A rapid UPLC-MS/MS method for therapeutic drug monitoring of bupivacaine: Validation and clinical application in perioperative analgesia

A rapid UPLC-MS/MS method for bupivacaine TDM was developed and validated (run time 2.5 min). • The assay meets FDA/Chinese Pharmacopoeia criteria with LLOQ of 1.0 μg/L using only 50 μL plasma. • Simple protein precipitation ensures high recovery (> 90%) and negligible matrix effects. • Clozapine is introduced as a cost-effective internal standard with excellent reproducibility. • Clinical application in 40 patients revealed distinct PK profiles of conventional vs. liposomal bupivacaine. Background Bupivacaine (BPC) is a cornerstone of perioperative analgesia, yet its narrow therapeutic index and high interindividual pharmacokinetic variability complicate dosing and increase toxicity risks. A robust, high-throughput analytical platform is needed to support therapeutic drug monitoring (TDM) and individualized therapy. Methods A rapid ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantifying BPC in human plasma was developed and fully validated according to FDA and Chinese Pharmacopoeia guidelines. Sample preparation consisted of a simple protein precipitation step using acetonitrile containing clozapine as the internal standard. Chromatographic separation was achieved within 2.5 min on a C18 column using gradient elution, enabling high-throughput analysis suitable for routine TDM. Results The method demonstrated excellent linearity from 1.0 to 500.0 μg/L ( R 2  > 0.995) and a short run time of 2.5 min, allowing batch analysis of up to 50 samples within 3 h. Intra- and inter-day precision (coefficient of variation) were < 15%, and accuracy ranged between 85% and 115% across quality control levels. The lower limit of quantification was 1.0 μg/L. Extraction recovery was consistent (90.9%–94.5%) and matrix effects were negligible. In a pilot clinical study involving 40 patients, the method successfully differentiated the pharmacokinetic profiles of two formulations: conventional BPC (75 mg) showed rapid distribution and elimination, while liposomal BPC (133 mg) maintained sustained plasma concentrations over 24 h. Conclusion This validated UPLC-MS/MS method is a rapid, sensitive, and high-throughput tool for BPC TDM that represents a promising candidate for implementation in clinical laboratories following appropriate local validation. Its successful clinical application highlights its utility in characterizing formulation-dependent pharmacokinetics and could assist in the development of precision dosing in perioperative analgesia to optimize efficacy and safety.