title: Liquid chromatography-tandem mass spectrometry method for simultaneous quantification of bisoprolol, ramiprilat, propranolol and midazolam in rat dried blood spots.
abstract: Dried blood spot (DBS) sampling represents a suitable method for pharmacokinetic studies in rats, particularly if serial sampling is needed. To study the pharmacokinetics of drugs in a rat heart failure (HF) model, we developed and validated a method for the simultaneous determination of bisoprolol, ramiprilat, propranolol and midazolam in DBS samples. Bisoprolol and ramipril are widely used in the treatment of HF, and midazolam and propranolol are markers of hepatic metabolism, which can be altered in HF. A 20μL sample of rat blood was pipetted onto Whatman 903 Protein Saver Card and allowed to dry. The whole spot was excised and 300μL of solvent (methanol with 10% ultrapure water and 0.1% formic acid) was added. After mixing and incubating the sample in an ultrasonic bath, a mixture of isotopically labeled internal standards was added. After centrifugation, the extracts were cleaned on an Ostro™ plate and analyzed using liquid chromatography-tandem mass spectroscopy. The method was successfully validated. No significant interference was observed in the retention times of analytes or internal standards. The intraday and interday accuracy and precision were within a ±15% interval. The method was linear in the range 5-250μg/L and the lower limit of quantification was 5μg/L for all four analytes. The absolute matrix effect ranged from 98.7% for midazolam to 121% for ramiprilat. The recovery was lowest for ramiprilat and highest for propranolol. Samples were stable at all tested temperatures. The method has been used successfully in a real-time pharmacokinetic study in rats.
authors.affiliation: University Clinic of Respiratory and Allergic Diseases Golnk, Pharmacy Department, Golnik 36, 4204 Golnik, Slovenia. Electronic address: katja.trobec@klinika-golnik.si.
authors.forename: Katja
authors.initials: K
authors.lastname: Cvan Trobec
authors.affiliation: University of Ljubljana, Faculty of Pharmacy, Askerceva 7, 1000 Ljubljana, Slovenia. Electronic address: jurij.trontelj@ffa.uni-lj.si.
authors.forename: Jurij
authors.initials: J
authors.lastname: Trontelj
authors.affiliation: Applied Cachexia Research, Department of Cardiology, Charité Medical School, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany; Center for Cardiovascular Research, Charité Medical School, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany; Norwich Medical School, University of East Anglia, Norwich NR4 7TJ, UK. Electronic address: jochen.springer@charite.de.
authors.forename: Jochen
authors.initials: J
authors.lastname: Springer
authors.affiliation: Applied Cachexia Research, Department of Cardiology, Charité Medical School, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany; University Clinic of Respiratory and Allergic Diseases Golnik, Division of Cardiology, Golnik 36, 4204 Golnik, Slovenia. Electronic address: mitja.lainscak@guest.arnes.si.
authors.forename: Mitja
authors.initials: M
authors.lastname: Lainscak
authors.affiliation: University of Ljubljana, Faculty of Pharmacy, Askerceva 7, 1000 Ljubljana, Slovenia. Electronic address: mojca.kerec-kos@ffa.uni-lj.si.
authors.forename: Mojca
authors.initials: M
authors.lastname: Kerec Kos
mesh_terms: D000818:Animals; D000959:Antihypertensive Agents / Q000097:blood*; D017298:Bisoprolol / Q000097:blood*; D002853:Chromatography, Liquid / Q000379:methods; D059788:Dried Blood Spot Testing / Q000379:methods*; D006993:Hypnotics and Sedatives / Q000097:blood*; D057230:Limit of Detection; D008297:Male; D008874:Midazolam / Q000097:blood*; D011433:Propranolol / Q000097:blood*; D017257:Ramipril / Q000031:analogs & derivatives* / Q000097:blood; D051381:Rats; D017208:Rats, Wistar; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D000959:Antihypertensive Agents; D006993:Hypnotics and Sedatives; C052549:ramiprilat; D011433:Propranolol; D017257:Ramipril; D008874:Midazolam; D017298:Bisoprolol
keywords: Bisoprolol; Dried blood sample; Midazolam; Propranolol; Ramiprilat