1. Applications of LC-MS in Chemical and Biochemical Sciences
Presented By : Malaika Argade
Department of Medicinal Chemistry
Virginia Commonwealth University
Date : 25th March 2011

2. LC-MS? Separates components Component identification is superior
LIQUID CHROMATOGRAPHY
MASS SPECTROMETRY
Separates components
Identification from retention time is difficult
Component identification is superior
But, interference from other ions
Ardrey, R. E. Liquid Chromatography Mass spectrometry: an introduction, Wiley, West Sussex, England, 2003

3. HISTORY 1906 : Mikhail Tsvet invented chromatography
1930 : Edgar Lederer, Chromatographic
separation of carotenoids
1960s : Csaba Horvath, developed the first HPLC
1990s: Engineering developments in HPLC
2004: UPLC and monolithic columns
Henry, R. A. et al. In Liquid Chromatography In Clinical Analysis; Humana Press: USA, 1981; p

4. Mass Spectrometry 1897- Sir “J.J.” Thomson, charge/mass of electron
1919- Francis W. Aston with Thomson developed a “mass spectrometer” to separate isotopes of elements
1970s- Interfacing LC with MS
Moving Belt Interface & Direct Liquid Interface
1989- Electro Spray Ionization, John B. Fenn
Watson, J. T. et al. In Introduction to mass spectrometry, 3rd ed, Wiley: Wiltshire, 2007.

5. COMPONENTS OF LC LIQUID RESERVOIR SAMPLE INJECTOR PUMP RECORDER
DETECTOR
COLUMN
Henry, R. A. et al. In Liquid Chromatography In Clinical Analysis; Humana Press: USA, 1981; p

6. COLUMNS Backpressure overcome by, Elevated temperatures
Height equivalent to theoretical plate
(HETP)
Height
Backpressure overcome by,
Elevated temperatures
Monolithic columns
Swartz, M.E. J. Liq. Chromatogr. R. T. 2005, 28,

7. Basic components of MS ION SOURCE MASS ANALYSER DETECTOR
Time-of-Flight,
Quadrupole,
Magnetic Sector
Frit-Fast Atom Bombardment
(Frit FAB),
Electro Spray Ionization (ESI)
DETECTOR
Electron Multiplier tube
(accessed on 3/23/2011)

8. MASS SPECTROMETER Different molecular weights and sizes.
A mixture of molecules.
Different molecular weights and sizes.
Sorted by the mass spectrometer according to abundance and m/z.
(accessed on 3/21/2011)

9. TANDEM MS or MS/MS SAMPLE MS 2 MS 1 Product Precursor ion ion
ESI,FAB
CID
SAMPLE
MS 2
Product
ion
MS 1
Precursor
ion
Selected Reaction Monitoring (SRM)
Multiple Reaction Monitoring (MRM)
DETECTED!

10. APPLICATIONS In areas such as, Organic chemistry
Archaeological science
Toxicology studies
Forensic sciences and urinanalysis
Impurity detection or identification
Natural product dereplication
Identification of metabolites
Enzyme inhibition studies

11. APPLICATIONS LC-MS has wide applications in,
Screening botanical extracts.

12. DISCOVERING INHIBITORS FROM BIOLOGICAL EXTRACTS
Biological extracts are complex mixtures of compounds.
Difficult to isolate a particular compound.
Problems of co-extraction and interference.
So Ultrafiltration with LC-MS

13. INHIBITORS OF QR-2
Resveratrol, a natural product inhibitor of Quinone Reductase 2. 2
Extract
Ultrafiltration
Removal of unbound compounds
Incubation
QR-2
LC-MS
Dissociation
Liu, D. et al. Anal.Chem. 2007, 79,

14. Resveratrol Test: Resveratrol + active QR-2 (solid line)
Control: Resveratrol + denatured QR-2 (dotted line)
m/z : 227
Difference indicates active binding.
Choi, Y. et al. Anal. Chem. 2011, 83,

