Determination of IMM-H004, a novel neuroprotective agent, in rat plasma and brain tissue by liquid chromatography-tandem mass spectrometry

- The chromatographic behavior of an amphoteric coumarin compound with multiple peaks was optimized.
- A rapid and sensitive HPLC-MS/MS method for the quantitation of amphoteric coumarin has been developed and validated.
- This assay is being utilized to quantitate IMM-H004 in rat plasma and brain after intravenous injection at 6 mg/kg.

A rapid and sensitive liquid chromatography-tandem mass spectrometry method for determination of IMM-H004, a novel neuroprotective agent, in rat plasma and brain was developed. Plasma and brain tissue homogenate samples containing IMM-H004 and propranolol (internal standard, IS) were prepared by using a direct protein precipitation of acetonitrile. Separation was carried out in Zorbax SB-C18 column at a flow rate of 0.3 mL/min utilizing acetonitrile/water as mobile phases which contain 0.5% formic acid (v/v). Triple quadrupole mass spectrometer was used for detection with selective reaction monitoring. The mass transition ion-pairs were 305 → 248 for IMM-H004 and 260 → 183 for IS in positive ion mode. The linear ranges of IMM-H004 were 5-1000 ng/mL in plasma and 1-200 ng/mL in brain tissue homogenate. The intra- and inter-day precisions were within ±14.9% for analyte in both matrices (±17.0% at the lowest limit of quantification level), while the deviation of assay accuracy was within ±12.9%. No obvious matrix effect was observed. The recovery of the analyte was higher than 85.3%. IMM-H004 was stable during the whole analytic process. The method was applied successfully to the plasma and brain pharmacokinetic study of IMM-H004 in rats after a single intravenous administration.