas

introduced; collision of analyte with gas ionsPositive CI uses NH3

Negative CI uses CH3

FAB•Low volatility compds•Solid/liquids mixed

with non-Volatile matrix: glycerol

•Bombarded with Ar or Xe atoms

•Gives M+H+ or M+Na+

ions

TS•Used: LC/MS

•Polar compounds•Ionisation:LC:Sample

+ C2H3O2NH4

•Produces M+H+ or M -H- ions

•Not commercially available today MALDI

•Similar to FAB

•Ionisation: Sample dissolved in matrix; absorbs light

•Coupled to TOF/MS not LC

•High mass range achievable

•Reproducibility issuesESI

•Polar non-volatile compounds

•Coupled to LC•Produces M+H+ or M -

H- ions•High Mr can be

determined

APCI• Wide range polar

cmpds• Ionisation:

HPLC+Corona discharged needle

• Form either M+H+ or M-H- ions

• Thermal degradation

Types of Mass analysers

• There are six general types of

mass analyzers that can be used

for the separation of ions in a

mass spectrometry.

Quadrupole Mass Analyzer

A combination of Direct Current (DC) andRadio Frequency (RF) voltages are appliedto two pairs of metal rods to influence ionstrajectories.

Time of Flight Mass Analyzer

Ions are accelerated by an electric fieldand the times it takes for the ion to travelover a known distance is measured.

Magnetic Sector Mass Analyzer

An ion is accelerated into a curved flighttube where a magnet deflects the trajectorywrt its m/e ratio

Electrostatic Sector Mass Analyzer

Ion travels through the electric field and the force onthe ion is equal to the centripetal force on the ion.Here the ions of the same kinetic energy are focused,and ions of different kinetic energies are dispersed.

Quadrupole Ion Trap Mass Analyzers

Ions are stabilised in a ring electrode containingdevice (trap) by applying an Radio Frequency voltage

Ion Cyclotron Resonance

Mass-to-charge ratio (m/z) of ions are determined in afixed magnetic field. The ions are excited within aPenning trap(a magnetic field with electric trappingplates).

Types of detectors

Detectors

Faraday cup

Electron Multiplier

Photomultiplier

Types of detectorsDetector

s

Faraday cup

Ions strike dynode surface; electrons emitted=current

induced

Electron Multiplier

+ve & -ve ions detected on same

instrument

Electron emitted focussed

magnetically

Photomultiplier

Photons emitted

Converted to current

2. Tandem Mass Spectrometry

• Tandem Mass Spectrometry, usually referred to as MS/MS, involves the use of 2 ormore mass analyzers.

• It is often used to analyze individual components in a mixture.

• This technique adds specificity to a given analysis.

• This is a powerful way of confirming the identity of certain compounds and determiningthe structure of unknown species.

• So MS/MS is a process that involves 3 steps: ionization, mass selection, mass analysis.

• MS/MS could be performed on instruments such as triple quadrupole (QQQ), ion trap,time of flight, fourier transform, etc.

• The triple quadrupole is the most frequently used mass spectrometer for MS/MS,perhaps because of the cost and ease of use among other factors.

Interpretation of Mass spectra

Analysis of Mass Spectra

Ester

Not aldehyde, ketone or carboxylic

acid

Mr=88

M: 44

Rest of ester: 88 - 44 = 44

CH3 and C2H5

15 + 29 = 44

M:15CH3

+

M:29CH3CH2

+15+44= 59CH3COO+ or

COOCH3+

m/z= 57 ?

M:31O-CH3

+

M:43CH3CO+

M:57C2H5CO+

Application of MS in Pharmacognosy• A crude plant extract may contain up to hundreds of different secondary metabolites of

considerably differing chemical nature and spectroscopic parameters.

• Therefore chromatographic, purification or isolation steps of separation are crucial prior todetection, identification and quantification.

• Characterization and identification of unknown constituents requires a more informative,selective and sensitive analytical tool.

• Some of the MS tools used are:

• Chromatographic techniques combined with mass spectrometry: GC-MS, LC-MS,HPLC-MS..

• Common mass spectrometer configurations and techniques: MALDI, MALDI-TOF..

• TANDEM MS: MS/MS, GC/MS/MS, LC/MS/MS..

