Mass Spectrometry

Quantitative Mass Spectrometry

Chiral Mass Spectrometry

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

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.)

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.

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

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

Quantitation of Modified Tyrosineby 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), 17415-17427, (2002)

Triple Quadrupole Mass Analyzer

Sample Inlet

Q2(Collision cell)

Q3

Ion Guide

Q1

EMDetector

Multiple Reaction Monitoring in a Triple Quadrupole

LCcolumn

Q1 (227) Q2 collision cell

Fragment parent ion

Q3 (181)

Set on mass ofparent ion

Transmit only diagnosticproduct ion

highest duty cycle triple quadrupole scan type!

OH

NH3+

NO2

O OH

OH

NH2+

NO2

MRM transitions

immonium loss of OHtyrosine 182 136 165

nitrotyrosine 227 181 21013C6 nitrotyrosine 233 187 216

chlorotyrosine 216 170 19913C6 chlorotyrosine 222 176 205

bromotyrosine 260 214 24313C6 bromotyrosine 266 220 249

diagnostic product ionsparent ion

4000

3000

2000

1000

0

1210864

Time, min

216/170 chlorotyrosine (immonium) 216/199 chlorotyrosine (-OH) 222/176

13C6 chlorotyrosine (immonium)

222/205 13

C6 chlorotyrosine (-OH) 13

C6 chlorotyrosine= 50.0 ng/mlchlorotyrosine= 15.1 ng/ml

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

Gas-Phase Ion Structure• Can chiral selectivity be observed in the gas-

phase?• First observation was by Fales et. al.

– JACS, 99(7), 2339-2340, (1977)

OH

OHO

O

OCH3

H3CO

OH

OHO

O

OCD3

D3CO

Ratio of protonated dimers was 1:1:1, not 1:2:1

Racemic mixture of labeled/unlabeled D/L enantiomers

Chiral Crown Ether Host-Guest Chemistry

• Sawada et. al. JACS, 117, 7726-7736, (1995)

• Used FAB to determine chiral selectivity of various crown ethers towards amino acids

O

O

O

OO OMe

OMe

Ph

H

Ph

H

NH3

CO2CH3

Et NH3

CO2CD3

Et

5:1 selectivity forone enantiomer

Mixture of labeled/unlabeled amino acidenantiomers

Thermochemical Measurement of Selectivity

• Dearden et. al JACS, 115, 4318-4320, (1993)– Measured equilibrium constant for chiral host guest reaction in the

gas phase (FT-ICR-MS)

O

N

O

OO

O

18O

O

O

OO

O

18

O O

NH3*+

KR=130KS=567∆G=4.2kJ/mol

∆G in CH2Cl2=4.6

Kinetic Evidence of Chirality

• Lebrilla et. al. JACS, 118, 8751-8752, (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

Requirements for Chiral MS Methodto 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

Host-Guest Exchange Reaction

• Lebrilla et. al. Anal. Chem., 73, 1684-1691, (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

Chiral Selectivity in Host-Guest Exchange

OH

NH2

CO2H

HO

DOPA

Cyclodextrin

NH2H2N

* L-DOPA Exchanges5x faster than D

Has been extended to other drugs (amphetamine,ephedrine, etc.) analysis onFTMS and Ion Trap

Kinetic Method Using CID

• Cooks et. al. Anal. Chem., 73, 1692-1698, (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.

Kinetic Method for Determination of %ee

CuLLA C IDA r* **+ CuLA** + CuLL* *+O R

CuL L

A

CIDAr

* *

*

+

CuL

A

*

*

+

CuL L* *

+OR

• 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