High-performance liquid chromatographic separation of penicillamine enantiomers labelled with N-[4-(6-dimethylamino-2-benzofuranyl)phenyl] maleimide on a chiral stationary phase.

Penicillamine enantiomers derivatized with N-[4-(6-dimethylamino-2-benzofuranyl)phenyl]maleimide (DBPM) were separated and determined by high-performance liquid chromatography. A fluorogenic reagent, DBPM easily reacted with D- or L-penicillamine to give each two kinds of strong fluorescent derivatives (D1-, D2-, L1- and L2-DBPM), which could be separated on a Pirkle-type chiral stationary phase using an eluent of 75% aqueous methanol solution containing 0.15 M CH3COONH4 and 0.05 M tetra-n-butylammonium bromide. Two of the peaks (D1- or L1-DBPM), having a shorter retention time than the others, had almost the same retention times (25 min for D1-DBPM and 25.7 min for L1-DBPM). The retention times of the peaks eluted later were 28 min and 31.6 min for D2- and L2-DBPM respectively. Linear calibration curves over the range of 2-50 pmol per injection were obtained for D- and L-penicillamines with a detection limit of 290 and 350 fmol at respectively at a signal-to-noise ratio of 3. Using the proposed method, the absence of contamination of L-penicillamine in a commercially available D-penicillamine preparation (capsule) was confirmed.