Un procedimiento de medición de referencia candidato basado en ID-HPLC-MS/MS para la cuantificación de imipenem,...

Carbapenems require precise plasma levels for optimal therapy, yet no LC-MS/MS method offered SI-traceability or reference-grade performance. In this study, a candidate reference measurement procedure (RMP) using isotope dilution high-performance liquid chromatography-tandem mass spectrometry (ID-HPLC-MS/MS) was developed and validated for accurate quantification of imipenem (IPM), meropenem (MEM) and ertapenem (ETP) in human plasma.

To ensure traceability to SI units, the absolute content of the IPM, MEM and ETP primary reference materials (PRMs) were determined by quantitative nuclear magnetic resonance (qNMR) spectroscopy. Samples were prepared by protein precipitation followed by dilution. Method validation was performed according to guidances from the Clinical and Laboratory Standards Institute (CLSI), including selectivity/specificity, matrix effect, carryover, linearity, limit of quantification (LOQ) and limit of detection (LOD), trueness and precision, and stability. Measurement uncertainty was evaluated according to the Guide to the Expression of Uncertainty in Measurement (GUM). Moreover, the candidate RMP was compared between two independent laboratories.

The method showed high selectivity and stability. Linear ranges were 0.208 to 52.087 μg/mL (IPM, r = 0.9997), 0.412 to 103.427 μg/mL (MEM, r = 0.9999) and 0.083 to 20.791 μg/mL (ETP, r = 0.9999). Mean recoveries were 100.05 to 104.80 %, 92.34 to 94.77 % and 95.55 to 100.04 %, respectively. The method demonstrated high sensitivity, with limits of LOQ of 0.103, 0.103 and 0.021 μg/mL and corresponding LOD of 0.0077, 0.0018 and 0.0006 μg/mL for IPM, MEM and ETP, respectively. No obvious matrix effect and carryover were observed. The expanded uncertainties (k = 2) were ≤ 6.12 %.

The qNMR-based candidate RMP provides SI-traceable, accurate simultaneous quantification of IPM, MEM and ETP, enabling standardization of clinical assays and reliable patient-sample measurement.