An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of piperacillin in human serum and plasma

Abstract

Abstract Objectives Piperacillin is a broad-spectrum antibiotic used to treat critically ill patients with severe infections, requiring therapeutic drug monitoring (TDM). We report the development of an isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure (RMP) to quantify piperacillin in human plasma and serum. Methods Primary reference material was characterized by quantitative nuclear magnetic resonance (qNMR) to ensure traceability to the International System of Units (SI). Piperacillin was analyzed using LC-MS/MS operating in positive electrospray ionization and multiple reaction monitoring mode. Method validation evaluated selectivity, matrix effects, precision, accuracy, and measurement uncertainty (MU) according to GUM guidelines. Results This RMP allowed quantification of piperacillin within the range of 0.773 µmol/L (0.400 µg/mL) to 464 µmol/L (240 µg/mL), with selectivity, sensitivity and matrix-independence. Intermediate precision was <2.3 % and <1.1 % for spiked analyte in free human serum and native patient pools, respectively. The repeatability CV ranged from 0.7 to 2.0 % and relative mean bias ranged from −1.5 to 2.9 % across matrices and concentrations. Single measurement expanded MU ranged from 2.8 % to 5.0 %. Expanded MU (k=2) for target value assignment ranged from 2.0 % to 3.4 % across the primary therapeutic range, reaching 7.1 % total error at the lower limit of the measuring interval (LLMI). Conclusions This candidate RMP enables accurate determination of piperacillin in human serum and plasma, providing a standardized platform to facilitate reliable clinical TDM and routine assay harmonization.