The method development was performed with a High-pressure liquid chromatographic system consisting of an autosampler Perkin Elmer LC ISS Series 200, an ultraviolet diode array detector (Perkin Elmer LC 235 C). The system was controlled, and data analysis were performed with the software package Turbochrom Version 4.1. Plus, and UV-spectrometric data were produced by TurboScan Version 2.0. The detector was set at 280 nm, and peak areas were integrated automatically by using the software. Chromatographic separation was carried out using LiChroCart LiChrospher 60 RP select B (4.0 mm x 250 mm, 5 µm).
A series of parameters, including composition and pH of mobile phase and flow rate, were tested concerning the location and shape of the peaks of pantoprazole and the internal standard in the corresponding chromatograms. The final choice of the mobile phase giving satisfying resolution and run time was 0.2 % (V/V) triethylamine in water with pH = 7 and acetonitrile (58:42, V/V). The triethylamine solution was prepared by adding 200 μL triethylamine in 100 mL water and pH of this solution [7 (link)] was adjusted by concentrated o-phosphoric acid. The mobile phase was filtered and degassed with helium and delivered at a flow rate of 1.2 mL/min. The injection volume was 50 µL.
A series of parameters, including composition and pH of mobile phase and flow rate, were tested concerning the location and shape of the peaks of pantoprazole and the internal standard in the corresponding chromatograms. The final choice of the mobile phase giving satisfying resolution and run time was 0.2 % (V/V) triethylamine in water with pH = 7 and acetonitrile (58:42, V/V). The triethylamine solution was prepared by adding 200 μL triethylamine in 100 mL water and pH of this solution [7 (link)] was adjusted by concentrated o-phosphoric acid. The mobile phase was filtered and degassed with helium and delivered at a flow rate of 1.2 mL/min. The injection volume was 50 µL.