Development and full validation of an UPLC-MS/MS method for the quantification of 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™ 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 h at RT, for 96 h in the autosampler at 10 °C, and for 84 days when stored below −65 °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 (t1/2) of 35 min, and a relatively high clearance (CL) of 2.71 L/h/kg.