title: 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.
abstract: 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 25mM ammonium acetate buffer (pH 4)/acetonitrile (45%/55%, flow rate 200μl/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 25-250ng/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.
authors.affiliation: Department of Clinical Pharmacology, Ernst-Moritz-Arndt University Greifswald, Greifswald, Germany. stefan.oswald@uni-greifswald.de
authors.forename: Stefan
authors.initials: S
authors.lastname: Oswald
authors.forename: Jette
authors.initials: J
authors.lastname: Peters
authors.forename: Monica
authors.initials: M
authors.lastname: Venner
authors.forename: Werner
authors.initials: W
authors.lastname: Siegmund
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000097:blood / Q000378:metabolism / Q000493:pharmacokinetics*; D001711:Biotransformation; D055745:Bronchioles / Q000166:cytology / Q000378:metabolism; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry* / Q000166:cytology; D002851:Chromatography, High Pressure Liquid / Q000662:veterinary; D017291:Clarithromycin / Q000031:analogs & derivatives / Q000097:blood / Q000378:metabolism / Q000493:pharmacokinetics*; D004347:Drug Interactions; D004355:Drug Stability; D006734:Horse Diseases / Q000097:blood* / Q000188:drug therapy*; D006736:Horses; D057230:Limit of Detection; D011650:Pulmonary Alveoli / Q000166:cytology / Q000378:metabolism*; D015203:Reproducibility of Results; D012141:Respiratory Tract Infections / Q000097:blood / Q000188:drug therapy / Q000662:veterinary*; D012293:Rifampin / Q000031:analogs & derivatives / Q000097:blood / Q000378:metabolism / Q000493:pharmacokinetics*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000662:veterinary; D053719:Tandem Mass Spectrometry / Q000662:veterinary
chemical_list: D000900:Anti-Bacterial Agents; C056228:14-hydroxyclarithromycin; D017291:Clarithromycin; C032393:25-deacetylrifampicin; D012293:Rifampin