Determination of rifalazil in dog plasma by liquid-liquid extraction and LC-MS/MS: quality assessment by incurred sample analysis.

Incurred dog plasma samples were utilized for method quality assessment in this study, where a sensitive LC-MS/MS method was used for determination of the antibacterial agent rifalazil (KRM-1648 or ABI-1648). Reproducibility was estimated by repeated analysis of samples from a pharmacokinetic study, where 23 out of 864 study samples were reassayed during the course of the study. Precision for same-day duplicates was %R.S.D. 3.1 (concentration range 0.7-149 ng/ml), and over the whole study %R.S.D. 11.0 (concentration range 0.5-52 ng/ml). Moreover, standard addition experiments with incurred samples (concentration range 0.30-45 ng/ml) are described, where the recovery of spiked rifalazil amount was measured as a surrogate parameter for accuracy. The mean recovery of the added rifalazil amount was 103% (%R.S.D. 26.2). It was concluded that the described method is robust and reproducible for incurred samples. Liquid-liquid extraction was used for isolation of rifalazil and an isotope labeled internal standard from plasma. A manual procedure, based on an 8 x 12 array format, was used for sample extraction. Extracts were analyzed by reversed-phase liquid chromatography using octylsilica column with gradient elution (1mM ammonium acetate+0.01% (v/v) acetic aid in water and methanol). Mass spectrometric detection was made with positive ion electrospray ionization and LC-MS/MS analysis in a triple-quadrupole mass spectrometer. The lower limit of quantification was 50 pg/ml. MS characteristics of rifalazil are presented. In particular, two different sets of ionization and selected reaction monitoring (SRM) conditions, where in-source fragmentation was used for precursor ion formation in one of the sets, were compared. The good correlation found between the two sets of results for authentic sample extracts indicated that either condition could be used for quantification of rifalazil in dog plasma.