Five-Step Plan for Proposing a Balanced HPLC Method via Fishbone Diagram: Untangling the Synergy of Efficiency and...

A five-step plan is presented to propose an HPLC method with the aid of the fishbone diagram to balance efficiency and sustainability. The five steps include method development, method optimization, method validation, greenness assessment, and sustainability profiling. A sensitive, accurate, robust, and rapid HPLC method has been developed for simultaneous determination and separation of anti-tubercular medications: rifampicin (RIF), isoniazid (INH), and pyrazinamide (PYR) in a fixed dose combination (FDC). The separation was attained by using a stationary phase Kromasil C₁₈ column (250 mm, 4.6,5 µm), accompanied by a gradient mode of the mobile phase in which the first solution was made up by using acetonitrile and phosphate buffer in a ratio of 4:96 v/v, and the second solution consisted of phosphate buffer and acetonitrile in ratio 45:55 v/v. The method was optimized by applying a fractional factorial design (2²), where the flow rate was set at 1.5 mL/min, the injection volume was 10 µL, and the detection was performed at 238 nm at a temperature of 35°C. The retention time for INH, PYR, and RIF were 3.82, 5.36, and 8.72 min (±0.02), respectively. The method was validated per the ICH guidelines, and the proposed method was found to be accurate, robust, precise, and selective. A design of experiment was established to assess the robustness of the study, where the obtained results proved that the developed method is robust. Two greenness tools, namely moGAPI and AGREE, were used to evaluate the method's greenness. In addition, the method was subjected to sustainability profiling using the EVG framework, where the suggested method was compared to other reported methods. The proposed method was found to be more sustainable and green, with a low negative impact on the environment.