TRACE ANALYSIS OF IMPURITIES IN 3'-AZIDO-3'-DEOXYTHYMIDINE BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AND THERMOSPRAY MASS-SPECTROMETRY

An analytical method has been developed for the detection of trace amo unts of impurities in 3'-azido-3-'deoxythymidine referred to herein as AZT (Zidovudine). A sample extract was preconcentrated by normal-phas e high-performance liquid chromatography (HPLC) with subsequent on-lin e reversed-phase HPLC-thermospray mass spectrometry (TSP-MS). During t he sample extraction and concentration step, carried out by semiprepar ative normal-phase chromatography, the preliminary separation of the i mpurities from the AZT takes place. The organic solvent (dichloroethan e-acetonitrile, 40:60) is evaporated from the collected fractions and the compounds are redissolved in a smaller volume of the reversed-phas e mobile phases for a further degree of concentration. The collected f ractions are then subjected to reversed-phase HPLC-TSP-MS. The influen ce of acetonitrile concentration and pH on the reversed-phase separati on together with the sensitivity of the TSP-MS detection have been exa mined to maximise detection levels. The 3'-azido-3'-deoxy-5'-O-tritylt hymidine, triphenyl methanol and 3'-chloro-3'-deoxythymidine, which ar e route-indicative impurities formed during the synthesis can be detec ted in the 50-100 ppb (w/w) range.