Development and validation of a stability-indicating HPLC method for the simultaneous determination of anticoccidial drugs in veterinary formulations: greenness and whiteness assessment

Intestinal coccidiosis is a significant parasitic disease affecting poultry, resulting in substantial economic losses for the industry. It compromises the nutrition absorption, leading to weight loss and elevated mortality rates. Furthermore, the stress caused by the infection can compromise the immune system, making poultry more susceptible to secondary infections and reducing overall productivity. As a result, simple analytical techniques are critical for determining anticoccidial drugs. A new, sensitive, and environmentally friendly HPLC method was developed for determining amprolium (AMP), sulfaquinoxaline (SUL), diaveridine (DIV), and vitamin K3 (VIT K3) in their formulations for the first time. Stability tests were performed under diverse stress conditions to verify the safety and efficiency of the formulation throughout its designated shelf time. These investigations ascertain the influence of various environmental conditions on a drug's chemical stability and physical characteristics. A Supelcosil C18 column was used as the stationary phase, and 0.05 M KH₂PO₄ and acetonitrile were mixed in a ratio of 80:20 (v/v) as the developing system with a flow rate of 2.0 mL min^-1. The proposed drugs were quantified at 260 nm. It was tested and found that the novel analytical method was linear for AMP and SUL between 20.0 and 60.0 µg mL^-1, 2.0-6.0 µg mL^-1 for VIT K3, and 2.1-6.3 µg mL^-1 for DIV. The anticipated method was validated according to ICH guidelines. Advanced evaluation tools, such as GAPI, Red Green Blue (RGB 12, whiteness), Blue Applicability Grade Index (BAGI), the Analytical Eco-Scale, and (AGREE) assessed the sustainability profile of the proposed method, illustrating its enhanced environmental friendliness and sustainability.