Quantitative determination of acetylshikonin in macaque monkey blood by LC-ESI-MS/MS after precolumn derivatization with 2-mercaptoethanol and its application in pharmacokinetic study1


  • 1

    This work was supported by Beijing Jin BenCao Chinese Herbal Medicine Technology Development.

Correspondence to Prof Gui-fang DOU. Phn/Fax 86-10-6693-2951. E-mail douguifang@vip.163.com


Aim: To develop and validate a novel precolumn derivatization method for the quantitative determination and pharmacokinetic application of acetylshikonin in macaque monkeys by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Methods: 2-Mercaptoethanol was added to the blood sample as the derivatization reagent. The derivatization reaction formed 1 major derivation product, which was well correlated with acetylshikonin. The acetylshikonin concentrations in the biological samples were calculated by quantitative determination of the major derivation product using LC-ESI-MS/MS. Separation was achieved using a C18 column (2 mm×50 mm, 5 μm) at room temperature and a linear gradient elution with a mobile phase containing methanol (1.96% acetic acid) and 10% methanol in water (1.96% acetic acid and 10 mmol/L ammonium acetate) at a flow rate of 0.2 mL/min. In addition, the major derivative, named derivative III, was identified by UV spectra, MS, and the 1H-NMR and 13C-NMR spectra. Results: Good linearity was obtained within the range of 5 and 2000 ng/mL (r>0.99 using a linear regression model with 1/x2 weighting) for acetylshikonin. The interday and intraday precisions were found to be less than 12.3%, with the exception of the lowest concentration, which was less than 17.2%. The interday and intraday accuracies, which were between –3% and 0.6%, were also observed. After the administration of acetylshikonin (80 mg/kg, po) in macaque monkeys, the pharmacokinetic parameters were obtained through the non-compartmental analysis, where the area under the concentration-time curve to the last measurable concentration, the terminal elimination half-life, and the mean residual time were 615.4±206.5 ng h/mL,12.3±1.6 h, and 10.2±0.7 h, respectively. Conclusion: The method was validated and applied to the quantitative determination and pharmacokinetic study of acetylshikonin in the blood samples of macaque monkeys.