The application of an LC-MS/MS method in a pharmacokinetic study for the determination of the concentration of nifekalant in human plasma

• An LC-MS/MS method for the determination of Nifekalant in human plasma.
• The method is a simple, sensitive and rapid.
• Successfully applied to a pharmacokinetic study in healthy volunteers.

In the present study, a simple, rapid, and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of the concentration of Nifekalant in human plasma was developed and validated. The analyte and the internal standard were extracted from human plasma using dichloromethane and analyzed using an ultra-fast liquid chromatographer (UFLC) coupled to an electrospray ionization (ESI) tandem mass spectrometer in the positive mode. The chromatographic analysis was performed isocratically on an Inertsil ODS-SP column (150 mm × 4.6 mm I.D., 5 μm). The mobile phase was a mixture of 15% 10 mM aqueous ammonium formate and 85% methanol (the pH was adjusted to 3.5 with formic acid) at a flow rate of 0.8 mL/min with a split ratio of 1:1 to the ionization source. The lower limit of quantification (LLOQ) was 5.05 ng/mL, and a linear calibration curve was obtained over the concentration range of 5.05 to 3030 ng/mL. The intra-day and inter-day assay variations were less than 9.06%, and the accuracy values (relative error) were in the range of −10.95% to 2.27%. The essential pharmacokinetic parameters of the intravenously injection of Nifekalant were found to be the following: t1/2 = 1.26 ± 0.16 h, Cmax = 1.943 ± 0.411 mg/L, and AUC0–12h = 4.600 ± 0.756 mg/L·h.