Analytical Chemistry: Identification and Quantitation of Compounds in Complex Mixtures

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Analytical Chemistry Identification and
Quantitation of Compounds in Complex Mixtures

• The General Analytical Strategy
• Spectroscopic Methods
• Mass Spectrometry
• Sample Preparation Methods
• Quantitation
• Application to the Analysis of Flavonoids Mass
  Spectrometry

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Analytical Chemistry Identification and
Quantitation of Compounds in Complex Mixtures
Example Common Flavonoid Structures



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The complexity of the problem Many flavonoids
are glycosylated

• Sugar linkage O-glycosylflavonoidsgtgtC-glycosylfl
  avonoids
• Positions of glycosylation 3-OHgt7-OHgtgt3, 4,
  or 5-positions
• Level of glycosylation
• Number of different sugars involved

flavonoid aglycone
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THE ANALYTICAL STRATEGY 1) Evaluate the
problem --pure component or mixture --solid,
liquid or gas --organic, inorganic,
elemental --sample size and number of
samples --quantitative or qualitative
analysis --type of matrix --requirements for
accuracy and precision
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• 2) Select the appropriate method.
• Select a sample preparation method
• extraction? dilution? acid/base conditions?
  filtration?
• If it is a mixture, select
• GC (Gas Chromatography)
• HPLC (High Performance Liquid Chromatography)
• CZE (Capillary Zone Electrophoresis)
• For analysis, select
• Classical "wet chemistry" methods (titrimetry,
  gravimetry)
• Electrochemical methods UV/Vis Molecular
  Absorption Spectroscopy
• Infrared Absorption Spectroscopy Molecular
  Fluorescence Spectroscopy
• Raman Scattering Spectroscopy Microscopy
• Nuclear Magnetic Resonance X-Ray Spectroscopy
• Electron Spectroscopy Atomic Spectroscopy
• Mass Spectrometry

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Analytical Strategy continued

• 3) Characterize the method with reference
  compounds and standards.
• Run controls. Establish accuracy and precision
  for quantitative applications.
• 4) Construct a calibration curve and/or standard
  addition method or use internal standard for
  quantitative analysis.
• 5) Evaluate samples of interest. Repeat as
  necessary.

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Overview of Spectroscopic Methods
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The absorption of energy.
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Molecular UV-Vis Absorption
Absorption of UV or visible light causes
electronic transitions in which electrons are
excited to antibonding orbitals.
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Absorption in Spectroscopy
Electromagnetic Radiation
Sample
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UV-Vis Spectra of Four Flavonoids
Rutin
Quercetin glycoside
Anthocyanidin
Ploridzin
Adapted from FEBS Letters, 401 (1997) 78-82,
Paganga and Rice-Evans.
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• Beers Law
• A a b c Absorbance
• where a absorptivity in L/(g-cm)
• b pathlength of radiation through sample in
  cm
• c concentration of sample in g/L

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• Absorptivity the probability that an analyte
  will
• absorb a particular wavelength of energy
• (also known as extinction coefficient)
• --range from 0 100,000
• --units of L/(g-cm) or L/(mol-cm)
• --depends on presence of chromophores in the
  analytes

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Absorbance ? concentration
Absorbance
Increasing concentration of analyte
A a b c
Wavelength in nm
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• Instrumentation for Spectroscopy
• Source of radiation
• Sample container
• Energy selector
• Radiation detector
• Signal processor

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UV-Vis Spectrometer
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IR Absorption
Formaldehyde Vibrational modes include
stretching and bending (twisting, rocking,
scissoring, wagging) Stretching change in
distance between atoms along interatomic
axis Bending change in angle between two bonds
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Characteristic IR Absorption Frequencies
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Infrared Absorption Spectrum of Naringin
aromatic
C-O stretch
CO stretch
OH
Adapted from Sadtler Index, 1973.
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FTIR Spectrometer
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Nuclear Magnetic Resonance
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Typical NMR Proton Chemical Shifts
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Proton NMR Spectrum of Morin
H6
H3
H8 or H6
H5
H8 or H6
Adapted from Biochem. Pharm., 59 (1995) 537-543,
Wu et al.
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NMR Spectrometer
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Overview of the Mass Spectrometer
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Common Ionization Method for GC-MS
M analyte e electron F fragment
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Electron Ionization Mass Spectrum of Chalcone
Molecular Weight 208 amu
Adapted from Rapid Commun. Mass Spectrom., 12
(1998) 139-143, Ardanaz et al.
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Fragmentation of Chalcone Ion
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Electrospray ionization for Larger, Involatile
Molecules
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Collisional Activation Dissociation to Fragment
Ions
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Collisional Activated Dissociation of Protonated
Nomilin
455 -- loss of CH3COOH 411 -- loss of CH3COOH and
CO2
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Solid-Phase Extraction
Contaminants
Compounds of Interest
2. Elute Compounds of Interest
1. Add Sample/Wash Contaminants
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Gas chromatography for separation of volatile
analytes in mixtures
Capillary column
Intensity
Retention time
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Derivatization Reactions for GCMS
Adapted from Current Practice of GC-MS, 2001,
Marcel Dekker, Ch. 15, Brodbelt et al., p.
369-386.
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HPLC Apparatus for involatile analytes in
mixtures

