Analytical Chemistry: Identification and Quantitation of Compounds in Complex Mixtures

1. 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

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

3. Common sugars found in flavonoid glycosides arabinose xylose galactose rhamnose glucose The complexity of the problem: Many flavonoids are glycosylated • Sugar linkage: O-glycosylflavonoids>>C-glycosylflavonoids • Positions of glycosylation: 3-OH>7-OH>>3’, 4’, or 5-positions • Level of glycosylation • Number of different sugars involved flavonoid aglycone

4. 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

5. Analytes of interest…. (a protein)

6. 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

7. 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.

8. Overview of Spectroscopic Methods

9. The absorption of energy….

10. Spectrophotometer

11. Molecular UV-Vis Absorption Absorption of UV or visible light causes “electronic transitions” in which electrons are excited to antibonding orbitals.

12. Absorption in Spectroscopy Electromagnetic Radiation Sample

13. UV-Vis Spectra of Four Flavonoids Rutin Quercetin glycoside Anthocyanidin Ploridzin Adapted from FEBS Letters, 401 (1997) 78-82, Paganga and Rice-Evans.

14. Beer’s 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

15. 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

16. Absorbance  concentration Absorbance Increasing concentration of analyte  higher absorbance A = a b c Wavelength in nm

17. UV-Vis Spectrometer

18. 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

19. Characteristic IR Absorption Frequencies

20. Infrared Absorption Spectrum of Naringin aromatic C-O stretch C=O stretch OH Adapted from Sadtler Index, 1973.

21. FTIR Spectrometer

22. Nuclear Magnetic Resonance

23. Typical NMR Proton Chemical Shifts

24. (Hydroxyl protons not shown, from 9 - 13 ppm) 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.

25. NMR Spectrometer

26. Overview of the Mass Spectrometer

27. Electron Ionization: “EI” Common Ionization Method for GC-MS M = analyte e = electron F = fragment

28. 77 207 208 193 131 105 89 179 165 152 Electron Ionization Mass Spectrum of Chalcone: Molecular Weight 208 amu Adapted from Rapid Commun. Mass Spectrom., 12 (1998) 139-143, Ardanaz et al.

29. Fragmentation of Chalcone Ion

30. EI-Quadrupole MS

31. ring electrode detector octapole ion guides heated capillary sample capillary ESI capillary sheath gas skimmer entrance end-cap exit end-cap Electrospray ionization for Larger, Involatile Molecules

32. Collisional Activation Dissociation to Fragment Ions

33. 411 455 (M + H)+ 515 393 231 m/z 200 300 400 500 Collisional Activated Dissociation of Protonated Nomilin 455 -- loss of CH3COOH 411 -- loss of CH3COOH and CO2

35. Solid-Phase Extraction Contaminants Compounds of Interest 2. Elute Compounds of Interest 1. Add Sample/Wash Contaminants

36. Gas chromatography for separation of volatile analytes in mixtures Capillary column Intensity Retention time

37. HPLC Apparatus: for involatile analytes in mixtures Regulated He Supply Pump Solvent Reservoirs Column Detector Injector Valve

38. C18 Stationary Phase C H H C 3 3 S i S i H C 3 S i i S H C 3 H C 3 S i

39. Quantitation

40. Typical Calibration Curves for Flavonoids Adapted from: FEBS Letters, 401 (1997) 78-82, Paganga and Rice-Evans.

41. Flavonoids in Kale: An LC-MS/MS Study quercetin (302) kaempferol (286)

42. supernatant N2 (l) Kale leaves kale powder 1:1 acetone vortex centrifuge powder 2:1 chloroform 1:1 30:70 acetone: water extract 1 aqueous phase collected separation filter • Kale extraction • Liquid extraction of flavonoids from kale Adapted from: Zhang, Satterfield, Brodbelt, Britz, Clevidence, Novotny Anal. Chem., 75 (2003) 6401-6407.

43. 1 ml kale extract 1 mix 100°C 2h + kale extract 2 5 ml 2N HCl in 50:50 water:methanol condition kale extract 2 wash elute C18 cartridge kale extract 3 (final) Kale extraction (cont’d) • Acid hydrolysis for cleaving flavonoid glycosides to their aglycone forms • Solid phase extraction for cleanup and concentration LC-MS/MS analysis

44. Flavonoid separation by HPLC • 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

45. Identification of flavonoids by ESI-MS/MS: CAD spectrum of quercetin 179: - C7H6O2 100 80 151: - C7H6O2 – CO 60 C15H10O7 Mw=302 Relative Abundance 40 273: - CO 20 257: - CO2 301 (M - H+) 107: C7H6O2 - CO - CO2 193 229 239 0 100 140 180 220 260 300 340 m/z

46. 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 concentrationrange: 0.03-90 g/ml Detection limit by HPLC-ESI-MS: quercetin-~10 pg; kaempferol-~3 pg

47. 0.040 int. std. 0.030 quercetin AU 0.020 kaempferol 0.010 0.000 4.00 8.00 16.00 20.00 24.00 12.00 Minutes Analysis of kale samples by LCMS A. HPLC-UV chromatogram B. TIC-MS chromatogram 100 int. std. 80 60 Relative Abundance 40 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.

48. Flavonoids in Grapefruit: Monitoring metabolites by LC-MS/MS • Identification of metabolites • Pharmacokinetics • Bioavailability

50. Analysis of urine by LCMS after consumption of grapefruit juice 100 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.