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.