A Critical Comparative Review of Analytical Techniques for Cloxacillin and Oxacillin Across Diverse Matrices

Cloxacillin (CLX) and oxacillin (OXA), two isoxazolyl β-lactam antibiotics widely used against penicillinase-producing Staphylococcus aureus, have attracted increasing attention due to their environmental persistence, residue occurrence, and analytical complexity across diverse matrices. This review provides a critical and integrated comparison of analytical techniques for the determination of CLX and OXA in pharmaceutical, biological, food, and environmental samples, addressing key challenges, including β-lactam instability, low intrinsic detectability, strong protein binding, and matrix interferences. Chromatographic methods, particularly HPLC and LC-MS/MS, remain the most reliable approaches, offering high selectivity and stability-indicating capability, with LC-MS/MS achieving superior sensitivity at ng/mL levels suitable for trace analysis, albeit at higher cost and with greater operational demands. Electrochemical techniques, especially nanomaterial- and molecularly imprinted-based sensors, demonstrate promising sensitivity and rapid response, though limitations in reproducibility and standardization persist. In contrast, spectroscopic methods are economical and simple but restricted by low sensitivity and poor selectivity in complex matrices. Overall, no single technique is universally optimal, and analytical performance is strongly matrix-dependent. Future perspectives should prioritize the development of hybrid, standardized, and green analytical platforms that balance sensitivity, robustness, and cost-effectiveness, alongside harmonized validation strategies to enable reliable and sustainable monitoring of CLX and OXA in real-world applications.