Simultaneous determination of binary diffusion coefficients from multiple response curves by chromatographic measurements

A validated two-dimensional HPLC method for the comprehensive analysis of small quantities of branched aliphatic D-amino acids in the presence of large amounts of their L-congeners in mammalian tissues and physiological fluids is described. The quantitative analysis of these aliphatic amino acids (Val, allo-Ile, Ile, and Leu) is important for the diagnosis of various inherent metabolic disorders of amino acids, and the D-enantiomers are expected to be of particular interest from a pharmacological point of view. Target analytes were determined as their fluorescent derivatives, pre-column labeled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), using an automated two-dimensional column-switching high-performance liquid chromatographic system combining a narrow bore reversed-phase column and an enantioselective column connected with an integrated multi-loop peak fraction storage device. The described two-dimensional analysis concept proved to be successful for the given task in biological samples taken from mammals. Total analysis time for the reversed-phase separation of the four target NBD-amino acids is 60 min, and the integrated enantiomer separation of each of the four analytes is completed in approximately 5 min. In the rat, significant amounts of D-Leu were found in all tissues and physiological fluids tested (trace-1.3 nmol/g tissue), and in the urine, the presence of high amounts of D-allo-Ile (D-isomer of a non-proteinogenic amino acid, 22.2 nmol/ml) was demonstrated. D-allo-Ile was also found in the urine of dog and mouse, which indicates the ubiquitous presence of this unusual D-amino acid and the potential need to clarify its unique metabolism in mammals.