LAUSR

Abstract Objective To develop and validate an UPLC–MS/MS assay for simultaneous determination of the total concentration of ceftazidime, ciprofloxacin, flucloxacillin, piperacillin, tazobactam, sulfamethoxazole, N-acetyl sulfamethoxazole and trimethoprim, and the protein-unbound concentration of flucloxacillin, in human plasma to be used for research and clinical practice. Methods Sample pretreatment included protein precipitation with methanol. For the measurement of protein-unbound flucloxacillin, ultrafiltration was performed at physiological temperature. For all compounds, a stable isotopically labelled internal standard was used. Reliability of the results was assessed by participation in an international quality control programme. Results The assay was successfully validated according to the EMA guidelines over a concentration range of 0.5–100 mg/L for ceftazidime, 0.05–10 mg/L for ciprofloxacin, 0.4–125 mg/L for flucloxacillin, 0.2–60 mg/L for piperacillin, 0.15–30 mg/L for tazobactam, 1–200 mg/L for sulfamethoxazole and N-acetyl sulfamethoxazole, 0.05–10 mg/L for trimethoprim and 0.10–50 mg/L for unbound flucloxacillin. For measurement of total concentrations, the within- and between-day accuracy ranged from 90.0% to 109%, and 93.4% to 108%, respectively. Within- and between-day precision (variation coefficients, CVs) ranged from 1.70% to 11.2%, and 0.290% to 5.30%, respectively. For unbound flucloxacillin, within-day accuracy ranged from 103% to 106% and between-day accuracy from 102% to 105%. The within- and between-day CVs ranged from 1.92% to 7.11%. Results of the international quality control programme showed that the assay is reliable. Conclusions The method provided reliable, precise and accurate measurement of seven commonly prescribed antibiotics, including the unbound concentration of flucloxacillin. This method is now routinely applied in research and clinical practice.