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-microL-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 microL-volumes were injected onto the HPLC system. Separation was achieved on a Gemini C18 column (150 mm x 2.1 mm ID, particle size 5 microm) 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 microL 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.
Vainchtein
Liia D
LD
Department of Pharmacy & Pharmacology, Slotervaart Hospital/The Netherlands Cancer Institute, Louwesweg 6, 1066 EC Amsterdam, The Netherlands. liia.vainchtein@slz.nl
Rosing
Hilde
H
Maier
Armin
A
Fiebig
Heinz-Herbert
HH
Schellens
Jan H M
JH
Beijnen
Jos H
JH
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D007211:Indoles / Q000097:blood*; D051379:Mice; D047428:Protein Kinase Inhibitors / Q000097:blood*; D012015:Reference Standards; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
C524511:5-methylindirubine; D007211:Indoles; D047428:Protein Kinase Inhibitors; D018844:Cyclin-Dependent Kinases