A rapid, LC-MS/MS assay for quantification of piperacillin and tazobactam in human plasma and pleural fluid; application to a clinical pharmacokinetic study

摘要

Piperacillin, in combination with tazobactam is a common first-line antibiotic used for the treatment of pleural infection, however its pleural pharmacokinetics and penetration has not previously been reported. The objective of this work was to develop and validate a rapid and sensitive liquid chromatography with tandem mass spectrometry (LC-MS/MS) assay for quantification of piperacillin (PIP) and tazobactam (TAZ). PIP and TAZ were extracted from both human plasma and pleural fluid samples by protein precipitation in methanol containing the internal standards (IS) piperacillin-d5(PIP-d5) and sulbactam (SUL). Briefly, 5?μL of sample was mixed with 125?μL of methanol containing IS, vortexed and centrifuged. Supernatant (50?μL) was diluted into 500?μL of mobile phase containing 10?mM of ammonium bicarbonate in LCMS grade water and transferred to the autosampler tray. Electrospray ionization in positive mode and multiple reaction monitoring (MRM) were used for PIP and PIP-d5at the transitionsm/z518.2?→?143.2 andm/z523.2?→?148.2 respectively, and electrospray ionization in negative mode and MRM were used for TAZ and SUL at the transitionsm/z299.1?→?138.1 andm/z232.4?→?140.1. The chromatographic separation was achieved using an Acquity BEH C-18 column with gradient elution of mobile phase containing 10?mmol/L ammonium bicarbonate in water and methanol. A linear range was observed over the concentration range of 0.25–352?mg/L and 0.25–50.5?mg/L for PIP and TAZ respectively. Complete method validation was performed according to US FDA guidelines for selectivity, specificity, precision and accuracy, LLOQ, matrix effects, recovery and stability, with all results within acceptable limits. This method was successfully applied to two patients with pleural infection and is suitable for further pharmacokinetic studies and therapeutic drug monitoring.