| 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 1/(c + ee), where c was calculated with the MATLAB software, or by plotting 1/(r - r 0) versus 1/ee, where r0 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. This study suggested that ESI-MS/MS can be a promising approach for the chiral analysis of other compounds. (Abstract shortened by UMI.). |
