A 2D HPLC-MS/MS method for several antibiotics in blood plasma, plasma water, and diverse tissue samples

An analytical method using 2D high-performance liquid chromatography followed by tandem mass spectrometry for the quantification of the beta-lactam antibiotics amoxicillin, flucloxacillin, piperacillin, benzylpenicillin, the beta-lactamase inhibitors clavulanic acid, and tazobactam, as well as the macrolide antibiotic clindamycin, is presented. All analytes were measured in human plasma, while amoxicillin, clavulanic acid, flucloxacillin, and clindamycin were also analyzed in human tissue samples. Because of its high-protein binding, additionally, the free fraction of flucloxacillin was measured after ultrafiltration. As internal standards, deuterated forms of the beta-lactams were used. Sample preparation for all matrices was protein precipitation followed by online extraction on a TurboFlow MAX column, while sample separation was performed on an Accucore XL C18 column. Calibration curves were linear over 0.2-25 mg/kg for the tissue samples and 0.05-20 mg/l for the free fraction of flucloxacillin. In plasma, the calibration curves for amoxicillin and piperacillin were linear over 3.125-125 mg/l, for clavulanic acid and tazobactam over 1-40 mg/l, for benzylpenicillin 0.25-40 mg/l, and for flucloxacillin and clindamycin over 1.5-60 mg/l and 0.05-8 mg/l respectively. In plasma and plasma ultrafiltrate, inaccuracy and imprecision for any analyte were always less than 15%. In tissue, the accuracy and precision varied up to 16%, respectively, 20%, when various tissues were analyzed using a calibration in water. Graphical abstract.