A. Almudaris et al., TRACE ANALYSIS OF IMPURITIES IN 3'-AZIDO-3'-DEOXYTHYMIDINE BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AND THERMOSPRAY MASS-SPECTROMETRY, Journal of chromatography, 689(1), 1995, pp. 31-38
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.