Development of an online two-dimensional high-performance liquid chromatographic system in combination with tandem mass spectrometric detection for enantiomeric analysis of free amino acids in human physiological fluid.

An automated two-dimensional HPLC-MS/MS system was designed and developed for the highly selective determination of trace levels of d-amino acids. As the targets, frequently observed ones in mammalian physiological fluids, Ala, Asp, Glu, Leu, Pro and Ser, were selected because these d-amino acids are the potential biomarkers for the early and sensitive diagnoses of various diseases. The target analytes were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), then isolated by a reversed-phase column in the first dimension. The NBD-amino acid fractions were automatically collected into the multi-loop device, and introduced into the enantioselective column in the second dimension to separate the d- and l-forms followed by detection by an MS/MS. The obtained resolution values of the enantiomers were 1.87-5.17 and the calibration lines, precision and accuracy were practically sufficient. By using the present 2D HPLC-MS/MS system, trace levels of d-amino acids in complex biological matrices were determined without disturbance by the intrinsic interfering compounds, and successfully applied to the analysis of the human clinical samples (plasma and urine).