Development and full validation of an UPLC-MS/MS method for the quantification of the plant-derived alkaloid indirubin in rat plasma

An UPLC-MS/MS method for the quantification of indirubin in lithium heparinized rat plasma was developed and validated according to current international guidelines. Indirubin was extracted from rat plasma by using Waters Ostro (TM) pass-through sample preparation plates. The method was validated with a LLOQ of 5.00 ng/mL and an ULOQ of 500 ng/mL. The calibration curve was fitted by least-square quadratic regression, and a weighting factor of 1/X was applied. Recoveries of indirubin and I.S. were consistent and >= 75.5%. Stability studies demonstrated that indirubin was stable in lithium heparinized rat plasma for at least 3 freeze/thaw cycles, for 3 hat RT, for 96 h in the autosampler at 10 degrees C, and for 84 days when stored below -65 degrees C. Preliminary pharmacokinetic (PK) data were obtained from Sprague Dawley rats after intravenous administration of indirubin (2 mg/kg b.w.) and blood sampling up to 12 h after injection. PK parameters were determined by non-compartmental analysis. Indirubin had a half-life (t(1/2)) of 35 min, and a relatively high clearance (CL) of 2.71 L/h/kg. (C) 2016 Elsevier B.V. All rights reserved.