Chiral ligand-exchange chromatography for diastereo-enantio separation of exametazime

Abstract

The diastereo-enantio separation of isomeric mixtures of exametazime (HM-PAO) by liquid chromatography is described using an achiral sorbent (RP-18). A chiral eluent with the initial complex of Cu(II) and the optically active selector N,N-dimethyl-l-phenylalanine (l-DM-PhA), based on the ligand-exchange principle, has been applied. The separation is based on the presence of the immobilized binary complex Cu(l-DM-PhA)(2) and formation of mixed ternary complex. The optimal mole ratio of Cu(II):l-DM-PhA is 1:4, the pH should be between 4.1 and 4.2 and up to 0.8 mM of triethylamine is added for column presaturation with the initial complex. The elution order has been defined using isolated l-HM-PAO via l-HM-PAO L(+)tartrate and tneso-HM-PAO obtained by repeated recrystallization from the isomeric mixture of HM-PAO. Complete resolution between all isomers (R-S from 2.14 to 3.91) and partial resolution for meso(EE)/l-HM-PAO (R-S = 0.83) has been obtained. This means that the proposed chiral ligand-exchange chromatography (CLEC) can be used for determination of the isomeric purity of HM-PAO. This as an alternative method for resolution measurements with chiral columns.