Determination of Total and Free Concentrations of Flucloxacillin and Cefazolin in Human Plasma by Liquid Chromatography/Tandem Mass Spectrometry

Abstract

A rapid, simple and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay was developed for the determination of total and free concentrations of flucloxacillin and cefazolin in human plasma. Free flucloxacillin and cefazolin were separated from bound drug by ultra filtration. Samples of plasma and plasma ultra filtrate were pre-treated with acetonitrile (sample: acetonitrile = 1:4). Flucloxacillin, cefazolin and butobarbitone, the internal standard, were then resolved on a C18 (2)column using gradient elution of 0.05% formic acid and methanol. The three compounds were detected using electrospray ionisation in the positive mode. Standard curves were linear over the concentration range 0.2 to 100 mg/L (r>0.99) in plasma and 0.005 to 10 mg/L (r>0.99) in plasma ultra filtrate for both flucloxacillin and cefazolin. For both total and free flucloxacillin and cefazolin, bias was <±10%, and intra- and inter-day coefficients of variation (imprecision) were <10%. The limit of quantification was 0.2 mg/L for both flucloxacillin and cefazolin in plasma. The limit of quantification was 0.005 mg/L for both flucloxacillin and cefazolin in plasma ultra filtrate. The assay has been used successfully in a clinical pharmacokinetic study of flucloxacillin, and in clinical practice to enhance the effective use of flucloxacillin and cefazolin.