15. Actinomyces sp. EXTRACT
Test : Extract of Actinomyces +
active QR-2
Control : Extract + denatured QR-2
m/z of 317
Structure of TME determined by NMR
Choi, Y. et al. Anal. Chem. 2011, 83,

16. HOPS EXTRACT Test : Extract + active QR-2
Control: Extract + denatured QR-2
m/z : 353 & 369
Structure confirmed by LC-MS, co-elution with standard.
But are they binding at the same pocket as that of Resveratrol ?
Choi, Y. et al. Anal. Chem. 2011, 83,

17. COMPETITIVE BINDING STUDIES
Difference in the peaks obtained indicate active binding.
Extract + QR-2 + Resveratrol
Choi, Y. et al. Anal. Chem. 2011, 83,

18. RESULTS
TME, xanthohumol and xanthohumol D bind at QR-2 and compete with resveratrol.
Enzyme inhibition assay determined,
X-ray Crystallography confirmed xanthohumol and X-D binding to active pocket of QR-2.
Compound
IC50
Resveratrol
5.1μM
Tetrangulol methyl ether
0.16μM (most potent)
Xanthohumol
196μM
Xanthohumol D
110μM
Choi, Y. et al. Anal. Chem. 2011, 83,

19. APPLICATIONS LC-MS has wide applications in,
Identification of natural products
Structural characterization of peptides

20. ESI-MS of Bovine Serum Albumin
Molecular weight by ESI-MS : Da
Average molecular weight calculated from 582 residues: Da
Difference: Da
An undetected residue?
Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173,

21. Determining Amino Acid Sequence
At DNA level
Base sequencing technique
Chain terminators used
Errors
Time consuming
At Amino Acid Level
Edman degradation
Cleaving of peptide from N-terminal side
One peptide at a time
Not for more than 50 amino acids
Frit-FAB LC-MS/MS
MS/MS gives valuable daughter ion information
Very quick

22. COMPARISON WITH HSA AND RSA
156
94 95

23. PROCEDURE + BSA Cleaved sequence Frit-FAB MS HPLC Trypsin
Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173,

24. RESULTS FROM HPLC 75 peaks found
Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173,

25. Two cases encountered during comparison,
Peaks from HPLC
Matching Tryptic Sequences
Peak 12
Arg144 – Tyr147
Gln195 – Arg198
Peak 36
Ala128 – Lys136
Glu564 – Lys573
Peak 66
Unmatched.
Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173,

26. Peak 12 : Daughter ions from HPLC-MS/MS indicating RHYP sequence .
Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173,

27. Two cases encountered during comparison,
Peptides
Matching Sequences and Results
Peak 12
Arg144 – Tyr147
Confirmed.
Similarly,
Peak 36
Found to contain –VEG-
Glu564 – Lys573
Peak 66
Edman degradation confirmed sequence
FYAPELLYY sequence
Confirmed by MS/MS
Y detected in 156th position!!!
Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173,

28. POSITION 94th AND 95th
Some peptides were not identified by Frit-FAB LC-MS after trypsin digestion like sequence
So, BSA was digested with lysyl endopeptidase which matched the sequences,
VASLRETYGDMADC*C*EK
QEPERNEC*FLSHK
Glu82 to Arg98 was established
94th and 95th : -QE- was established
77-93
94-106
Hirayama, K. et al. Biochem. Biophys. Res. Commun. 2006, 173,

29. COMPARISON OF RESULTS BSA Previous findings New findings
Molecular weight Da Da
156th position
No residue
Tyrosine
Residue on 94th and 95th position
-EQ-
-QE-
Total amino acid residues
582 by Edman Degradation
583

30. APPLICATIONS LC-MS has wide applications in,
Identification of natural products
Structural characterization of peptides
Measuring enzyme activity

31. Angiotensin Converting Enzyme
ACE is a target for anti-hypertensive drugs because,
ACE
Angiotensin 1
Vasoconstriction
Angiotensin 2
AND
Bradykinin
Vasodilatation
ACE
Geng, F. et al. Biomed. Chromatogr. 2010, 24,

