title: A validated HPLC-FLD method for analysis of intestinal absorption and metabolism of capsaicin and dihydrocapsaicin in the rat.
abstract: A sensitive and selective reverse-phase high performance liquid chromatographic method with fluorescence detection has been developed for determination of capsaicin (8-methyl-N-vanillyl-(trans)-6-nonenamid) and dihydrocapsaicin (8-methyl-N-vanillylnonanamide) in samples generated in rat small intestine luminal perfusion experiments. The experiments were designed to study the biotransformation of capsaicinoids in the small intestine in the rat. The chromatographic separation was performed at room temperature on a ZORBAX Eclipse(®) XDB-C8 column using isocratic elution with a mobile phase consisting 0.05M orthophosphoric acid solution and acetonitrile (60:40, v/v; pH 3.0) with a flow rate of 1.5mL/min. Fluorescence detection was performed at excitation and emission wavelengths of 230 and 323nm, respectively. The method was evaluated for a number of validation characteristics (accuracy, repeatability and intermediate precision, limit of detection, limit of quantification and calibration range). The limit of detection (LOD) was 50ng/mL and the limit of quantification (LOQ) was 100ng/mL for both capsaicin and dihydrocapsaicin reference standards dissolved in blank perfusate. The method was successfully applied for investigation of intestinal absorption of capsaicin and dihydrocapsaicin while 30μg/mL standardized Capsicum extract - containing capsaicin and dihydrocapsaicin - was luminally perfused for a 90min period. The structure of the glucuronide metabolites of capsaicin and dihydrocapsaicin appeared in the perfusate was identified by mass spectrometry.
authors.affiliation: Institute of Pharmaceutical Chemistry, University of Pécs, Rókus str. 2, H-7624 Pécs, Hungary.
authors.forename: Mónika
authors.initials: M
authors.lastname: Kuzma
authors.affiliation: Institute of Pharmaceutical Chemistry, University of Pécs, Rókus str. 2, H-7624 Pécs, Hungary.
authors.forename: Krisztina
authors.initials: K
authors.lastname: Fodor
authors.affiliation: Department of Biochemistry and Medical Chemistry, University of Pécs, Szigeti str. 12, H-7624 Pécs, Hungary.
authors.forename: Gábor
authors.initials: G
authors.lastname: Maász
authors.affiliation: Department of Biochemistry and Medical Chemistry, University of Pécs, Szigeti str. 12, H-7624 Pécs, Hungary.
authors.forename: Péter
authors.initials: P
authors.lastname: Avar
authors.affiliation: First Department of Medicine, Medical and Health Center, University of Pécs, Ifjúság str. 13, H-7624 Pécs, Hungary.
authors.forename: Gyula
authors.initials: G
authors.lastname: Mózsik
authors.affiliation: First Department of Medicine, Medical and Health Center, University of Pécs, Ifjúság str. 13, H-7624 Pécs, Hungary.
authors.forename: Tibor
authors.initials: T
authors.lastname: Past
authors.affiliation: Department of Pharmacology and Pharmacotherapy, University of Pécs, Szigeti str. 12, H-7624 Pécs, Hungary.
authors.forename: Emil
authors.initials: E
authors.lastname: Fischer
authors.affiliation: Institute of Pharmaceutical Chemistry, University of Pécs, Rókus str. 2, H-7624 Pécs, Hungary. Electronic address: pal.perjesi@aok.pte.hu.
authors.forename: Pál
authors.initials: P
authors.lastname: Perjési
mesh_terms: D000818:Animals; D019540:Area Under Curve; D002138:Calibration; D002211:Capsaicin / Q000031:analogs & derivatives* / Q000378:metabolism / Q000493:pharmacokinetics*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007408:Intestinal Absorption; D057230:Limit of Detection; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D012015:Reference Standards; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D002211:Capsaicin; C012906:dihydrocapsaicin
keywords: Capsaicin and dihydrocapsaicin; Fluorescent detection; Glucuronide metabolites; High performance liquid chromatography; Intestinal metabolism