Critically ill patients are often affected by several pathophysiological conditions requiring
antibiotic administration and, frequently, extracorporeal
therapy that significantly alter the normal pharmacokinetics of drugs. Therapeutic drug monitoring (TDM) may assist to establish the correct
antibiotic dosage, but a TDM service is usually available only for some
aminoglycosides and
glycopeptides. The aim of this study is the validation of an HPLC-UV method for the simultaneous quantification of
meropenem,
vancomycin,
piperacillin and
tazobactam in human plasma samples. The analytes were extracted from 250 μL of human plasma by the addition of
acetonitrile for
protein precipitation. After evaporation to dryness of the
solvent, samples were reconstituted with 250 μL of mobile phase, and 100 μL were injected in HPLC. Chromatographic analysis was performed using a Kinetex C18 column and an UV/Vis detector set at 220 and 298 nm. The mobile phase was a mixture of
phosphate buffer 0.1 M pH 3.15 and
methanol in gradient, delivered at 1 mL/min. The method was validated over clinical concentration ranges. For all the analytes, the lower limit of quantification was 1 μg/mL, and the calibration curves were linear between 1 and 100 μg/mL, with coefficients of determination ≥ 0.999. Intra-day precision was < 4%, while inter-day precision was < 7% for each analyte. The applicability of the method has been evaluated by analysing plasma samples collected from 4
critically ill patients undergoing
continuous renal replacement therapy. Moreover, the analysis of
vancomycin with VANC Flex® confirmed a good correlation between the results of HPLC-UV and commercially available kits usually used by TDM service. The method we developed only requires a small volume of plasma and uses the same sample preparation protocol, stationary phase and elution conditions for all analytes. This method offers the additional advantages of simple and rather inexpensive sample preparation and instrumentation, features that make this method an easy implementation for a general TDM laboratory.