Details

Autor(en) / Beteiligte

• Hroch, Miloš
• Mičuda, Stanislav
• Cermanová, Jolana
• Chládek, Jaroslav
• Tomšík, Pavel

Titel

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

Ist Teil von

• Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, 2013-10, Vol.936, p.48-56

Ort / Verlag

Netherlands: Elsevier B.V

Erscheinungsjahr

2013

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• •Major boldine metabolites were identified in rats for the first time.•A universal method for assay in plasma, bile and urine was developed.•Fluorescence and mass spectrometric detection were used.•Validation demonstrated the high precision and accuracy of the method.•Extended pharmacokinetic data for boldine were obtained. 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–MSn were boldine-O-glucuronide, boldine-O-sulphate and disulphate, boldine-O-glucuronide-O-sulphate and N-demethyl-boldine-O-sulphate.