Simultaneous Determination Of Donafenib And Its N-oxide Metabolite In Human Plasma By Liquid Chromatography-tandem Mass Spectrometry

Background Donafenib is the deuterium derivative of sorafenib,and is developed as an anti-tumor drug in China.It is currently in clinical trials.Objective To develop and validate an accurate and sensitive liquid chromatography-tandem mass spectrometry?LC-MS/MS?method for the simultaneous determination of donafenib and its N-oxide metabolite in human plasma.The method was to be validated and applied to the pharmacokinetics study of donafenib tosylate tablets in volunteers.Method Since donafenib is the deuterium derivative of sorafenib,so choosing sorafenib as the internal standard of donafenib is preferable.Similarly,sorafenib N-oxide was selected as the internal standard of donafenib N-oxide.The analytes and internal standards were extracted from plasma by protein precipitation with acetonitrile,and separated on a Gemini C₁₈?50 mm×2.0 mm,5?m?column using a gradient elution procedure.The mobile phase used for gradient elution were 5 mmol·L^-1ammonium acetate-formic acid?100/0.2,v/v?and acetonitrile.The total chromatographic run time was 5.0 min.Positive electrospray ionization was performed using multiple reaction monitoring?MRM?with transitions of m/z 468.2?273.2 for donafenib and m/z 465.2?270.2 for its internal standard sorafenib,m/z 484.2?289.2 for donafenib N-oxide and m/z481.2?286.2 for its internal standard sorafenib N-oxide.Results The standard curves were linear in the range of 5.00 to 5000ng·mL^-1 for donafenib,and 1.00 to 1000 ng·mL^-1 for donafenib N-oxide.The precision and accuracy values were within the acceptable rang.Stability of donafenib and donafenib N-oxide in human plasma were evaluated after storage at room temperature for 6 h,after storage at-70?for 51 days and 302 days,after storage at-20?for 245 days.The post-preparative stability was measured after exposure of the processed samples at room temperature for 24 h.Stability of plasma samples were evaluated after three freeze-thaw cycles from-70?to room temperature.The results showed that the plasma samples were stable under the above storage conditions.The matrix effect of donafenib were 106%and 108%at the low and high concentration,and the RSD was below 2.5%.The matrix effect of donafenib N-oxide were 93.7%and 101%at the low and high concentration,and the RSD was below 2.8%.These results showed that the method is not affected by variations in the sample matrix.The method was validated and successfully applied to the pharmacokinetics study of donafenib tosylate tablets in patients.Conclusion An accurate and sensitive liquid chromatography-tandem mass spectrometry?LC-MS/MS?method was developed and validated for the simultaneous determination of donafenib and its N-oxide metabolite in human plasma.The method was then successfully applied to the first-in-human pharmacokinetics study of donafenib tosylate tablets in volunteers.The results showed that it is beneficial for biological sample analysis by selecting nondeuterium compounds as internal standard of their deuterium derivatives.