Highly specific and sensitive liquid chromatography–tandem mass spectrometry method for the determination of 3-desmethylthiocolchicine in human plasma as analyte for the assessment of bioequivalence after oral administration of thiocolchicoside

A sensitive method for the determination of 3-desmethylthiocolchicine in plasma was developed, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. The plasma samples were extracted with ethyl acetate and separated on a Phenomenex Luna C18(2) 5 μm, 150×2 mm column with a mobile phase consisting of acetonitrile–0.005% formic acid (350:650, v/v) at a flow rate of 0.35 ml/min. Detection was achieved by an Applied Biosystems API 2000 mass spectrometer (LC–MS–MS) set at unit resolution in the multiple reaction monitoring mode. TurboIonSpray ionisation was used for ion production. The mean recovery for 3-desmethylthiocolchicine was 70%, with a lower limit of quantification set at 0.39 ng/ml. The increased selectivity of mass spectrometric (MS–MS) detection allowed us to distinguish between thiocolchicoside and its primary metabolite 3-desmethylthiocolchicine in human plasma, thereby giving more insight about the pharmacokinetics of the drug in humans.