Simultaneous quantification of cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid and piperacillin in human serum using an isotope-dilution HPLC–MS/MS method

• Published in: Journal of pharmaceutical and biomedical analysis 2018-04, Vol.152, p.102-110
• Main Authors: Paal, M., Zoller, M., Schuster, C., Vogeser, M., Schütze, G.
• Format: Article
• Language: English
• Subjects: Anti-Bacterial Agents - blood; Antibiotics; Cephalosporins - blood; Chromatography, High Pressure Liquid - methods; Ciprofloxacin - blood; Drug Monitoring - methods; Fluoroquinolones - blood; High performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS); Humans; Isotopes - chemistry; Limit of Detection; Linezolid - blood; Pharmacokinetic/pharmacodynamics (PK/PD); Piperacillin - blood; Reproducibility of Results; Serum - chemistry; Tandem Mass Spectrometry - methods; Therapeutic drug monitoring (TDM); Thienamycins - blood
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2018.01.031

•A multi-analyte HPLC–MS/MS method for antibiotic TDM in human serum is described.•Cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid and piperacillin are quantified.•The method was validated according to the EMA bioanalytical method validation protocol.•Sample cleanup and analysis is done within a total process time of 30 min. The aim of the current study was to develop and validate a robust multi-analyte high performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) method for simultaneous quantification of cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid and piperacillin, which are the most commonly used antibiotics in intensive care units. Sample clean-up included a protein precipitation protocol, followed by chromatographic separation on a C8 reverse phase HPLC column within 4 min, using a formic acid-ammonium formiate methanol step-elution gradient. All compounds were detected with electrospray ionization (ESI+) mass spectrometry in multiple reaction time monitoring. The method was validated according to the protocol from the European Medicines Agency and was thoroughly evaluated for interferences and quantification linearity. Linear relationships between peak area responses and drug concentrations were obtained in the range of 0.25–200 mg/l for cefepime, 0.25–120 mg/l for meropenem, 0.05–10 mg/l for ciprofloxacin, 0.125–10 mg/l for moxifloxacin, 0.125–50 mg/l for linezolid and 0.5–400 mg/l for piperacillin with an R2 > 0.997. Imprecision and inaccuracy values (both intra- and inter-assay) were ≤ 6.8% and ≤10.9% for all analytes in quality control samples, respectively. The assay proved to be selective for the study antibiotics, and the internal standards consistently compensated for matrix effects. The described simple and reliable HPLC–MS/MS assay is a powerful tool for routine TDM of cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid and piperacillin in human serum in clinical laboratories. With a total process time of approximately 30 min, it allows for accurate and selective quantification up to the expected pharmacokinetic peak concentrations