Determination of IMM-H004 and its active glucuronide metabolite in rat plasma and Ringer's solution by ultra-performance liquid chromatography-tandem mass spectrometry

IMM-H004 is a novel neuroprotective agent and its glucuronide metabolite IMM-H004G has similar protective effects against cerebral ischemic injury in vivo and in vitro. A specific and sensitive ultra-performance liquid chromatography-tandem mass spectrometry method was established and validated for determination of IMM-H004 and IMM-H004G simultaneously in rat plasma and Ringer's solution. Plasma samples containing IMM-H004, IMM-H004G and internal standard propranolol were prepared by direct protein precipitation in a sample-to-solvent ratio of 1:2:6 (plasma: water: acetonitrile), whereas no protein precipitation was required for Ringer's solution samples. Separation was performed with a gradient mobile phase of methanol/water with 0.5% formic acid (v/v) on Eclipse Plus C18 column (2.1 × 50 mm, 3.5 μm) at a flow rate of 0.3 mL/min. The detection was operated on a triple quadrupole mass spectrometer in positive ion multiple reaction monitoring (MRM) mode. The monitored transitions were 305.1 → 248.1 for IMM-H004, 481.3 → 305.1 for IMM-H004G and 260.1 → 183.1 for propranolol. The linear ranges of IMM-H004 and IMM-H004G were 5 to 3000 ng/mL and 10 to 3000 ng/mL for plasma method and 0.5 to 500 ng/mL for Ringer's solution method. All the intra-day and inter-day precision and accuracy for the two analytes in rat plasma were below 7.5% and the intra-day precision and accuracy for analytes in Ringer's solution were within ± 14.7%. There was no obvious matrix effect and the recoveries of the analytes were higher than 94.2%. IMM-H004 and IMM-H004G were stable during one analytic process. The established method was applied successfully to plasma pharmacokinetic and brain microdialysis studies of IMM-H004 and IMM-H004G in rats after a single intravenous administration of IMM-H004.