1. Mass Spectrometry Quantitative Mass Spectrometry
Chiral Mass Spectrometry

2. Quantitation by MS
Goal is to develop methodology to sensitively, specifically, accurately and rapidly measure one or more compounds in a sample
LCMS and GCMS are well suited to achieve this goals

3. External Standards
Standard curve: best when sample matrix is uncomplicated and when only one analyte is to be quantitated. Need linear instrument response.
Standard addition: same considerations as standard curve, but better when matrix is complicated.
In either case it is necessary to add a known amount of an internal standard that can be used to account for sample handling variations. (losses during preparations, variations in injection volume, etc.)

4. Isotopically Labeled Internal Standards
Add a known quantity of isotopically labeled internal standard, quantitate analyte by peak area ratio
Need isotopically labeled standard(s) (13C, 15N) that can be well resolved from the isotope peaks of the analyte (+4 amu or more)
Need to avoid 1° KIEs that will cause the analyte and standard to have different retention times.
More efficient than external standard methods:
eliminates need for separate analysis of standards.
multiple analytes can be rapidly quantitated.
Corrects for sample handling variations, instrument variations and matrix effects.

5. Mass Spec Scans
Any mass spectrometer/scan type can be used for quantitation, however:
Quantitation by GCMS is usually done by sector instruments or single quads using selected ion monitoring (SIM) of parent/ fragment ions
SIM is 1000x more sensitive than full scan
For LCMS, the triple quadrupole mass spectrometer has significant advanatges
MS/MS can be used to increase specificity
The MRM scan performed by a triple quad is the highest duty cycle scan available and is especially useful for quantitating multiple analytes in a complex matrix

6. GC/MS of Dioxin
Routine EPA method uses high resolution magnetic sector operated in SIM mode.
For each compound of Interest, several EI fragment/parent ions analyzed
SIM window can be very narrow with sector
peak ratios must match expected values
Isotopically labeled (per 13C) dioxins are used as internal standards

7. Quantitation of Modified Tyrosine by LC/MS
Levels of Nitration, Chlorination, and Bromination of Tyrosine in Biological systems may correspond to inflammation/disease.
LC/MS/MS can be used as a tool to quantitate levels of each modification.
Hazen et. al. J. Biol. Chem., 277(20), , (2002)

8. Triple Quadrupole Mass Analyzer
Sample Inlet Q2
(Collision cell) Q3
Ion Guide Q1 EM
Detector

9. Multiple Reaction Monitoring in a Triple Quadrupole
Q2 collision cell
Q3 (181) LC
column
Set on mass of
parent ion
Fragment parent ion
Transmit only
diagnostic
product ion
highest duty cycle triple quadrupole scan type!

10. MRM transitions

12. Stereospecific Mass Spectrometry
To observe chiral specificity in MS
need an optically active probe reagent
reagent must differentially complex to analyte
reaction can occur in the source or analyzer
Two primary methods
Two enantiomers with different isotopic labels
Only useful for determining selectivity (screening)
MS/MS of diastereomeric complexes
Can be used to determine %ee

13. Gas-Phase Ion Structure
Can chiral selectivity be observed in the gas- phase?
First observation was by Fales et. al.
JACS, 99(7), , (1977)
Racemic mixture of labeled/unlabeled D/L enantiomers
Ratio of protonated dimers was 1:1:1, not 1:2:1

14. Chiral Crown Ether Host-Guest Chemistry
Sawada et. al. JACS, 117, , (1995)
Used FAB to determine chiral selectivity of various crown ethers towards amino acids
Mixture of labeled/
unlabeled amino acid
enantiomers
5:1 selectivity for
one enantiomer

15. Thermochemical Measurement of Selectivity
Dearden et. al JACS, 115, , (1993)
Measured equilibrium constant for chiral host guest reaction in the gas phase (FT-ICR-MS)
KR=130
KS=567
∆G=4.2kJ/mol
∆G in CH2Cl2=4.6

16. Kinetic Evidence of Chirality
Lebrilla et. al. JACS, 118, , (1996)
Reaction of multiply-charged ions of cytochrome c with R and S enantiomers of 2-butylamine
Multiple rate constants measured, indicating several reactive sites of deprotonation (or multiple conformations of the protein)
All rates were ~10x faster for the R enantiomer

17. Requirements for Chiral MS Method to Determine %ee
Employs instruments which are commercially and broadly available
Experimental protocol should be simple
Isotopic labeling should not be required
Large chiral selectivity is desired to achieve accurate quantitation
Two kinetic methods have emerged

18. Host-Guest Exchange Reaction
Lebrilla et. al. Anal. Chem., 73, , (2001)
Used cyclodextrins of varying sizes to form complexes with chiral amino acids and pharmaceuticals
Diastereomeric complexes were isolated by FT-ICR and allowed to react with various bases
Calibration curve can be constructed for a compound, then one measurement can determine %ee of mixture

19. Chiral Selectivity in Host-Guest Exchange
L-DOPA Exchanges
5x faster than D
Has been extended to
other drugs (amphetamine,
ephedrine, etc.) analysis on
FTMS and Ion Trap

20. Kinetic Method Using CID
Cooks et. al. Anal. Chem., 73, , (2001)
Formed Diastereomeric Cu(II) complexes of amino acids and pharmaceuticals by ESI
Isolated desired complex in an ion trap
Subsequent fragmentation yields loss of amino acid or drug.
Fragment ratio depends on stereochemistry of analyte.

21. Kinetic Method for Determination of %ee
Product Ratio is Determined by Configuration of A.
Calibration Curve yields %ee of unknowns with 2-4% error
Method has been used for amino acids, drugs, and sugars