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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
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.