Development and validation of an LC-MS-MS method for the simultaneous determination of sulforaphane and its metabolites in rat plasma and its application in pharmacokinetic studies.

A highly sensitive and simple high-performance liquid chromatographic-tandem mass spectrometric (LC-MS-MS) assay is developed and validated for the quantification of sulforaphane and its metabolites in rat plasma. Sulforaphane (SFN) and its metabolites, sulforaphane glutathione (SFN-GSH) and sulforaphane N-acetyl cysteine (SFN-NAC) conjugates, are extracted from rat plasma by methanol-formic acid (100:0.1, v/v) and analyzed using a reversed-phase gradient elution on a Develosil 3 μm RP-Aqueous C(30) 140Å column. A 15-min linear gradient with acetonitrile-water (5:95, v/v), containing 10 mM ammonium acetate and 0.2% formic acid, as mobile phase A, and acetonitrile-water (95:5, v/v), containing 10 mM ammonium acetate and 0.2% formic acid as mobile phase B, is used. Sulforaphane and its metabolites are well separated. Sulforaphene is used as the internal standard. The lower limits of quantification are 1 ng/mL for SFN and 10 ng/mL for both SFN-NAC and SFN-GSH. The calibration curves are linear over the concentration range of 25-20,000 ng/mL of plasma for each analyte. This novel LC-MS-MS method shows satisfactory accuracy and precision and is sufficiently sensitive for the performance of pharmacokinetic studies in rats.