An UPLC-PDA assay for simultaneous determination of seven antibiotics in human plasma

Abstract

Appropriate antibiotic dosing in critically ill patients requires concentration monitoring due to the occurrence of pathophysiological changes and frequent extracorporeal therapy that could significantly alter the normal pharmacokinetics of drugs. Herein, we describe an ultra-performance liquid chromatography with photodiode array (UPLC-PDA) for the simultaneous concentration determination of seven frequently used antibiotics (meropenem, cefotaxime, cefoperazone, piperacillin, linezolid, moxifloxacin, and tigecycline) in plasma from critically ill patients. The analytes were extracted from 200 μL human plasma by the addition of methanol for protein precipitation. The chromatographic separation was achieved using an ACQUITY UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm) with a water (containing 0.1% trifluoroacetic acid)/acetonitrile linear gradient at a flow rate of 0.5 mL/min in a 4.5 min turn-around time. PDA detection wavelength was set individually for the analytes. The method was fully validated according to the European Medicines Agency (EMA) guideline. The lower limits of quantification for the analytes were between 0.05 and 0.8 μg/mL. The method is accurate (intra/inter-assay bias -8.4 to +12.4%) and precise (intra/inter-assay coefficient of variations 0.9-10.1%) over the clinically relevant plasma concentration ranges (upper limits of quantification 5-400 µg/mL). The applicability of the method has been successfully demonstrated by analyzing plasma samples collected from critically ill patients undergoing continuous renal replacement therapy.

Keywords: Antibiotics; Human plasma; Method validation; UPLC.