Metabolite characterization of a novel anti-cancer agent, icotinib, in humans through liquid chromatography/quadrupole time-of-flight tandem mass spectrometry.

Icotinib is a novel anti-cancer drug that has shown promising clinical efficacy and safety in patients with non-small-cell lung cancer (NSCLC). At this time, the metabolic fate of icotinib in humans is unknown. In the present study, a liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (LC/Q-TOF MS) method was established to characterize metabolites of icotinib in human plasma, urine and feces. In addition, nuclear magnetic resonance (NMR) detection was utilized to determine the connection between side-chain and quinazoline groups for some complex metabolites. In total, 29 human metabolites (21 isomer metabolites) were characterized, of which 23 metabolites are novel compared to the metabolites in rats. This metabolic study revealed that icotinib was extensively metabolized at the 12-crown-4 ether moiety (ring-opening and further oxidation), carbon 15 (hydroxylation) and an acetylene moiety (oxidation) to yield 19 oxidized metabolites and to further form 10 conjugates with sulfate acid or glucuronic acid. To our knowledge, this is the first report of the human metabolic profile of icotinib. Study results indicated that significant attention should be paid to the metabolic profiles of NSCLC patients during the development of icotinib.

Quantitative determination of icotinib in human plasma and urine using liquid chromatography coupled to tandem mass spectrometry.

We developed a rapid, specific and sensitive method based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) to determine icotinib concentrations in human plasma and urine. Liquid-liquid extraction (LLE) and direct dilution were firstly used to isolate icotinib from plasma and urine followed by injection of the extracts onto a C(18) column with gradient elution. Ionization of icotinib and midazolam (internal standard, IS) was performed using an electrospray ionization source in positive mode and detection was carried out in multi-reaction monitoring (MRM) mode. The lower limits of quantitation (LLoQ) of icotinib in human plasma and urine by this method were 0.1 and 1.00ng/mL, respectively. The accuracy, precision, specificity, recovery, matrix effect, linearity and several of stabilities have been validated for icotinib in human plasma and urine. In conclusion, the validation results showed that this method is robust, specific and sensitive and it can successfully fulfill the requirement of clinical pharmacokinetic study of icotinib hydrochloride in Chinese healthy subjects.