32. DETECTING ACE ACTIVITY
ACE activity is usually determined by formation of a product from a substrate.
Hippuric acid formed indicates ACE activity.
ACE
Hippuryl-Histidine-Leucine
Hippuric acid
Geng, F. et al. Biomed. Chromatogr. 2010, 24,

33. METHOD
Standard solutions of HA were analyzed by UPLC-MS and peak area plotted against known concentration.
Inhibitors were added with HHL and ACE.
After incubation HA was analyzed by UPLC-MS and compared with standard solutions.
HHL HA
Geng, F. et al. Biomed. Chromatogr. 2010, 24,

34. RESULTS Advantages; Quick screening Lower limits of detection
Where,
C0 = HA concentration without inhibitor
C = HA concentration with inhibitor
Advantages;
Quick screening
Lower limits of detection
Lesser analysis time
Peak area
Standard. HA curve
Concentration
Geng, F. et al. Biomed. Chromatogr. 2010, 24,

35. APPLICATIONS LC-MS has wide applications in,
Identification of natural products
Structural characterization of peptides
Measuring enzyme activity
Forensic analysis

36. Detection Of Steroids For drug-free competitions
To avoid false positives
Sensitive method to detect drugs in small amounts from hair samples
E.g. Stanzolol and Nandrolone.
ELISA
Traditional method for steroid detection.
Based on competition between drug and drug-enzyme conjugate.
Deshmukh, N. et al. Steroids. 2010, 75,

37. ELISA Less drug More drug Y – Antibody - Drug - Drug enzyme conjugate
Y Y Y Y
Y Y Y Y
Y Y Y Y S S S
Tetra methyl
benzidine S
Y Y Y Y
Y Y Y Y
Less
drug
More
drug
Voller, A. et al. J. Clin. Pathol. 1978, 31,

38. UPLC-MS/MS RESULTS Nandrolone Stanzolol m/z transition 275.2 109.2
Nandrolone
Stanzolol
m/z transition
Deshmukh, N. et al. Steroids. 2010, 75,

39. ELISA Vs. UPLC-MS/MS METHOD NANDROLONE STANZALOL ELISA 3 16 UPLC-MS/MS
Number of participants : 160
METHOD
NANDROLONE
STANZALOL
ELISA 3 16
UPLC-MS/MS 1 12
UPLC-MS/MS more sensitive than ELISA
Deshmukh, N. et al. Steroids. 2010, 75,

40. APPLICATIONS LC-MS has wide applications in,
Identification of natural products
Structural characterization of peptides
Forensic analysis
Measuring enzyme activity
Wine Chemistry

41. CONTENTS OF SHEDEH Of religious importance in Ancient Egypt
Blood of God Osiris, symbolizes rebirth of the dead
Contents were unknown
Very small samples
LC-MS/MS in MRM mode: highly specific.
Guasch-Jané, M.R. et al. J. Archaeol. Sci. 2004, 33,

42. DETECTION Tartaric acid: Wine marker Syringic acid: Red wine marker
So, wine it is!
White or red ?
Guasch-Jané, M.R. et al. J. Archaeol. Sci. 2006, 33,
Guasch-Jané, M.R. et al. Anal. Chem. 2004, 76,

43. Why alkaline fusion? Syringic acid Alkaline Malvidin-3-glucoside
Guasch-Jané, M.R. et al. J. Archaeol. Sci. 2006, 33,

44. RESULTS Shedeh is indeed red wine.
Successful detection of syringic and tartaric acid in trace amounts.
MRM mode is a confirmation in itself.

45. Summary LC-MS applications are wide
Over the years, MS has been replaced by MS/MS and even MSn ; LC by UPLC .
The technique offers a lot of flexibility and adaptability.
Each engineering aspect plays an important role.

46. ACKNOWLEDGEMENTS Dr. Umesh Desai The Desai Group
Department of Medicinal Chemistry, School of Pharmacy
Virginia Commonwealth University
Friends and family