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

• Published in: Analytical biochemistry 2003, Vol.312 (1), p.66-72
• Main Authors: Morikawa, Akiko, Hamase, Kenji, Zaitsu, Kiyoshi
• Format: Article
• Language: English
• Subjects: Alanine - analysis; Animals; Anterior pituitary gland; Central Nervous System - chemistry; Chiral separation; Chromatography, High Pressure Liquid - instrumentation; Chromatography, High Pressure Liquid - methods; d-Amino acid; Male; Rats; Rats, Wistar; Spectrometry, Fluorescence
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/S0003-2697(02)00432-3

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.