Total Phenolic by MS• Phenolic compounds are plant secondary metabolites, which play important roles in disease

resistance

• The interest on these compounds is related with their antioxidant activity and promotion of health benefits

• Virgin olive oil is an important dietary oil, rich in natural antioxidants

• Olives and leaves of ten olive tree cultivars (Olea europaea L.) from the region of Trás-os-Montes e AltoDouro (Portugal) were studied: ‘Bical’, ‘Borrenta’, ‘Cobrançosa’, ‘Coimbreira’, ‘Lentisca’, ‘Madural’, ‘Negrinha de Freixo’, ‘Redondal’, ‘Santulhana’ and ‘Verdeal Transmontana’.

• HPLC Coupled with Atmospheric Pressure Chemical Ionisation (APCI) MS

• Total phenolic was determined colorimetrically at 760nm after reacting with Folin reagent; Expressed as tannic acid.

S. Silva et al, 2006, Phenolic Compounds and Antioxidant Activity of Olea europaea L. Fruits and Leaves, Food Sci TechInt 2006; 12(5):385–396

• The type of phenolic compounds detected in leaf, fruit and seed varied

markedly.

• The high antioxidant activity of seed extracts is due to nüzhenide and related

compounds, suggesting the possible application of olive seeds as sources of

natural antioxidants.

S. Silva et al, 2006, Phenolic Compounds and Antioxidant Activity of Olea europaea L. Fruits and Leaves, Food Sci TechInt 2006; 12(5):385–396

Total Phenolic by MS

GC-MSFour Greek endemic Boraginaceae plants, Onosma erecta Sibth. & Sm., Onosma kaheirei Teppner, Onosma leptantha Heldr., and Cynoglossum columnae L. (aerial parts), were screened for their content of pyrrolizidine alkaloids (PAs)- present in the form of N-Oxide; highly polar; possess hepatotoxic, hemolytic, antimitotic, teratogenic, mutagenic, and carcinogenic effects.

• Qualitative tests by TLC

• Quantitative test by GC-MS

• Extraction of dry plant material done by using MeOH

• Liquid-liquid extraction done by CH2Cl2

• CH2Cl2 was condensed and analysed

• Results:

• 23 peaks were obtained and their structures were identified

• 100% PAs and PA-N-Oxides from C. columnae

• 100% PA-N-Oxides from O. leptantha

• No free PAs were obtained from O. erecta and O. kaheirei (Reason: Thermally decomposed)

Damianakos et al., 2014, The Chemical Profile of Pyrrolizidine Alkaloids from Selected Greek Endemic BoraginaceaePlants Determined by Gas Chromatography/Mass Spectrometry : Journal of AOAC International Vol. 97, No. 5

LC-TOF/MS• LC-TOF/MS method was developed for qualitative and quantitative

analysis of the major chemical constituents in Andrographispaniculata. (Used for common cold, fever and non-infectiousdiarrhea)

• Ultrasonic Extraction: 0.2 g plant sample extracted with 10 mL of70% ethanol and extraction for 30 min at under 50 kHz ultrasonicirradiation.

• LC performed; fifteen compounds, including flavonoids andditerpenoid lactones, were unambiguously or tentatively identified in10 min by comparing their retention times and accurate masses withstandards or literature data.

• TOF-MS done to identify the compound (eg C6: Andrographolide)

• This study would facilitate the quality evaluation of A. paniculata forsafe and efficacious use and be a powerful reference for theidentification of similar compounds presented here by MS spectra.

Yong-Xi Song, 2013, Qualitative and Quantitative Analysis of Andrographis paniculata by Rapid Resolution LiquidChromatography/Time-of-Flight Mass Spectrometry, Molecules 2013, 18, 12192-12207

HPLC-MS• β-sitosterol is an important component in food and herbal products and beneficial inhyperlipidemia.• Higher conc. in serum may lead to coronary artery disease in case of sitosterolemia.• Quantity of β-sitosterol in food and herbal drugs needs to be determined.• Quantitative estimation of β-sitosterol present in hot and cold water extracts of bark,regenerated bark, leaves and flowers of the S. asoca and Ashokarista drugs were carried outfirst time using high performance liquid chromatography coupled (HPLC) with quadrupoleTOF-MS• Extraction was done with deionised water for 3days; centrifuged, lyophilised and filtered.• Different concentrations of β-sitosterol and crude extracts were estimated by HPLC andmass spectrometry.• The results showed significant differences in the distribution of β-sitosterol amongdifferent organs of S. asoca.• This type of approaches could be helpful for the quality control of herbal medicines andprovides necessary information for the rational utilization of plant resources.

