Preliminary pharmacokinetic study of the anticancer 6BIO in mice using an UHPLC-MS/MS approach

Abstract

Indirubins represent a group of natural and synthetic products with bio-activities against numerous human cancer cell lines acting by inhibiting protein kinases. The natural sources of indirubins are plants of Isatis sp., Indigofera sp., and Polygonum sp., recombinant bacteria, mammalian urine and some marine mollusks. Specifically, the halogenated derivative 6-bromo indirubin-3′-oxime (6BIO) possesses increased selectivity against GSK-3. However, to our knowledge, no analytical method to determine 6BIO in biological fluids has been developed till now. Therefore, a rapid, sensitive and high throughput UHPLC-MS/MS methods were developed and validated to evaluate the concentrations of 6BIO in mice plasma. Plasma samples were pre-treated by protein precipation using cold mixture of methanol: acetonitrile (9:1, v/v) and separations were carried out on a Hypersil Gold C18 column (50 × 2.1 mm i.d.; 1.9 μm p.s.) using 0.1% acetic acid and methanol as mobile phase at a flow rate of 500 mL/min in a gradient mode. For quantitation, a hybrid LTQ-Orbitrap MS equipped with an electro-spray ionization source was used applying a selected reaction monitoring (SRM) option. The monitored transitions were m/z 354.0 → 324.0 for 6BIO and 297.1 → 282.1 for afromorsin (used as the internal standard) in the negative mode. Following the EMA, ICH and FDA guidelines for validation of analytical procedures, the assay method was fully validated in terms of selectivity, linearity, recovery, matrix effect, accuracy, precision, stability, and robustness. The validated methods were successfully applied to the pharmacokinetic studies of 6BIO following an oral administration to mice at the dose of 50 mg/kg. The results indicated that 6BIO possesses a Tmax of 30 min, a half-life of 1 h, and low plasma bioavailability.