Design of experiment-oriented development of solvent-free mixed micellar chromatographic method for concomitant determination of metronidazole and ciprofloxacin hydrochloride.

A green, fast and robust solvent-free chromatographic method has been developed for concomitant analysis of ciprofloxacin HCl and metronidazole in bulk powder as well as in dosage form using levofloxacin as internal standard (I.S.). Two different designs including fractional factorial (FFD) and Box-Behnken (BBD) designs were implemented for screening and optimization steps, respectively. The optimum chromatographic separation was accomplished using mobile phase composed of 0.13 M sodium dodecyl sulfate and 0.02 M Birij-35 solution adjusted to pH 2.5 using phosphoric acid at a flow rate of 1.3 mL/min and column oven temperature of 40 °C. Chromatographic analysis was performed on X-Bridge (150 mm × 4.6 mm, 5 µm) column with UV detection at 280 nm. A linear response was acquired over the range of 0.4-50 µg/mL for both drugs. The developed method was applied for quantitation of cited drugs in commercially available tablet with mean percent recovery ± SD of 99.45 ± 0.72 and 100.13 ± 0.81 for metronidazole and ciprofloxacin respectively. The method was proven to be green as evaluated by three greenness assessment tools. The run time was 8 min, thus saving time and reagent.

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.

Quality by design paradigm for optimization of green stability indicating HPLC method for concomitant determination of fluorescein and benoxinate.

A green, robust and fast stability indicating chromatographic method has been developed for concomitant analysis of fluorescein sodium and benoxinate hydrochloride in the presence of their degradation products within four minutes. Two different designs including fractional factorial and Box-Behnken designs were implemented for screening and optimization steps, respectively. The optimum chromatographic analysis was achieved using a mixture of isopropanol and 20 mM potassium dihydrogen phosphate solution (pH 3.0) in the ratio 27:73 as mobile phase. The flow rate was 1.5 mL/min and column oven temperature was 40 °C. Chromatographic analysis was performed on Eclipse plus C18 (100 mm × 4.6 mm × 3.5 µm) column with DAD detector set at 220 nm. A linear response was acquired over the range of 2.5-60 µg/mL and 1-50 µg/mL for benoxinate and fluorescein respectively. Stress degradation studies were executed under acidic, basic, and oxidative stress conditions. The method was implemented for quantitation of cited drugs in ophthalmic solution with mean percent recovery ± SD of 99.21 ± 0.74 and 99.88 ± 0.58 for benoxinate and fluorescein respectively. The proposed method is more rapid and eco-friendly compared to the reported chromatographic methods for determination of cited drugs.

Experimental design approach for development of spectrofluorimetric method for determination of favipiravir; a potential therapeutic agent against COVID-19 virus: Application to spiked human plasma.

The present work describes development of rapid, robust, sensitive and green spectrofluorimetric method for determination of favipiravir (FAV). Different factors affecting fluorescence were carefully studied and Box Behnken Design was applied to optimize experimental parameters. The proposed method is based on measuring native fluorescence of FAV in 0.2 M borate buffer (pH 8.0) at 432 nm after excitation at 361 nm. There was a linear relationship between FAV concentration and relative fluorescence intensity over the range 40-280 ng/mL with limit of detection of 9.44 ng/mL and quantitation limit of 28.60 ng/mL. The method was successfully implemented for determination of FAV in its pharmaceutical formulation with mean % recovery of 99.26 ± 0.87. Moreover, the high sensitivity of the method allowed determination of FAV in spiked human plasma over a range of 48-192 ng/mL. The proposed spectrofluorimetric method was proved to be eco-friendly according to analytical eco-scale.