Quantitative and selective assay of 5-methylindirubine, an inhibitor of cyclin-dependent kinases, in murine plasma using coupled liquid chromatography and electrospray tandem mass spectrometry

A sensitive and rapid LC–MS/MS assay for the quantitative determination of 5-methylindirubine (5-MI) in murine plasma is described. A 50-μL-murine plasma aliquot was spiked with an internal standard, indirubine-3-monoxime (IMO), and extracted with 1.25 mL diethyl ether. Dried extracts were reconstituted in methanol–water (8:2, v/v) and 10 μL-volumes were injected onto the HPLC system. Separation was achieved on a Gemini C18 column (150 mm × 2.1 mm ID, particle size 5 μm) using an alkaline eluent (10 mM ammonium hydroxide–methanol (5:95, v/v)). Detection was performed by negative ion electrospray followed by tandem mass spectrometry. The assay quantifies 5-MI in a range from 1 to 500 ng/mL using 50 μL of murine EDTA plasma samples. Validation results demonstrate that 5-MI concentrations can be accurately and precisely quantified in murine plasma. This assay is used to support pre-clinical pharmacologic studies with 5-MI.