Refinement and validation of a reverse phase-hplc methodology for piperacillin quantification in therapeutic drug monitoring

Piperacillin is a semisynthetic ureidopenicillin with enhanced activity against resistant Gram-negative bacteria and is commonly administered in combination with the β-lactamase inhibitor tazobactam. This combination exhibits broad-spectrum antibacterial activity and is widely used in the treatment of severe polymicrobial infections. Accurate quantification of piperacillin in biological matrices is essential for pharmacokinetic evaluation and therapeutic drug monitoring (TDM). Although previous studies have established optimized HPLC methods for piperacillin analysis, further refinement of chromatographic parameters is necessary to enhance method performance and suitability for application in complex biological matrices such as ultrafiltrate and plasma. This study aimed to refine and validate a reversed-phase high- performance liquid chromatography (RP-HPLC) method for the quantification of piperacillin in these matrices. Calibration curves were constructed over a concentration range of 5–100 µg/mL. Chromatographic separation was achieved using a C18 column with an isocratic phosphate buffer–acetonitrile mobile phase and diode array detection at 230 nm. The method demonstrated excellent linearity in standard solution and ultrafiltrate (R² = 0.995), while lower linearity was observed in plasma (R² = 0.893), likely due to matrix effects. Precision values (%RSD < 2%) confirmed acceptable repeatability and reproducibility. The refined method is suitable for quantifying piperacillin in ultrafiltrate and demonstrates acceptable performance in plasma, with further optimisation required to improve analytical sensitivity for tazobactam.