title: Development of an HPLC fluorescence method for determination of boldine in plasma, bile and urine of rats and identification of its major metabolites by LC-MS/MS.
abstract: Boldine belongs to the group of aporphine alkaloids isolated from Boldo tree. In contrast with numerous reports on the pharmacological effects of boldine, the data about its pharmacokinetics and biotransformation are scarce. No validated bioanalytical method of sufficient sensitivity has so far been described in the literature which could be used for quantification of boldine in various body fluids collected in pharmacokinetic studies. This work presents, for the first time, the assay for boldine in the plasma, bile and urine of rats. It includes liquid-liquid extraction/back-extraction of boldine, its chromatographic separation and sensitive fluorescence detection. Separation was carried out on a pentafluorophenyl core-shell column (Kinetex PFP, 150×3mm, 2.6μm) in gradient elution mode with solvent system consisting of an acetonitrile-ammonium formate buffer (5mM, pH=3.8). Fluorimetric detection (λEX=320nm, λEM=370nm) was used for quantitative work. Validation according to the EMEA guideline proved the assay LLOQ (0.1μmolL(-1)), linearity over a broad range of 0.1-50μmolL(-1), precision (intra- and inter-day CVs less than 4.5% and 6.1%, respectively) and accuracy (relative errors between -5.8% and 4.8%). In a pilot pharmacokinetic experiment, the concentration-time profiles were described for boldine (single i.v. bolus 50mgkg(-1)) in plasma and bile and cumulative excretion in urine was investigated. The major metabolites identified by means of LC-MS(n) were boldine-O-glucuronide, boldine-O-sulphate and disulphate, boldine-O-glucuronide-O-sulphate and N-demethyl-boldine-O-sulphate.
authors: Charles University in Prague, Department of Medical Biochemistry, Faculty of Medicine in Hradec Králové, Šimkova 870, 500 38 Hradec Králové, Czech Republic. Electronic address: hrochm@lfhk.cuni.cz.
authors: Miloš
authors: M
authors: Hroch
authors: Stanislav
authors: S
authors: Mičuda
authors: Jolana
authors: J
authors: Cermanová
authors: Jaroslav
authors: J
authors: Chládek
authors: Pavel
authors: P
authors: Tomšík
mesh_terms: D000818:Animals; D001060:Aporphines / Q000032:analysis* / Q000737:chemistry / Q000493:pharmacokinetics; D001646:Bile / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004355:Drug Stability; D016018:Least-Squares Analysis; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
chemical_list: D001060:Aporphines; C011686:boldine
keywords: 7-methoxytacrine; BOL; Boldine; CID; CV; Core–shell column; DCM; EMEA; ESI; European Medicines Agency; FLD; Fluorescence; IS; LC/MS/MS; LLOQ; MEOTA; MTBE; Metabolites; Pharmacokinetics; RE; RPM; boldine; coefficient of variation; collision induced dissociation; dichloromethane; electrospray ionization; fluorescence detection; internal standard; lower limit of quantification; methyl tert-butyl ether; relative error; revolutions per minute