The resolution of free DL-amino acids in human nail was carried out by combination of the R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R(-)-DBD-PyNCS] derivatives and UPLC-ESI-TOF-MS.The reaction of the reagent with amino acids effectively proceeds at 55℃ for 20 min in the presence of 1% triethylamine (TEA) to produce the corresponding diastereomers.Each pair of the resulting derivatives was efficiently separated by a gradient program (a mixture of H2O and CH3CN containing 0.1% formic acid (HCOOH) or 5 mM CH3COONH4 and CH3CN) using a reversed-phase ACQUITY UPLCTM BEH C18 (1.7 μm, 100 mm × 2.1 mm i.d.) column and sensitively detected by TOF-MS.