International Journal of Antimicrobial Agents 40 (2012) 416–422 Contents lists available at SciVerse ScienceDirect International Journal of Antimicrobial Agents j our na l ho me p age: http://www.elsevier.com/locate/ijantimicag Quantification of seven -lactam antibiotics and two -lactamase inhibitors in human plasma using a validated UPLC-MS/MS method Mieke Carlier a,∗ , Veronique Stove a , Jason A. Roberts b , Eric Van de Velde a , Jan J. De Waele c , Alain G. Verstraete a a Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, De Pintelaan 185, Building 2P8, 9000 Ghent, Belgium b University of Queensland, Burns, Trauma and Critical Care Research Centre, Brisbane, Australia c Department of Critical Care Medicine, Ghent University, De Pintelaan 185, Building 2K12-IC, 9000 Ghent, Belgium a r t i c l e i n f o Article history: Received 3 April 2012 Accepted 30 June 2012 Keywords: -Lactam antibiotics Therapeutic drug monitoring UPLC-MS/MS a b s t r a c t There is an increasing interest in monitoring plasma concentrations of -lactam antibiotics. The objec- tive of this work was to develop and validate a rapid ultra-performance liquid chromatographic method with tandem mass spectrometric detection (UPLC-MS/MS) for simultaneous quantification of amoxicillin, ampicillin, cefuroxime, cefazolin, ceftazidime, meropenem, piperacillin, clavulanic acid and tazobac- tam. Sample clean-up included protein precipitation with acetonitrile and back-extraction of acetonitrile with dichloromethane. Six deuterated -lactam antibiotics were used as internal standards. Chromato- graphic separation was performed on a Waters ACQUITY UPLC system using a BEH C 18 column (1.7 m, 100 mm × 2.1 mm) applying a binary gradient elution of water and acetonitrile both containing 0.1% formic acid. The total run time was 5.5 min. The developed method was validated in terms of precision, accuracy, linearity, matrix effect and recovery. The assay has now been successfully used to deter- mine concentrations of amoxicillin/clavulanic acid, cefuroxime and meropenem in plasma samples from intensive care patients. © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. 1. Introduction Infections are an extremely important problem in critical care medicine. Sepsis alone is the leading cause of mortality in non- cardiac Intensive Care Units (ICUs) [1]. Adequate antibiotic therapy is one of the mainstays in treatment, with the emphasis on timely administration and appropriateness of the spectrum [2]. The - lactam antibiotics are central to the treatment of sepsis and life-threatening infections in the ICU. They are generally consid- ered as time-dependent antibiotics, which means that they exert an optimum bactericidal effect when drug concentrations at the site of infection are maintained above the minimum inhibitory concentration (MIC) [3]. Although timely administration and appropriateness of spec- trum are important, antibiotic dosing is also highly likely to affect clinical efficacy [4]. Recent data demonstrate that the concentra- tion of antibiotics in plasma and at the site of infection is highly variable and that standard doses of antibiotics lead to underdosing in a considerable number of patients [5,6]. This is most commonly caused by pharmacokinetic changes in the volume of distribution ∗ Corresponding author. Tel.: +32 9 3326 733; fax: +32 9 3324 985. E-mail address: Mieke.Carlier@ugent.be (M. Carlier). and increased clearance compared with non-critically ill patients and may result in decreased efficacy. On the other hand, overdos- ing is also possible, leading to toxicity without increased efficacy [4]. This wide spectrum of pharmacokinetics makes empirical dos- ing choices highly challenging and likely to result in suboptimal antibiotic exposures or toxicity. Based on these considerations, monitoring antibiotic concen- trations and subsequent dose adaptation might offer a solution to maximise efficacy and minimise toxicity, especially in patients with considerable pharmacokinetic variability such as intensive care patients [7,8]. As current research is primarily focused on the study of broad-spectrum antibiotics, pharmacokinetic study of narrower- spectrum -lactams has received little attention. Patient-tailored antibiotic therapy may allow reliable use of the full spectrum of antibiotics available, including narrow-spectrum antibiotics that are only rarely used in most ICUs. If these antibiotics can be adequately monitored, physicians may be more confident to pre- scribe them in severe infections. With this in mind, we believe a method capable of simultaneous quantification both of narrow- and broad-spectrum antibiotics is required. At this time, the concentration–effect relationship for the -lactamase inhibitors is poorly described. We therefore decided to incorporate these compounds in this method because, if this assay was to be used 0924-8579/$ – see front matter © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. http://dx.doi.org/10.1016/j.ijantimicag.2012.06.022