Antibacterial drugs are receiving the biggest attention due to multiple hazards associated with their uncontrolled usage such as hypersensitivity and evolution of antibiotic-resistant bacterial mutants such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), and multidrug-resistant Tuberculosis (MDR-TB). The restrictions regarding the usage of antimicrobials in humans play an important role in decreasing the unwanted effects, but the case is not the same when it comes to the usage of antimicrobials in animals. The effect of uncontrolled usage of antimicrobials in animals is not limited to animals’ lives but reaches humans in different ways. One of these ways is the consumption of poultry products of birds that have been treated with antimicrobials. Some estimations reported that poultry products represent around 40 % of global meat production according to data published by the Food and Agriculture Organization (FAO) [1]. In the year 2021, poultry meat yearly consumption per capita in the Middle East region was estimated to reach its maximum in Saudi Arabia (41.5 kg), then Libya (38.4 Kg), Egypt (21.3 Kg), and finally Sudan (1.75 Kg) [2]. This huge percentage and the tremendous increase in demand required a parallel development in the industry to satisfy the demand. Thus, the use of antibiotics like tetracycline, bacitracin, sulphonamides, and others is common in the poultry industry for treatment, prophylaxis or even growth promotion [3].

To control the leakage of antibiotics to humans, Codex Alimentarius Commission (CAC), the central part of the Joint FAO/WHO Food Standards Program, places food guidelines known as Codex Alimentarius [4]. These guidelines determine the maximum residue limit (MRL) of different compounds, including pharmaceuticals, which are allowed to be present in food and contribute to the quality, safety, and equity of global food trading.

Coccimix® is a veterinary drug formulation that consists of four drugs, two sulfonamide antibiotics, sulfadimidine sodium (SDM) and sulfaquinoxaline sodium (SQX), which inhibit the bacterial synthesis of folic acid by competing with p-aminobenzoic acid for dihydrofolate synthetase in metabolism, one antiprotozoal namely diaveridine (DVR), and vitamin K3 (VK3), also known as menadione. Coccimix is indicated for both the prevention and treatment of a disease known as coccidiosis, which is a parasitic infection that may occur as a result of the coccidian protozoa that infects poultry. The chemical structures for the four drugs are shown in Supplementary material Fig. 1SM.

Several chromatographic methods have been reported for the determination of different sulfonamides in chicken [5] such as HPLC-UV [6], [7], [8], [9], HPLC with fluorescence detection [10], UPLC [11,12], and gas chromatography [13,14]. Moreover, a few chromatographic methods have been reported for the analysis of DVR alone [15,16] or in combination with other veterinary drugs in chicken [17], [18], [19]. For VK3, it has been quantified in eggs using liquid chromatography tandem mass spectrometry [20], and in feed premixes using gas chromatography [21]. The analysis of the dosage form (Coccimix ®) quaternary mixture has been reported using spectrophotometry [22] and liquid chromatography [23], but both methods could not be applied for the analysis of the mixture of the four drugs in poultry tissues due to matrix interference and critical residual concentrations that cannot be detected by either of these methods. Thus, a new bioanalytical method was needed.

This decline curve study aims to analyze the critical concentration of the residues of the studied drugs in the tissues of poultry treated with Coccimix in order to decide the time required to reach the MRL, then the poultry product will be safe for human use. Bioanalytical method was successfully developed using UPLC-MS-MS. The design of experiment with response surface methodology ensured the development of optimal extraction procedure of the studied drug residuals from such a complex matrix. The greenness of the proposed sample preparation and analytical method was assessed by applying Analytical Eco-scale (AES) and AGREE couplesd with AGREEprep. The competence of the whole study was evaluated via a three-pillar framework known as Efficiency, validation, and greenness (EVG) framework to achieve a balance point represented by a radar chart.