A HPLC–MS/MS method for the quantitation of free, conjugated, and total HDND-7, a novel hesperetin derivative, in rat plasma and tissues: Application to the pharmacokinetic and tissue distribution study

• HDND-7 is a novel hesperetin derivative which possesses hepatoprotective activity in vitro and in vivo.
• It is the first systematic report of pharmacokinetics and tissue distribution study of HDND-7 in rat.
• HDND-7 is widely distributed in various tissues, and transfer across the blood–brain barrier.
• The glucuronides and sulfates of HDND-7 are the major forms in systematic circulation and tissues.

A sensitive and reliable HPLC–MS/MS method was developed and validated for the determination of free (unconjugated), glucuronidated, sulfated, and total (free and conjugated) HDND-7 in rat plasma and tissues. Plasma and tissues samples were treated prior to and after the enzyme hydrolysis. Chromatographic separation was achieved on a Phenomenex Luna C18 column (150 × 4.6 mm, 3 μm), using isocratic mobile phase consisting of 0.1% formic acid–acetonitrile (50:50, v/v) at a flow rate of 300 μl/min. The detection was performed on a triple quadruple tandem mass spectrometer using positive electrospray ionization (ESI) source with a chromatographic run time of 5.0 min. The detection was operated by multiple reaction monitoring (MRM) of the transitions of m/z 429.3 → 223.9 for HDND-7 and 272.9 → 152.9 for naringenin (IS), respectively. This method was validated in terms of specificity, linearity, precision, accuracy, and stability. The calibration curves for plasma and tissues were linear over a wide concentration range of 0.02–40 μg/ml with a lower limit of quantification (LLOQ) of 0.02 μg/ml. Mean extraction recoveries in plasma and tissues ranged from 87.4 to 97.1% and from 54.2 to 70.5%, respectively. The intra- and inter-day precision values were below 15% and the accuracy was within ±15%. The samples were stable under all the tested conditions. This method has been successfully applied to the pharmacokinetic study following oral doses of 25, 50 and 100 mg/kg and intravenous dose of 25 mg/kg, and tissue distribution study following oral dose of 50 mg/kg.

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