title: Sensitive quantification of roflumilast and roflumilast N-oxide in human plasma by LC-MS/MS employing parallel chromatography and electrospray ionisation.
abstract: A high throughput bioanalytical method based on semi-automated liquid extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the sensitive quantification of roflumilast and its metabolite roflumilast N-oxide, a phosphodiesterase (PDE) inhibitor in human plasma and serum. The sample work-up procedure comprised liquid extraction using penta-deuterated analogues of both analytes as internal standards. Chromatography was performed on C18 revered phase analytical columns at a flow rate of 0.5 mL/min in the dual column mode employing a column switching technique and a linear gradient from 18% to 54% acetonitrile in 0.005 M aqueous ammonium acetate containing 0.006% formic acid. Mass spectrometry was performed on an API 4000 instrument in the positive ion SRM-mode (selected reaction monitoring) with the Turbo-V ionspray interface. The method showed linear detector responses over the entire calibration range between 0.1 ng/mL (lower limit of quantification (LLOQ)) and 50 ng/mL (upper limit of quantification (ULOQ)) for both analytes. Linear regression analysis with concentration-squared weighting (1/x(2) for roflumilast and 1/x for roflumilast N-oxide) yielded inaccuracy and precision values <15% and coefficients of correlation (r) for the calibration curves >0.99 for both analytes.
authors.affiliation: Department of Bioanalytics, Nycomed GmbH, Byk-Gulden Strasse 2, 78467 Konstanz, Germany. norbert.knebel@nycomed.com
authors.forename: Norbert G
authors.initials: NG
authors.lastname: Knebel
authors.forename: Rolf
authors.initials: R
authors.lastname: Herzog
authors.forename: Felix
authors.initials: F
authors.lastname: Reutter
authors.forename: Karl
authors.initials: K
authors.lastname: Zech
mesh_terms: D000631:Aminopyridines / Q000097:blood* / Q000493:pharmacokinetics; D001549:Benzamides / Q000097:blood* / Q000493:pharmacokinetics; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003521:Cyclopropanes / Q000097:blood / Q000493:pharmacokinetics; D006801:Humans; D016014:Linear Models; D059625:Liquid-Liquid Extraction; D008297:Male; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000631:Aminopyridines; D001549:Benzamides; D003521:Cyclopropanes; C424423:Roflumilast; C517734:roflumilast N-oxide