Gahlaut, et al, 2013, β-sitosterol in different parts of Saraca asoca and herbal drug ashokarista: Quali-quantitativeanalysis by liquid chromatography-mass spectrometry, Journal of Advanced Pharmaceutical Tech.| Jul-Sep|Vol 4:3

MALDI-MS• A mass spectrometric imaging (MSI) was performed to localize

ginsenosides (Rb1, Rb2 or Rc, and Rf) in cross-sections of the Panaxginseng root at a resolution of 100 m using matrix-assisted laserdesorption/ionization mass spectrometry (MALDI-MS).

• MALDI-MSI confirmed that ginsenosides were located more in thecortex and the periderm than that in the medulla of a lateral root.

• In addition, it revealed that localization of ginsenosides in a root tip(diameter, 2.7 mm) is higher than that in the center of the root(diameter, 7.3 mm).

• A quantitative difference was detected between localizations ofprotopanaxadiol-type ginsenoside (Rb1, Rb2, or Rc) andprotopanaxatriol-type ginsenoside (Rf) in the root.

• This imaging approach is a promising technique for rapid evaluationand identification of medicinal saponins in plant tissues.

S. TAIRA et al., 2010, Mass Spectrometric Imaging of Ginsenosides Localization in Panax ginseng Root, The AmericanJournal of Chinese Medicine, Vol. 38, No. 3, 485–493

Conclusion• Natural products (also known as secondary metabolites) have always been a

significant source of new lead compounds in pharmaceutical industries.

• Mass spectrometry has long been used in medicines.

• There are several types of MS that are use depending on the nature of the

plant extract to be analysed i.e. Volatile, polarity, temperature sensitive etc

• It is a good method for detecting, identifying and quantifying expected

metabolites using ESI, MALDI, TOF/MS, Tandem etc

• Also detect, identify and relatively quantify unexpected metabolites

• Used for Standardisation of phenolics for its antioxidants activities

(Universal protocol should be used)

• It can be used in Quality control

• Overall, MS is great tool to use in pharmacognosy: small sample size

required, fast, can be combined with GC, LC to run mixtures.

REFERENCES

• ‘Introduction to Mass Spectrometry’ (no date). chemwiki.ucdavis.edu. Available at:http://chemwiki.ucdavis.edu/Analytical_Chemistry/Instrumental_Analysis/Mass_Spectrometry/Introductory_Mass_Spectrometry/Introduction_to_Mass_Spectrometry (Accessed: 1 March 2015).

• Mass Spectrometry Tutorial | Chemical Instrumentation Facility (no date). Available at: http://www.cif.iastate.edu/mass-spec/ms-tutorial (Accessed: 1 March 2015).

• S. Silva et al, 2006, Phenolic Compounds and Antioxidant Activity of Olea europaea L. Fruits and Leaves, Food Sci Tech Int 2006;12(5):385–396

• Damianakos et al., 2014, The Chemical Profile of Pyrrolizidine Alkaloids from Selected Greek Endemic Boraginaceae PlantsDetermined by Gas Chromatography/Mass Spectrometry : Journal of AOAC International Vol. 97, No. 5

• Yong-Xi Song, 2013, Qualitative and Quantitative Analysis of Andrographis paniculata by Rapid Resolution LiquidChromatography/Time-of-Flight Mass Spectrometry, Molecules 2013, 18, 12192-12207

• S. TAIRA et al., 2010, Mass Spectrometric Imaging of Ginsenosides Localization in Panax ginseng Root, The American Journal ofChinese Medicine, Vol. 38, No. 3, 485–493

• Mass Spectrometry in Biotechnology by Gary Siuzdak , Academic Press 1996 SiuzdakBiotechnology”

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• The Ideal Mass Analyzer: Fact or Fiction?" (Curt Brunnee, Int. J. Mass Spectrom. Ion Proc. 76 (1987), 125-237

• M. Careri et al, 2002,r ecent advances in the application of mass spectrometry in food-related analysis, Journal of ChromatographyA, 970 (2002) 3–64