Determination of henatinib in human plasma and urine by liquid chromatography–tandem mass spectrometry and its pharmacokinetic application

• We develop a simple, sensitive, rapid LC–MS/MS for henatinib in biofluids.
• Retention and peak feature of henatinib get much improved with high pH mobile phases.
• Our methods show higher sensitivity and wider linear range than previously reported.
• Elimination half-life of henatinib is as long as over 20 h.
• Henatinib accumulates to a certain extent after daily administration for 28 days.

Henatinib is a novel oral small-molecule multikinase inhibitor that has demonstrated broad and potent antitumor activities in preclinical studies. In support of a clinical pharmacokinetic study, a simple, sensitive and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantification of henatinib in human plasma and urine. The sample preparation procedure involved a simple protein precipitation with methanol and the addition of midazolam as the internal standard. The chromatographic separation was achieved on an XBridge C18 column (50 mm × 2.1 mm, 2.5 μm) using a mixture of 5 mM ammonium formate (pH 9.5)–acetonitrile–methanol (60:30:10, v/v/v) as the mobile phase. The MS/MS detection was performed in the positive ion multiple reaction monitoring (MRM) mode by monitoring the precursor → product ion transitions at m/z 469.1 → 382.2 for henatinib and m/z 326.2 → 291.3 for the internal standard. Assays were validated over the concentration range of 0.100–400 ng/mL and 1.00–2500 ng/mL for plasma and urine, respectively. The established method was highly sensitive with the lower limit of quantification (LLOQ) of 0.100 ng/mL and 1.00 ng/mL for plasma and urine, respectively. The intra- and inter-day precisions were <8.6% and <9.2% for plasma and urine, respectively. The mean assay accuracy was within ±6.8% of nominal values for both plasma and urine. The analytical runtime within 3.5 min per sample made this method suitable for high-throughput determination. The validated method was successfully applied to a phase I dose escalation pharmacokinetic study in Chinese cancer patients.

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