development of a validated rp-hplc method for separation - farmacia

FARMACIA, 2009, Vol. 57, 5 537
Impurities standard solutions. 0.5mg/mL impurities stock
standard solutions were prepared in methanol. These solutions were kept at
+4°C, protected from light. In these conditions the solutions were stable for
two months. Working solutions were prepared by dilution to a concentration
of 1 µg/mL and injected into the system.
Omeprazole standard solution. 1.015mg/mL omeprazole stock
standard solution was prepared in methanol and was kept at +4°C, protected
from light. In these conditions it was stable for two months. Omeprazole
working solutions were prepared by dilution using previously prepared
impurities solutions at concentrations between 40-200µg/mL and injected
into the system.
Sample solution. 2mg/mL solution of omeprazole sample was
prepared in methanol and diluted to a concentration of 0.200µg/mL and
injected into the system.
Forced degradation samples for the specificity study.
Omeprazole was kept in a climatic chamber at 40±5°C, 75±5%
humidity and was exposed to artificial light for 6 months to study the
formation of the degradation products. Every two weeks, an omeprazole
sample was withdrawn from the climatic chamber to perform the analyse.
Chromatographic conditions. A Zorbax Extend C 18 analytical
column (150mm×4.6 mm, 5µm packing) (Agilent) was used for analysis at
25°C. The mobile phase was pumped through the column at a flow rate of
0.8 mL/min. Gradient program was: 0min(18%B), 7.2min(18%B),
10.5min(50%B), 12min(50%B), 16min(18%B) and 18min(18%B). The
sample injection volume was 20µL. The UV detector was set to a
wavelength of 280 nm for the detection.
Results and discussion
Method development
In order to develop a suitable and robust LC method for the
determination of omeprazole and its process-related impurities, different
mobile phases and columns were employed to achieve the best separation
and resolution. Finally, the mobile phase consisting of water:triethylamine
1% pH adjusted to 9.5 with 85% H 3 PO 4 and acetonitrile at a flow rate of 0.8
mL/min, using a ZorbaxExtend C 18 (150mm×4.6mm, 5µm) column was
found to be appropriate, allowing good separation of omeprazole and its
process-related impurities. Because, the impurity D and the impurity G were
not separated in the described chromatographic conditions, we choose to use
only impurity D along with others impurities in future analysis. The reason