Chiral analysis by electrospray ionization mass spectrometry/mass spectrometry. 2. Determination of enantiomeric excess of amino acids

The determination of enantiomeric excess (ee) of amino acids was achieved by investigating the collision-induced dissociation spectra of protonated trimers that were formed by electrospray ionization of amino acids in the presence of one of the following chiral selectors: L- or D-N-tert-butoxy-tert-carbonylphenlalanine, L- or D- N-tert-butoxycarbonylproline, and L- or D-N-tert-butoxycarbonyl-O-benzylserine, The protonated trimers were dissociated to form protonated dimers, and the observed dissociation efficiency r (i.e., the intensity ratio of protonated dimers to protonated trimers) for an enantiomeric mixture was found to be related to its ee value by the following equation: r = a + b/(c + ee), where a, b, and c were constants, A linear calibration plot was obtained by plotting r versus 1/(e + ee), where c was calculated with the MATLAB software, or by plotting 1/(r - r(0)) versus 1/ee, where r0 was 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{1/(r(L) - r(0)) - 1/(r(D) - r(0))}/{2/(r - r(0)) - 1/(r(L) - r(0)) - 1/(r(D) - r(0))}, With r(L) and rD being the r values for the enantiomerically pure Land 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 similar to5.3 nmol of sample, and by the three-point method, giving an error of 0.4\% ee and consuming only similar to2.3 nmol of sample, In the latter case, it took 27 min for the mass spectrometric measurements of the three calibration standards and an additional 9 min for the unknown sample. The direct ee determination of more than one amino acid in a mixture was also demonstrated in the study