Reversed-Phase Liquid Chromatographic Separation of Enantiomeric and Diastereomeric Bases Related to Chloramphenicol and Thiamphenicol

Abstract

The important antimicrobial agents Chloramphenicol and thiamphenicol are N-acylated amines whose chemical structures include two chiral centers. Each drug is the single enantiomer of R,Rconfiguration. The N-deacylated bases of the drugs are important intermediates in their synthesis and optical resolution. In this report, reversed-phase HPLC methods are described for the separation of enantiomeric and diastereomeric bases of the two drugs and of two closely related bases used in some syntheses of the drugs. The stereoisomeric bases were derivatized with a homochiral isothiocyanate and the resulting diastereomeric thioureas were separated on C18 columns with methanol:water mixtures as mobile phases and detection at 254 nm. The four stereoisomeric bases of Chloramphenicol and those of its unnitrated analogue were thus separable after derivatization with 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate. This reagent also allowed the separation of the D-threoisomer of the p-mercaptomethyl analogue of thiamphenicol base from its stereoisomers. The stereoisomers of thiamphenicol base were similarly separated with (R)-α-methylbenzyl isothiocyanate as the derivatizing agent. The diastereomers of chloramphenicol base and of thiamphenicol base were chromatographically separable after derivatization with the nonchiral reagent benzyl isothiocyanate. The procedures developed may be useful in the determination of the stereoisomeric composition of the drugs in research and in quality control, and may be applicable to other similar agents whose chemistry and pharmacology are receiving considerable attention.