Validation of a HPLC-MS/MS assay for the determination of total and unbound concentration of temocillin in human serum

OBJECTIVES: The aim of this study was to develop and validate a HPLC-MS/MS assay to determine total and unbound concentrations of temocillin in serum samples. DESIGN AND METHODS: Methanolic protein precipitation and ultrafiltration were used for total and unbound concentration extraction, respectively. Extract was injected into a LC-MS/MS system. Reversed phase chromatography was performed on a phenyl grafted column in gradient mode. Temocillin and internal standard (ticarcillin) were identified in positive electrospray ionization mode using ion transitions of m/z 415.34>339.1 and 385.31>160.3, respectively. RESULTS: Temocillin total and unbound concentration quantification assays were linear over concentrations ranging from 1 to 500mg/L and from 0.5 to 300mg/L, respectively. Both assays presented acceptable intra and inter-assay precision and accuracy <13.9%. Limits of quantification and detection were of 1 and 0.10mg/L, and 0.5 and 0.05mg/L for total and unbound concentration respectively. Total temocillin concentration recovery ranged from 85.80 to 99.40%. Temocillin ion suppression effect was <36.2 % in both assays. CONCLUSION: The method described is fast, sensitive and selective, with no interferences. This method may be used for both pharmacokinetic studies and therapeutic drug monitoring purposes.