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) ( 13 C, 15 N) 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 13 C) dioxins are used as internal standards
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), 17415-17427, (2002)
Triple Quadrupole Mass Analyzer Sample Inlet Ion Guide Q1 Q2 (Collision cell) Q3 EM Detector
Multiple Reaction Monitoring in a Triple Quadrupole Q1 (227) Q2 collision cell Q3 (181) LC colum n H N 2 NH 3 + H H N 2 NH 2 + Set on mass of parent ion Fragment parent ion Transmit only diagnostic product ion highest duty cycle triple quadrupole scan type!
MRM transitions diagnostic product ions parent ion immonium loss of H tyrosine 182 136 165 nitrotyrosine 227 181 210 13 C 6 nitrotyrosine 233 187 216 chlorotyrosine 216 170 199 13 C 6 chlorotyrosine 222 176 205 bromotyrosine 260 214 243 13 C 6 bromotyrosine 266 220 249
Intensity, cps 4000 3000 2000 216/170 chlorotyrosine (immonium) 216/199 chlorotyrosine (-H) 222/176 13 C 6 chlorotyrosine (immonium) 222/205 13 C 6 chlorotyrosine (-H) 13 C6 chlorotyrosine= 50.0 ng/ml chlorotyrosine= 15.1 ng/ml 1000 0 4 6 8 Time, min 10 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 nly 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 gasphase? First observation was by Fales et. al. JACS, 99(7), 2339-2340, (1977) Racemic mixture of labeled/unlabeled D/L enantiomers H H H 3 C CH 3 D 3 C CD 3 H H Ratio of protonated dimers was 1:1:1, not 1:2:1
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 Ph Ph H Me H Mixture of labeled/ unlabeled amino acid enantiomers Me 5:1 selectivity for one enantiomer Et NH 3 Et NH 3 C 2 CH 3 C 2 CD 3
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) N 18 18 K R =130 K S =567 G=4.2kJ/mol * NH 3 + G in CH 2 Cl 2 =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 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
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 H H DPA H 2 N NH 2 NH * 2 L-DPA Exchanges C 2 H 5x faster than D Cyclodextrin Has been extended to other drugs (amphetamine, ephedrine, etc.) analysis on FTMS 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.
+ L* + L* + CID L* L* L* Cu Ar Cu R Cu A* A* Kinetic Method for Determination of %ee L* L* Cu A * + CID Ar Cu A * * L + R L* L* Cu + 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