Development and validation of an RP-HPLC method for cefpodoxime proxetil epimers in human plasma with metabolite characterization, hydrolysis kinetics, and sustainability assessment

To develop and validate a reverse-phase high-performance liquid chromatography (RP-HPLC) bioanalytical method for quantifying cefpodoxime proxetil (CFP) epimers in human plasma and to integrate metabolite synthesis, hydrolysis kinetics, antibacterial evaluation, in silico toxicity prediction, and analytical sustainability assessment for its active metabolite, cefpodoxime acid (CFA). CFP was extracted from spiked human plasma by liquid–liquid extraction and quantified using an RP-HPLC–UV method validated conferring to US FDA (2018) bioanalytical guidance. CFA was synthesised by alkaline hydrolysis, structurally characterised, and chromatographically separated from CFP, although wide-ranging bioanalytical validation of CFA was not performed because of limited metabolite availability. Hydrolysis kinetics, antibacterial activity against Escherichia coli ATCC 25,922 and Bacillus subtilis ATCC 6633, TOPKAT toxicity prediction, and AGREE and White Analytical Chemistry (WECA) sustainability assessments were also performed. The method reached baseline separation within 15 min and was linear over 200–6400 ng/mL using weighted (1/X²) regression (r² > 0.999). Validation demonstrated satisfactory selectivity, accuracy, precision, recovery, dilution integrity, and stability. CFP underwent rapid first-order alkaline hydrolysis to CFA (k = 0.049 min⁻¹; t½ = 14.0 min). CFA retained antibacterial activity comparable to CFP against the tested reference strains. The method achieved an AGREE score of 0.65 and a WECA score of 61. The validated RP-HPLC technique provides a simple, reliable, and cost-effective platform for CFP bioanalysis and chromatographic monitoring of CFA, supporting pharmacokinetic, bioavailability, and bioequivalence studies while incorporating complementary physicochemical, biological, and sustainability evaluations.