Quantitative determination of acetylshikonin in macaque monkey blood by LC-ESI-MS/MS after precolumn derivatization with 2-mercaptoethanol and its application in pharmacokinetic study.
OBJECTIVE
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 x 50 mm, 5 microm) 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 (1)H-NMR and (13)C-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 halflife, and the mean residual time were 615.4+/-206.5 ng x dh/mL,12.3+/-1.6 h, and 10.2+/-0.7 h, respectively.


CONCLUSIONS
The method was validated and applied to the quantitative determination and pharmacokinetic study of acetylshikonin in the blood samples of macaque monkeys.
Sun
Dong-xiao
DX
Laboratory of Drug Metabolism and Pharmacokinetics, Beijing Institute of Transfusion Medicine, Beijing 100850, China.
Tian
Hui-fang
HF
Meng
Zhi-yun
ZY
Du
Alicia
A
Yuan
Dan
D
Gu
Ruo-lan
RL
Wu
Zhuo-na
ZN
Dou
Gui-fang
GF
D000818:Animals; D000880:Anthraquinones / Q000097:blood / Q000737:chemistry / Q000493:pharmacokinetics; D002227:Carbazoles / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D004365:Drugs, Chinese Herbal / Q000737:chemistry / Q000378:metabolism; D008251:Macaca; D008623:Mercaptoethanol / Q000737:chemistry*; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular; D011422:Propionates / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D000880:Anthraquinones; D002227:Carbazoles; D004365:Drugs, Chinese Herbal; D011422:Propionates; C515629:chiglitazar; D008623:Mercaptoethanol; C073944:acetylshikonin