THESIS
2000
xvii, 151 leaves : ill. ; 30 cm
Abstract
Chiral analysis of amino acids was achieved by investigating the collision-induced dissociation spectra of protonated trimers that were formed from the electrospray ionisation of amino acids in the presence of one of the following chiral selectors: L-or D- N-t-butoxycarbonylphenylalanine (BPhe), L- or D- N-t-butoxycarbonylproline (BPro) and L- or D- N-t-butoxycarbonyl-O-benzylserine (BBSer). The protonated trimers were dissociated to protonated dimers, and the intensity ratio of the protonated dimer (product ion) to the protonated trimer (precursor ion), i.e., the observed dissociation efficiency r, was found to be strongly dependent on the chirality of the amino acids with respect to that of the chiral selectors. The results showed that the chirality of all 19 common amino acids can be...[
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Chiral analysis of amino acids was achieved by investigating the collision-induced dissociation spectra of protonated trimers that were formed from the electrospray ionisation of amino acids in the presence of one of the following chiral selectors: L-or D- N-t-butoxycarbonylphenylalanine (BPhe), L- or D- N-t-butoxycarbonylproline (BPro) and L- or D- N-t-butoxycarbonyl-O-benzylserine (BBSer). The protonated trimers were dissociated to protonated dimers, and the intensity ratio of the protonated dimer (product ion) to the protonated trimer (precursor ion), i.e., the observed dissociation efficiency r, was found to be strongly dependent on the chirality of the amino acids with respect to that of the chiral selectors. The results showed that the chirality of all 19 common amino acids can be definitely differentiated. The method was demonstrated as rapid, sensitive, precise, robust, and requiring no reference standards and only minimal sample preparation. The chirality of all three amino acids in a mixture was determined without prior separation of the amino acids, consuming only 70 pmole of sample and requiring only about 14 minutes of mass spectrometric measurements. A cyclodipeptide with unknown chirality was determined to be cyclo-( L-Pro-L-Leu) by acid hydrolysis followed by the present method, and the results were consistent with the physiochemical properties and NMR data of the compound.
The relationship between an observed mass spectrometric signal and enantiomeric excess (ee) has been reported to be linear in other previous studies. In the present study, however, both our derivations and experimental results confirmed that the relationship between the r value for an enantiomeric mixture and its ee is hyperbolic, and r = a + b/(c + ee), where a, b and c are constants. A linear calibration plot was obtained by plotting r versus l/(c + ee), where c was calculated with the MATLAB software, or by plotting l/(r - r
0) versus 1/ee, where r
0 is the r value for the racemic mixture. The latter "Two-Reciprocal" method was more convenient for application. Another practical method for ee determination was the "Three-Point" method, whereby the ee of an unknown sample with a measured r value could be derived from the equation ee = 100 x {l/(r
L-r
0) - l/(r
D-r
0)} / {2/(r-r
0) - l/(r
L-r
0) - l/(r
D-r
0)}, with r
L and r
D being the r values for the enantiomerically pure L- and D- forms of the sample respectively. A calibration plot was not required. The ee determination was achieved with acceptable precision even for the worst case of acceptable chiral recognition with a particular chiral selector, suggesting that the ee determination of all 19 common amino acids could be achieved by the present method. The ee of a histidine sample was determined both by the "Two-Reciprocal" method, giving an error of 0.2% ee (1.1% relative error) and consuming only about 5.3 nmole of sample, and by the "Three-Point" method, giving an error of 0.4% ee and consuming only about 2.3 nmole of sample. In the latter case, it took 27 minutes for the mass spectrometric measurements of the three calibration standards and an additional 9 minutes for that of the unknown sample. The direct ee determination of more than one amino acids in a mixture was also demonstrated in the study.
This study suggested that ESI-MS/MS can be a promising approach for the chiral analysis of other compounds.
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