title: Quantitative LC-MS/MS determination of flupirtine, its N-acetylated and two mercapturic acid derivatives in man.
abstract: The non-opiate analgesic drug flupirtine was shown in vitro to undergo hydrolysis followed by N-acetylation to form D13223, glucuronidation and conjugation with glutathione to form the stable mercapturic acid derivatives M-424 and M-466. To quantify flupirtine and its metabolites in samples obtained in a clinical study in healthy subjects selected on their genotype of NAT2, UGT1A1 and GSTP1, two LC-MS/MS methods were developed. The validation range for flupirtine and D-13223 in serum was 0.5-500 ng/ml. For urine and feces, the validation ranges for flupirtine and D-13223 were 20-5000 ng/ml and 5.0-5000 ng/ml, respectively. M-424 and M-466 could be quantified in urine between 5.0 and 5000 ng/ml. Free flupirtine and D-13223 were separated from serum, urine and feces with liquid-liquid extraction. For flupirtine and D-13223, the chromatography was performed on a XTerra C18 column isocratically with a mobile phase consisting of ammonium formate buffer (pH 3.5mM) and acetonitrile (50:50; v/v), for M-466 and M-424 a Synergi(®) Fusion-RP column was used and a linear gradient method with water/HCOOH (pH 3) and acetonitrile. The mass spectrometer operated both with electro spray ionization in positive multiple reaction monitoring mode. The developed methods fulfilled the current FDA criteria on bioanalytical method validation for accuracy (error: -16.9 to 11.2%), precision (1.2-13.4%), recovery, stability and matrix effects over the observed analytical range. Thus, the methods were suitable to quantify flupirtine absorption and metabolic disposition in man after single intravenous and oral dosing (100mg) and repeated oral administration (400mg once daily).
authors.affiliation: Department of Clinical Pharmacology (ES, WS) of the Center of Drug Absorption and Transport (C_DAT), University Medicine, Greifswald, Germany. Electronic address: scheuch@uni-greifswald.de.
authors.forename: Eberhard
authors.initials: E
authors.lastname: Scheuch
authors.affiliation: Department of Pharmaceutical Chemistry (KM, PJB), University of Greifswald, Germany.
authors.forename: Karen
authors.initials: K
authors.lastname: Methling
authors.affiliation: Department of Pharmaceutical Chemistry (KM, PJB), University of Greifswald, Germany.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Bednarski
authors.affiliation: Department of Clinical Pharmacology (ES, WS) of the Center of Drug Absorption and Transport (C_DAT), University Medicine, Greifswald, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Oswald
authors.affiliation: Department of Clinical Pharmacology (ES, WS) of the Center of Drug Absorption and Transport (C_DAT), University Medicine, Greifswald, Germany.
authors.forename: Werner
authors.initials: W
authors.lastname: Siegmund
mesh_terms: D000107:Acetylation; D000111:Acetylcysteine / Q000032:analysis* / Q000378:metabolism*; D000631:Aminopyridines / Q000032:analysis* / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000631:Aminopyridines; C034161:flupirtine; D000111:Acetylcysteine
keywords: D-13223; Flupirtine; LC–MS/MS; Pharmacokinetics; Quantification