Evaluation of a series of prolylamidepyridines as the chiral derivatization reagents for enantioseparation of carboxylic acids by LC–ESI–MS/MS and the application to human saliva

Abstract

Mass spectrometry has become a popular analytical tool because of its high sensitivity and specificity. The use of a chiral derivatization reagent for the mass spectrometry (MS) detection seems to be efficient for the enantiomeric separation of racemates. However, the number of chiral reagents for the liquid chromatography (LC)-MS/MS analysis is very limited. According to these observations, we are currently in the process of developing novel labeling reagents for chiral molecules in MS/MS analysis. The derivatization reagent that is effective for enhancing not only the electrospray ionization-MS/MS sensitivity but also the reversed-phase LC resolution of carboxylic acid enantiomers should have a highly proton-affinitive moiety and an asymmetric structure near the reactive functional group. Furthermore, the resulting derivative has to provide a characteristic product ion suitable for the selected reaction monitoring. Based upon these considerations, a series of prolylamidepyridines ((S)-N-pyrrolidine-2-carboxylic acid N-(pyridine-2-yl)amide (PCP2), (S)-N-pyrrolidine-2-carboxylic acid N-(pyridine-3-yl)amide, and (S)-N-pyrrolidine-2-carboxylic acid N-(pyridine-4-yl)amide) was synthesized as ideal labeling reagents for the enantioseparation of chiral carboxylic acids and evaluated in terms of separation efficiency and detection sensitivity by ultra-performance LC (UPLC)-MS/MS. Among the synthesized reagents, PCP2 was the most efficient chiral derivatization reagent for the enantioseparation of carboxylic acid. The Rs values and the detection limits of the derivatives of non-steroidal anti-inflammatory drugs, which were selected as the representative carboxylic acids, were in the range of 2.52-6.07 and 49-260 amol, respectively. The sensitive detection of biological carboxylic acids (detection limits, 32-520 amol) was also carried out by the proposed method using PCP2 and UPLC-MS/MS. The PCP2 was applied to the determination of carboxylic acids in human saliva. Several biological carboxylic acids, such as lactic acid (LA), 3-hydroxybutylic acid, maric acid, succinic acid, α-ketoglutalic acid, and citric acid, were clearly identified in the saliva of healthy persons and diabetic patients. Furthermore, the ratio of D-LA in diabetic patients was higher than that in normal subjects. Judging from these results, PCP2 seems to be a useful chiral derivatization reagent for the determination not only of chiral, but also achiral, carboxylic acids in real samples.