A sensitive and selective LC–MS–MS method for simultaneous determination of picroside-I and kutkoside (active principles of herbal preparation picroliv) using solid phase extraction in rabbit plasma: Application to pharmacokinetic study

Abstract

A rapid, sensitive and selective LC–MS–MS method for the simultaneous quantitation of picroside-I and kutkoside (active constituents of herbal hepatoprotectant picroliv) was developed and validated in rabbit plasma. The analytes and internal standard (Amarogentin) were extracted using Oasis ® HLB solid phase extraction cartridges. Analysis was performed on Spheri RP-18 column (10 μm, 100 mm × 4.6 mm i.d.) coupled with guard column using acetonitrile:MilliQ water (50:50, %v/v) as mobile phase at a flow rate of 1 ml/min with a retention time of 1.39, 1.33 and 1.42 min for picroside-I, kutkoside and amarogentin, respectively. The quantitation was carried out using an API-4000 LC–MS–MS with negative electro spray ionization in multiple reaction monitoring (MRM) mode. The precursor to product ion transitions for picroside-I, kutkoside and amarogentin were m/ z 491 > 147, 199; 511 > 167, 235; 585 > 227, respectively. The method was validated in terms of establishing linearity, specificity, sensitivity, recovery, accuracy and precision (within- and between-assay variation), freeze–thaw (f–t), auto injector and dry residue stability. Linearity in plasma was observed over a concentration range of 1.56–400 ng/ml with a limit of detection (LOD) of 0.5 ng/ml for both analytes. The recoveries from spiked control samples were >60 and >70% for picroside-I and kutkoside, respectively. Accuracy and precision of the validated method were within the acceptable limits of <20% at low and <15% at other concentrations. The analytes were stable after three freeze–thaw cycles and their dry residues were stable at −60 °C for 15 days. The method was successfully applied to determine concentrations of picroside-I and kutkoside post i.v. bolus administration of picroliv in rabbit.