Determination of minute amounts of D-leucine in various brain regions of rat and mouse using column-switching high-performance liquid chromatography.
A highly sensitive method for the determination of minute amounts of D-Leu in biological samples was developed. For accurate and sensitive determination, a column-switching system using a micro ODS column and a chiral column was adopted. After pre-column derivatization of D- and L-Leu with NBD-F, the derivatives of the enantiomers were purified on a micro ODS column as a DL mixture. The eluted DL-Leu was then introduced to the chiral column, and each enantiomer was determined. The calibration curve for D-Leu, which was constructed by adding known amounts of D-Leu to a rat hippocampus, was linear from 1 to 1000 fmol (r>0.999), and the detection limit of added D-Leu was 1 fmol (S/N=5). Within-day and day-to-day precisions of D-Leu determination using the same homogenate of rat hippocampus were 5.11 and 5.25% (RSD), respectively. The content of D-Leu in rat hippocampus was 0.69 nmol/g wet tissue (the percentage of D-enantiomer for total Leu was 0.97%), which was consistent with the reported value. The distribution of D-Leu in mouse brain was also investigated, and the presence of D-Leu in various regions of the mammalian brain was first observed.
Inoue
T
T
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Hamase
K
K
Morikawa
A
A
Zaitsu
K
K
D000818:Animals; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006624:Hippocampus / Q000737:chemistry*; D007930:Leucine / Q000032:analysis* / Q000302:isolation & purification; D008297:Male; D051379:Mice; D051381:Rats; D017208:Rats, Wistar; D013237:Stereoisomerism
D007930:Leucine