Separation of 3'-azido-2',3'-dideoxythymidine pronucleotide diastereoisomers in biological samples by CZE with cyclodextrin addition

The possibility of using capillary electrophoresis as an alternative technique to HPLC for the separation of pronucleotide diastereoisomers of AZT was investigated. In the pH range 6.2-7.2 where the analytes are stable, a chiral additive, carboxymethyl-beta-CD, was found appropriate to enable the separation of the uncharged diastereoisomers. An experimental design strategy was used to study the influence of several parameters (CD and phosphate buffer concentration, methanol content of the electrolyte, injected volume, capillary length, electric field and separation temperature) on the separation and find suitable analytical conditions for monitoring the prodrugs in cell extracts. The diastereoisomers of the three tBuSATE phenylphosphotriester derivatives of AZT studied could be fully resolved within short analysis time (less than 10 min). Method validation results showed satisfactory results for linearity, accuracy and repeatability.