Regulated He Supply
Pump
Solvent Reservoirs
Column
Detector
Injector Valve
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Quantitation
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Typical Calibration Curves for Flavonoids
Adapted from FEBS Letters, 401 (1997) 78-82,
Paganga and Rice-Evans.
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Flavonoids in Kale An LC-MS/MS Study
quercetin (302)
kaempferol (286)
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• Kale extraction
• Liquid extraction of flavonoids from kale
  

Adapted from Zhang, Satterfield, Brodbelt,
Britz, Clevidence, Novotny Anal. Chem., 75 (2003)
6401-6407.
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Kale extraction (contd)

• Acid hydrolysis for cleaving flavonoid
  glycosides to
• their aglycone forms

• Solid phase extraction for cleanup and
  concentration

LC-MS/MS analysis
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Flavonoid separation by HPLC
B. With pH adjustment (w/0.3 HCOOH)
A. Without pH adjustment



kaempferol
0.040











0.030




int. std.



quercetin
0.020





AU




0.010








0.000



0.0

























4.0

8.0

12.0

16.0

20.0

24.0








Minutes

• Guard column Waters Symmetry C18, 2.1?10 mm,
  3.5 ?m
• Analytical column Waters Symmetry C18, 2.1?50
  mm, 3.5 ?m
• Mobile phase Solvent A-water, solvent
  B-acetonitrile. 0-13 min 30?-100? B
• 13-15 min 100?-30? B 15-25 min 30? B

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Identification of flavonoids by ESI-MS/MS CAD
spectrum of quercetin
179 - C7H6O2
100
80
151 - C7H6O2 CO
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C15H10O7 Mw302
Relative Abundance
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273 - CO
20
257 - CO2
301 (M - H)
107 C7H6O2 - CO - CO2
193
229
239
0
100
140
180
220
260
300
340
m/z
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Calibration curves of flavonoids by LCMS
Kaempferol y 0.0635x 0.0876 R2 0.9972
Quercetin y 0.0556x 0.199 R2 0.9785



Linear concentration range 0.03-90 ?g/ml

Detection limit by HPLC-ESI-MS quercetin-10 pg
kaempferol-3 pg
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Analysis of kale samples by LCMS
A. HPLC-UV chromatogram
B. TIC-MS chromatogram
100
int. std.

80
60
Relative Abundance
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Quercetin in kale 77 ppm Kaempferol in kale 235
ppm Recovery 65
kaempferol
quercetin
20
0
0
4
8
12
16
20
24
Time (min)

Adapted from Zhang, Satterfield, Brodbelt,
Britz, Clevidence, Novotny Anal. Chem., 75 (2003)
6401-6407.
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Flavonoids in Grapefruit Monitoring
metabolites by LC-MS/MS

• Identification of metabolites
• Pharmacokinetics
• Bioavailability

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Analysis of urine by LCMS after consumption of
grapefruit juice
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Urine at t 7.5 h
RT 6.4 m/z 447
RT 5.6 m/z 447
RT 9.7 m/z 253
Relative Abundance
RT 10.7 m/z 351
0
Time (minutes)
Zhang and Brodbelt, The Analyst., 129 (2004)
1227-1233.
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Fragmentation patterns of components from
previous LCMS chromatogram
A glucuronide A sulfate Another
sulfate Another sulfate A glucuronide-
sulfate Another sulfate
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Time plot of major metabolites in urine after
consumption of grapefruit juice (analysis by
LC-MS/MS)