Determination of impurities in azidothymidine by HPTLC densitometry and HPLC

Summary

HPTLC densitometry and HPLC have been developed for the determination of potential impurities of azidothymidine (theymine, isothymidine, Az-Tr-thymidine, Ms-Tr-thymidine and Tr-alcohol). In HPTLC the substances were separated on silica gel with fluorescence indicator in 5∶95 methanol-chloroform for the simultaneous determination of Az-Tr-thymidine, Ms-Tr-thymidine and Tr-alcohol and in 10∶90 methanolchloroform for the simultaneous determination of thymine and isothymidine. Absorbance measurement (detection of reflectance) of separated substances was carried out “in situ” at 268 and 220 nm using four-level calibration (external standard) in the range of 25–100 ng thymine and isothymidine per spot. 50–200 ng Ms-Tr-thymidine and Az-Tr-thymidine per spot and 250–1000 ng Tr-alcohol per spot. For the HPLC determination of impurities two procedure are described one for thymine and isothymidine and the other for Ms-Tr-thymidine, Tr-alcohol and Az-Tr-thymidine. The first procedure was carried out using RP-18 stationary phase and methanol+aqueous 0.03 mol/l KH₂PO₄ (18+82, v/v) as the mobile phase. The temperature was 50°C and the detection wavelength 266 nm. In the second procedure the methanol content of the mobile phase was increased to 81% and the detection wavelength was changed to 210 nm. The detection limit of all impurities was 0.05 %. The concentration range for thymine was 1–40μg/ml and for the other impurities 1–20 μg/ml (for an injection volume of 10 μl). The results were evluated by linear regression analysis.