Separation, Isolation, and Characterization of Isoform Impurities of Gemcitabine Formed During the Anomerization of Protected α‐Gemcitabine to Gemcitabine

Abstract

In the impurity profile of the gemcitabine active pharmaceutical ingredient, two process related impurities (0.12%) were detected other than the known α‐isomer of gemcitabine impurity in HPLC with respect to gemcitabine. The mass spectral data and UV spectrum of these two impurities were similar to that of gemcitabine. These impurities were referred as isoform impurity I and isoform impurity II. These isoform impurities were isolated from a crude sample of gemcitabine bulk drug using a reverse phase preparative high performance liquid chromatography (prep‐HPLC), and were characterized by LC‐MS, LC‐MS/MS, and FT‐NMR experimental techniques. Based on the results obtained from the different spectroscopic experiments, these isoform impurities were characterized as β‐anomer of 4‐amino‐1‐(3,3‐difluoro‐4,5‐dihydroxy tetrahydro‐2H‐pyran‐2‐yl)pyrimidin‐2(1H)‐one (isoform impurity I) and α‐anomer of 4‐amino‐1‐(3,3‐difluoro‐4,5‐dihydroxy tetrahydro‐2H‐pyran‐2‐yl)pyrimidin‐2(1H)‐one (isoform impurity II), respectively. Formation, separation, isolation, and characterization of these isoform impurities were discussed in detail.

Keywords:

• Gemcitabine
• Isoform impurities
• Isolation
• Prep‐HPLC
• Spectroscopy
• Characterization