Emerging quality-by-design optimized HPLC method for the concurrent determination of cefixime and ornidazole: a multi-criteria green and blue environmental footprinting

www.nature.com/scientificreports OPEN Emerging quality-by-design optimized HPLC method for the concurrent determination of cefixime and ornidazole: a multi-criteria green and blue environmental footprinting Yasmeen E. Mostafa, Fawzi Elsebaei & Mohammed El-Sayed Metwally Design of experiments has emerged as an influential and proactive approach that demonstrated favored applicability in optimizing chromatographic procedures. Quality-by-design guarantees the long-standing efficiency of the developed HPLC method reducing the need for costly and resourceintensive univariate approach. A low environmental footprint, sensitive, fast, and simple HPLC method was developed for concurrent estimation of cefixime (CFX) and ornidazole (ORN) in their raw materials, laboratory-prepared tablets, and pharmaceutical formulations using 23 full factorial design. The separation was performed on a cyano column through UV detection at 300 nm in less than 4 min. The elution was achieved using a mobile phase (pH = 6.0) composed of methanol and 0.3% triethylamine in ratio of (85%: 15%, v/v), respectively with a flow rate of 1 mL/min. The method’s validity was established with ICH Q2 R1 regulations. Excellent linear correlation (r = 0.9999) was established within concentration ranges lies between 1 and 50 µg/mL and 0.5–50 µg/mL with high percent found of 100.04 ± 0.82 and 99.99 ± 1.01 for CFX and ORN, respectively. The approach exhibited high precision (% RDS < 2) and sensitivity with detection limits of 0.07 and 0.09 µg/mL and quantification limits of 0.21 and 0.28 µg/mL for CFX and ORN, respectively. Several metrics evidenced the method’s greenness and sustainability profile. Keywords Full factorial design, HPLC, Cefixime, Ornidazole, Laboratory prepared tablet, Sustainability profile From the early 1900 s, chromatography has emerged as the most used analytical technique analysis tool in many domains either in organic chemistry, pharmaceutical chemistry, and biochemistry1. Nevertheless, one of the great challenges in chromatography has regularly been creating a robust and trustworthy chromatographic analysis procedure. Numerous parameters that impact the analysis findings must be investigated to reach the optimum analytical conditions. However, that stage was achieved through a one-variable-at-time approach. In this traditional approach, the impact of changing one variable was examined while keeping the level of other variables constant. As a result, it has numerous drawbacks, including being laborious, time and materialsconsuming, tedious and requiring large number of experiments. Additionally, neglecting the interaction between factors is considered a flaw at its core of that univariate approach2. Thus, there is an impressive need to overcome these disadvantages while developing a chromatographic method. The design of experiments (DOE) system plays a crucial role in analyzing and modelling how process variables affect response variables. In other words, it is a means to assess cause and effect interactions. Statistically designed experiments produce valid and significant findings through statistical data collection and analysis3. DOE has been effectively implemented in various fields such as pharmaceuticals4,5, biology6, environment7 and food8. Recently, DOE has been profoundly employed to optimize the parameters of analytical techniques, such as chromatography. The fundamental objective of using DOE is to reduce time, energy, cost, and materials. Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Mansoura University, P.O. Box 35516, Mansoura, Egypt. email: Scientific Reports | (2026) 16:16309 | https://doi.org/10.1038/s41598-026-51859-3 1 In our method, a full factorial design with two-levels (23 FFD) was applied as statistically significant substitute for univariate approach to ensure high-quality analyses. The most influential factors affecting chromatographic findings were accurately determined by concurrently investigating the impact of each factor on multiple levels of the other factors. Additionally, the acquired results represent the net consequence of the full experimental domain with regards to perspective interaction between the variables leading to a more precise and reliable draw up window opposing to a one-variable-at-time approach2. Cefixime (CFX) (Fig. 1a) is chemically named (6R,7R)−7-[[(Z)−2-(2-Aminothiazol-4-yl)−2[(carboxymethoxy)imino]acetyl]amino]−3-ethenyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid trihydrate9. CFX is an orally administered third generation cephalosporins. It is a bactericidal agent as it inhibits cell wall synthesis causing cell lysis10. It exhibited widespread spectrum activity towards gram positive and gram-negative bacteria11. It is administered to manage acute bronchitis, otitis media, uncomplicated urinary tract infections, pharyngitis/tonsillitis in addition to early and acute syphilis12,13. After oral intake, 40% to 50% of the drug is absorbed. About 50% is excreted unchanged in urine within 24 h14. Ornidazole (ORN) (Fig. 1b.) is chemically named 1-(3-chloro-2-hydroxypropyl)−2-methyl-5-nitroimidazole. It is a nitroimidazole derivative effective against protozoa and anaerobic bacteria given orally, vaginally or via intravenous route15. It acts by destructing DNA causes cell death16. It treats many vaginal, urinary and intestinal infections such as trichomoniasis, amebiasis, and giardiasis14,15. It is preferred over metronidazole due to its Fig. 1. The Chemical structure of a cefixime and b ornidazole. Scientific Reports | (2026) 16:16309 | https://doi.org/10.1038/s41598-026-51859-3 2 longer half-life (12–14 h vs. 6–8 h) permits fewer daily doses and a shorter overall treatment course, improving patient compliance and convenience15. Combination therapy of CFX and ORN treats bacterial and parasitic infections such as otitis media, pharyngitis, gonorrhea, urinary, gastrointestinal, and genital tract infections17. The literature reported various analytical methods for determining CFX and ORN either alone or combined with other drugs. The published methods for assaying CFX involving: HPLC18–24, spectrophotometry23,25–28, fluorimetry29–31, HPTLC32, voltammetry33,34 and capillary zone electrophoresis35. The published articles for assaying ORN include HPLC36–38, spectrophotometry39–41, HPTLC42–44, TLC45, voltammetry46–48 and capillary zone electrophoresis49. Besides, several methods are reported for the determination of the dual mixture including HPLC50–53, spectrophotometry53–55, HPTLC56. Our suggested method has an advantage over previous HPLC methods since it achieved separation of the studied drugs with higher sensitivity demonstrating low LOD and LOQ values in a faster analysis time. Moreover, our method did not rely on dangerous solvents50–53 or composite gradient elution53 or a long analysis time52,53. These published methods50–53 used acetonitrile which is less environmentally friendly and (...truncated)