A novel isocratic reverse phase high performance liquid chromatography (RP–HPLC) with photo diode array (PDA) detection method for the determination of dobutamine (DBT) in rat plasma was developed and validated after optimization of various chromatographic conditions and other experimental parameters. Homoveratrylamine was used as an internal standard. Methanol was used as the extracting solvent for the preparation of plasma samples. Samples were separated on a Symmetry C18 (250 mm×4.6 mm i.d., 5 μm) analytical column. Acetonitrile and 15 mM potassium dihydrogen phosphate (pH 5.0 with 0.3% TEA) (20:80, v/v) was used. The column oven temperature was optimized at 35 °C and the flow rate was 0.8 mL/min. The detection wavelength was fixed at 230 nm for entire analysis. The calibration curve was found to be linear over the concentration range of 50–2000 ng/mL (r2=0.9992). The limit of quantification (LOQ) of the method was 50 ng/mL. The % RSD values of accuracy and precision values for intra and inter days were <15% at quality control (QC) concentrations. Recovery, stability and robustness were studied within the acceptable range according to ICH guidelines. The method was efficiently applied to a pharmacokinetic study in healthy Wistar rats.

This paper describes the development of a rapid, novel, stability-indicating gradient reversed-phase high-performance liquid chromatographic method and associated system suitability parameters for the analysis of naproxcinod in the presence of its related substances and degradents using a quality-by-design approach. All of the factors that affect the separation of naproxcinod and its impurities and their mutual interactions were investigated and robustness of the method was ensured. The method was developed using an Ascentis Express C8 150 × 4.6 mm, 2.7 µm column with a mobile phase containing a gradient mixture of two solvents. The eluted compounds were monitored at 230 nm, the run time was 20 min within which naproxcinod and its eight impurities were satisfactorily separated. Naproxcinod was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation. Naproxcinod was found to degrade significantly in acidic and basic conditions and to be stable in thermal, photolytic, oxidative and aqueous degradation conditions. The degradation products were satisfactorily resolved from the primary peak and its impurities, proving the stability-indicating power of the method. The developed method was validated as per International Conference on Harmonization guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness.