LC–MS/MS method for the simultaneous determination of clarithromycin, rifampicin and their main metabolites in horse plasma, epithelial lining fluid and broncho-alveolar cells

Clarithromycin (CLA) is a well established macrolide antibiotic which is frequently used in therapy of airway diseases in foals. It is extensively metabolized by CYP3A4 resulting in the antimicrobial active metabolite 14-hydroxyclarithromycin (OH-CLA). Rifampicin (RIF) is often comedicated to prevent resistance and augment therapy. RIF is a known inducer for metabolizing enzymes and transporter proteins. Therefore, comedication might bare the risks of pharmacokinetic drug interactions which were investigated in a clinical trial. As no adequate method to determine CLA, RIF and their main metabolites OH-CLA and 25-O-desacetylrifampicin (DAc-RIF) were described so far, we developed a selective and sensitive assay to measure concentrations of all four substances simultaneously in plasma, epithelial lining fluid (ELF) and broncho-alveolar cells (BAC) of foals. Drugs were measured after extraction with methyl tert-butyl ether using roxithromycin as internal standard and liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) for detection. The chromatography was done isocratically using 25 mM ammonium acetate buffer (pH 4)/acetonitrile (45%/55%, flow rate 200 μl/min). The MS/MS analysis was performed in the positive ion mode (m/z transitions: CLA, 748.5–590.1; OH-CLA, 764.1–606.1; RIF, 823.1–791.2; DAc-RIF, 781.1–749.1 and 837.3–679.2 for the internal standard). The method was validated according to selectivity, linearity, accuracy, precision, recovery, matrix effects and stability. The validation ranges for all substances were 2.5–25 for the low and 25–250 ng/ml for the high validation range. The described assay was shown to be valid and successfully applied to measure disposition of CLA, OH-CLA, RIF and DAc-RIF in plasma, ELF and BAC of foals in a clinical trial.