Enantiomeric separation of D- and L-serine using high-performance liquid chromatography with electrochemical detection following pre-column diastereomer derivatization.

Enantiomeric separation of D- and L-serine on an octadecylsilica column was investigated using (2R)-2,5-dioxopyrrolidin-1-yl-2,5,7,8-tetramethyl-6-(tetrahydro-2H-pyran-2-yloxy)chroman-2-carboxylate (R-NPCA), which was developed for a pre-column derivatization reagent for electrochemical detection. In addition, (2S)-2,5-dioxopyrrolidin-1-yl-2,5,7,8-tetramethyl-6-(tetrahydro-2H-pyran-2-yloxy)chroman-2-carboxylate (S-NPCA) was newly synthesized from (S)-(-)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (S-alpha-CA), and the enantiomeric separation of D- and L-serine using S-NPCA was also examined. The enantiomeric separation of D,L-serine was achieved using the R- or S-NPCA as a chiral derivatization reagent, and the elution orders of the enantiomers were reversed between R- and S-NPCA. The elution orders of D- and L-serine unexpectedly reversed between the phosphate buffer at pH 4.0 and pH 2.2, both of which were used in the mobile phase. Separation factors obtained using R- and S-NPCA were similar-1.09 and 1.07, respectively. The detection limit was approximately 940 fmol on the column (signal-to-noise ratio 3) when the applied voltage was +650 mV.