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.
Zhao
Hua
H
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Hamase
Kenji
K
Morikawa
Akiko
A
Qiu
Zongyin
Z
Zaitsu
Kiyoshi
K
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives*; D000818:Animals; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D013237:Stereoisomerism; D013912:Threonine / Q000031:analogs & derivatives* / Q000032:analysis*; D014018:Tissue Distribution
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; C032930:7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole; D013912:Threonine; D009327:4-Chloro-7-nitrobenzofurazan