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

Abstract Objectives Therapeutic drug monitoring (TDM) of gentamicin is critical to ensure efficacy and minimize toxicity. We report a candidate reference measurement procedure (RMP) to quantify gentamicin in biological matrices, establishing an unbroken traceability chain to the International System of Units (SI). Methods An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method was validated using quantitative nuclear magnetic resonance (qNMR) spectroscopy to replace historical biological activity-based standardization with gravimetric mass. Strict ID-MS principles were applied to the major congeners (C1, C1a, and C2). Isomers C2a and C2b, eluting at distinct retention times, were quantified using C2-D 3 as a structural analog internal standard. Pharmacokinetic-based analytical performance specifications set the maximum allowable standard measurement uncertainty (k=1) at ≤6.7 %. Results The RMP demonstrated high analytical control, with trueness (bias 1.0–2.8 %) and within-run precision (CV 0.2–0.9 %) fulfilling clinical requirements. Expanded measurement uncertainties (k=2) for target value assignment ranged from 2.0 to 2.4 %. Comparison with the current JCTLM-listed RMP revealed a systematic negative bias of −9.2 %. A three-way method correlation confirmed these differences as a direct result of the metrological transition from activity to SI-traceable mass units. Conclusions This candidate RMP provides a robust, highly precise SI-traceable foundation for gentamicin quantification. While the resulting downward shift in reported concentrations may necessitate re-evaluating established clinical decision thresholds and TDM protocols, the clinical requirement for such adjustments remains to be fully determined.