mass spectrometry in pharmacognosy
TRANSCRIPT
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
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