Determination of D-alanine in the rat central nervous system and periphery using column-switching high-performance liquid chromatography.

A column-switching chiral HPLC system for the determination of minute amounts of D-Ala in mammalian tissues has been established. D-Ala and its L-enantiomer are purified as a DL mixture on a micro-ODS column after precolumn fluorescence derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and are introduced to a chiral column to determine each enantiomer. The calibration curve of D-Ala spiked into a rat cerebellum sample is linear from 5 to 5000 fmol with a correlation coefficient of 1.0000. The lower limit of quantitation of D-Ala is 5 fmol (S/N=5). Within-day and day-to-day precisions of spiked D-Ala (15 fmol) are 3.9 and 4.8% (R.S.D), respectively. With this system, the anatomical distribution of free D-Ala in the rat central nervous system and periphery has been investigated. Among the 22 examined tissues of the rat, the highest amount of D-Ala has been observed in the anterior pituitary gland (86.4+/-9.9 nmol/g wet tissue), and the second highest amount has been observed in the pancreas (29.2+/-5.0 nmol/g wet tissue). Postnatal and day-night changes in D-Ala amounts in the anterior pituitary gland have also been studied. The amount of D-Ala is highest at 6 weeks of age and significantly decreases with age, and the amount of D-Ala is significantly higher during the daytime than during the nighttime.