A rapid, automatic and accurate assay for quantifying temocillin in human serum and CSF using turbulent flow liquid chromatography coupled to high-resolution mass spectrometry. Clinical application.

Temocillin is a ß-lactamase-resistant penicillin used for the treatment of multiple drug-resistant Gram-negative bacteria. To maximize efficacy and avoid adverse effects, the dose regimen has to be quickly adjusted to the clinical situations. This necessitates the development of a rapid, reliable and accurate analytical method. Temocillin and the stable isotopically labeled internal standard ([13 C6 ]-amoxicillin) were extracted from either serum or cerebrospinal fluid by a turbulent flow liquid chromatographic method and eluted onto an octadecyl-silica phase with polar endcapping. Mass spectrometry was conducted using an exact mass determination method by electrospray positive ionization high-resolution mass spectrometry. The LLOQ and ULOQ of the present method were determined to be 0.4 and 200 µg/ml for serum and cerebrospinal fluid samples, respectively. The total analysis time was <7 min. The recovery ranged from 87.7 to 120.8%. Intra- and inter-day precision and trueness were tested at four concentration levels: 0.4, 8, 40 and 160 µg/ml. Values were 6.33 ± 1.53, 8.8 ± 1.3, 8.8 ± 0.36 and 2.1 ± 0.76%, and 5.0 ± 0.54, 9.9 ± 1.0, 5.8 ± 1.6 and 0.1 ± 1.1%, for inter- and intra-day analysis, respectively. Temocillin was found to be stable under all relevant laboratory conditions. The method was cross-validated with a microbiological assay. This method is suitable for accurate measurement of temocillin concentration in small volumes of serum or cerebrospinal fluid. Thanks to the online extraction procedure, the overall analytical time is compatible with high-throughput analysis for clinical application.