Determination of d- and l-enantiomers of threonine and allo-threonine in mammals using two-step high-performance liquid chromatography

A sensitive and selective method for the determination of four threonine (Thr) isomers (l-Thr, d-Thr, l-allo-Thr and d-allo-Thr) in mammalian tissues has been established using two-step high-performance liquid chromatography. This method includes the precolumn fluorescence derivatization of amino acids with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and the separation using a combination of a reversed-phase column and a chiral column. The calibration ranges of d-Thr, d-allo-Thr and l-allo-Thr spiked in the rat cerebellum sample are 2.5 fmol–5 pmol per injection, and that of l-Thr is 50 fmol–50 pmol. Within-day and day-to-day precisions of the determination of the four Thr isomers are approximately 5% in the rat cerebellum. By using this method, the tissue distributions of d-Thr, d-allo-Thr and l-allo-Thr in mammals have been demonstrated for the first time in rats, and found that significant amounts of d-Thr and d-allo-Thr are present in the frontal brain areas and urine. Among the 12 tissues tested, the highest amounts of d-Thr (0.85 ± 0.05 nmol/g wet tissue) and d-allo-Thr (5.01 ± 0.32 nmol/g wet tissue) were found in the corpus striatum. l-allo-Thr was not present in any of the tested tissues and physiological fluids.