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Simultaneous Determination of Cefalexin, Cefazolin, Flucloxacillin, and Probenecid by Liquid Chromatography–Tandem Mass Spectrometry for Total and Unbound Concentrations in Human Plasma

Zhang, Mei, PhD*,†; Moore, Grant A., BSc (Hons); Chin, Paul K. L., MBChB, FRACP, PhD*; Everts, Richard, FRACP, ABMM; Begg, Evan J., MD, FRACP*

doi: 10.1097/FTD.0000000000000555
Original Article

Background: Pharmacokinetic studies and therapeutic drug monitoring of antibiotics require a simple, rapid, and reliable analytical method for monitoring the concentrations in plasma, including unbound concentrations for highly protein-bound drugs. The aim of the current work was to develop and validate a liquid chromatography–tandem mass spectrometry method for the simultaneous determination of total and unbound concentrations of 3 widely used β-lactam antibiotics (cefalexin, cefazolin, and flucloxacillin) and the often coadministered drug probenecid in human plasma, suitable for pharmacokinetic studies and for routine use in ordinary, busy hospital laboratories.

Methods: Unbound drug was separated from bound drug by ultrafiltration. A simple 1-step protein precipitation was used for sample preparation. Cefalexin, cefazolin, flucloxacillin, probenecid, and their corresponding isotopically labeled internal standards were then resolved on a C18 (2) column. All the compounds were detected using electrospray ionization in the positive mode.

Results: Standard curves were linear for all compounds over the concentration range of 0.2–100 mg/L (r > 0.99) for total drug in plasma and 0.01–10 mg/L (r > 0.99) for unbound drug in plasma ultrafiltrate. For both total and unbound drugs, bias was <±10%, and intra- and interday coefficients of variation (imprecision) were <10%. The limit of quantification was 0.2 mg/L for total plasma concentrations and 0.01 mg/L for plasma ultrafiltrate concentrations of all drugs.

Conclusions: The method has proven to be simple, rapid, robust, and reliable and is currently being used in clinical pharmacokinetic studies and in the routine clinical service to enhance the effective use of the β-lactam antibiotics.

*Department of Medicine, University of Otago-Christchurch, Christchurch;

Toxicology, Department of Specialist Biochemistry, Canterbury Health Laboratories, Christchurch; and

Department of Medicine, Nelson Hospital, Nelson, New Zealand.

Correspondence: Mei Zhang, PhD, Department of Medicine, University of Otago-Christchurch, Christchurch 8140, New Zealand (e-mail:

The authors declare no conflict of interest.

Received March 05, 2018

Accepted June 20, 2018

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