Pharmacokinetics of indirubin in rat plasma by liquid chromatography-tandem mass spectrometric method

AIM To develop a sensitive and rapid liquid chromatography-tandem mass spectrometric method(LC-MS/MS) for the determination of indirubin in rat plasma,and pharmacokinetic study of indirubin. METHODS After a simple protein precipitation by acetonitrile,internal standard chrysophanol and indirubin in plasma were separated on an Agilent C_(18) column.The mobile phase consisted of acetonitrile-water(70:30,V/V), at a flow-rate of 0.30 mL·min~(-1).An API5000 tandem mass spectrometer equipped with electrospray ionization source was used as detector and was operated in the negative ion mode.Multiple reaction monitoring(MRM) using the precursor to product ion combinations of m/z 260.9→156.9 and m/z 253.0→225.0 was performed to quantity indirubin and the internal standard chrysophanol,respectively.Six rats were administrated with indirubin 20 mg·kg~(-1) by gavage.Rats' blood samples were collected before and at 0.25,0.5,0.75,1,2,3, 4,6,8,10,12,24,and 32 h after administration.Plasma drug concentrations were determined by LC-MS/ MS and drug concentration-time curve was drawn.The main pharmacokinetic parameters were calculated by statistical moment method.RESULTS The linear calibration curves of indirubin were obtained in the range of 0.1 - 10μg·L~(-1) in rat plasma.The lower limit of quantification(LLOQ) was 0.1μg·L~(-1).The intra-and interbatch precision(RSD) was below 8%calculated from quality control(QC) samples.The extraction recovery of low,medium,and high concentration QC samples and the internal standard were between 93.6%and 96.3%. The room temperature,freeze-thaw,and 30-day stability test results meet the requirements.After oral administration of 20 mg·kg~(-1) dose of indirubin,the pharmacokinetic parameters of indirubin were(4.2±1.0) h for t_(max),(3.7±0.7)μg·L~(-1) forρ_(max),(4.7±2.5) h for t_(1/22),(22.8±8.7)μg·h·L~(-1) for AUC_(0-t).CONCLUSION The method is simple,sensitive,as well as rapid,and is suitable for application in pharmacokinetic study of indirubin.