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

Penicillamine enatiomers 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 (D 1− , D 2− , L 1− and L 2 DBPM), which could be separated on a Pirkle‐type chiral stationary phase using an eluent of 75% aqueous methanol solution containing 0.15 M CH 3 COONH 4 and 0.05 M tetra‐ n ‐butylammonium bromide. Two of the peaks (D 1− or L 1 ‐DBPM), having a shorter retention time than the others, had almost the same retention times (25 min for D 1 ‐DBPM and 25.7 min for L 1 ‐DBPM). The retention times of the peaks eluted later were 28 min and 31.6 min for D 2 ‐ and L 2 ‐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.