Novel experimental design paradigm for development of eco-friendly gradient chromatographic method for simultaneous determination of metronidazole and spiramycin.

This work describes the innovative experimental design-assisted development of a green gradient chromatographic method for concomitant analysis of metronidazole (MTR) and spiramycin (SPR). Two different designs including fractional factorial and Box-Behnken designs were implemented for screening and optimization steps, respectively. The optimum chromatographic conditions involved a mobile phase consisting of ethanol and 20 mM sodium dihydrogen phosphate solution (pH adjusted to 2.5) in the ratio 2:98 (v/v) for 2 min then the ratio changed to 30:70 (v/v). The flow rate was 1.3 mL/minute. Separation and analysis were performed on X-bridge C18 (150 mm × 4.6 mm × 3.5 μm) column with diode array detector set at 230 nm. Column oven temperature was 40°C. A linear response was acquired over the range of 5-125 μg/mL for both drugs. Detection and quantitation limits were 0.86 and 2.62 μg/mL for MTR and 0.92 and 2.83 μg/mL for SPR, respectively. The method was implemented for determination of both drugs in three tablet formulations. The method was proved to be green as evaluated by three assessment tools. The application of experimental designs assists in development of a robust green chromatographic method in gradient elution mode for determination of both drugs within reasonable time.