DEVELOPMENT OF A LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY METHOD FOR THE DETERMINATION OF INDIRUBIN-5-NITRO-3′-MONOXIME, A NOVEL CDK INHIBITOR, IN RAT PLASMA

Abstract

A simple and rapid liquid chromatography-tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the quantitation of indirubin-5-nitro-3′-monoxime (5-NIO) in rat plasma using 5-chloro-5′-chloro-indirubinoxime as the internal standard. The sample preparation involved protein precipitation using acetonitrile. Separation was achieved on an Xterra column C₁₈ (2.1 mm × 50 mm, i.d. 5 µm) using a mixture of 0.1% formic acid-water/acetonitrile (18:82, v/v) as the mobile phase at a flow rate of 300 µL/min. Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization and multiple reaction monitoring (MRM) in negative ion mode. The total chromatographic run time was 2.5 min, and the calibration curves of 5-NIO were linear (correlation coefficient range: 0.9989–0.9999) over the concentration range of 0.2–2000 ng/mL in rat plasma. Also, the detection limit of the method was 0.05 ng/mL and it showed good accuracy and precision within the concentration range of 0.45–376 ng/mL. This validated method was successfully applied to estimating the pharmacokinetic parameters of 5-NIO following intravenous administration of 5-NIO to rats.

Keywords:

• cyclin-dependent kinase inhibitor
• indirubin-5-nitro-3′-monoxime
• LC-MS/MS
• pharmacokinetics
• plasma
• rat

ACKNOWLEDGMENT

This research was supported by the Graduate Research Assistantship of Dankook University.

Notes

^a Back-calculated following equation; y = 0.02023x + 0.00021, y: 5-NIO peak area divided by IS peak area, x: measured concentration; mean±standard deviation.

^b (Mean measure concentration/nominal concentration) × 100; mean.