Separation and quantification of D- and L-phosphoserine in rat brain using N alpha-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (Marfey's reagent) by high-performance liquid chromatography with ultraviolet detection.

D-Serine has recently been described to be present in the brain at high concentrations. However, while prior research has demonstrated that L-phosphoserine is the major precursor of L-serine in the brain, the possible role of D-phosphoserine as the direct precursor of D-serine is unknown. To address this problem, we developed an assay to separate and quantitate D- and L-phosphoserine. A very simple HPLC-UV procedure for the separation and quantification of D- and L-phosphoserine is presented using precolumn derivatization with a chiral reagent, N alpha-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (Marfey's reagent), and a conventional C18 reversed-phase column. The procedure is sensitive to 11 pmol on-column and derivatives are stable for at least two weeks at room temperature. Rat brain regions (cortex, hippocampus, striatum, and cerebellum) were analyzed for the presence of D- and L-phosphoserine. It was determined that the brain regions studied contained exclusively L-phosphoserine.