Development of a liquid chromatographic–tandem mass spectrometric method with precolumn derivatization for the determination of dencichine in rat plasma

Abstract Dencichine is a nonprotein amino acid used for hemostasis. A very sensitive method was developed for the determination of dencichine concentration in rat plasma using a derivatization step to enhance signal intensity. The method consisted of a protein precipitation extraction followed by derivatization with 10 M hydrochloric acid–methanol (10:90, v/v) and analysis by liquid chromatography coupled with tandem mass spectrometry (LC–MS–MS) via atmospheric pressure chemical ionization (APCI) source. The separation was achieved using a XDB C 8 analytical column with an isocratic mobile phase composed of methanol–water–formic acid in the ratio of 35:65:0.5 (v/v/v). Signal intensity of the dencichine derivative was increased up to 50-fold as compared to the underivatized dencichine in positive APCI mode. The matrix effect was also investigated. The method has a lower limit of quantification of 20 ng/ml for a 100 μl plasma aliquot. The intra- and inter-day precision (R.S.D.), calculated from quality control (QC) samples, was less than 6.3%. The accuracy (R.E.), determined using QC samples, ranged from −0.2 to 3.9%. The method offered increased sensitivity, selectivity and speed of analysis over existing method. The method was utilized to support clinical pharmacokinetic studies of dencichine in rat following intravenous administration. It indicated that this LC–MS–MS method is useful for pharmacokinetic studies of dencichine in animals, because it enabled the serial determination of plasma concentration of dencichine in rats.