Simultaneous quantification of phencynonate and its active metabolite N‐demethyl phencynonate in human plasma using liquid chromatography and isotope‐dilution mass spectrometry

A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed to simultaneously quantify phencynonate (PCN) and its major metabolite N‐demethyl phencynonate (DM‐PCN) in human plasma. Following one‐step liquid‐liquid extraction, the analytes were separated on a reversed‐phase C₁₈ column. Methanol and 0.02% formic acid in 10 mM ammonium acetate (62:38, v/v) was used as isocratic mobile phase at a flow‐rate of 0.3 mL/min. An API 5000 tandem mass spectrometer equipped with a Turbo IonSpray ionization source was used as the detector and was operated in the positive ion mode. Multiple reaction monitoring using the transition of m/z 358.4 → m/z 156.2, m/z 344.4 → m/z 142.2, and m/z 361.3 → m/z 159.2 was performed to quantify PCN, DM‐PCN, and the internal standard (D₃‐PCN), respectively. This approach showed a lower limit of quantification of 10 pg/mL and 25 pg/mL for PCN and DM‐PCN in plasma, respectively. This sensitivity was at least 50‐fold superior to previously reported ones and thus enabled the approach well applicable to low‐dose pharmacokinetic studies. The intra‐ and inter‐day precisions were less than 14.2 % at each QC level for both PCN and DM‐PCN. The inter‐day relative errors ranged from ‐1.9% to ‐4.9% for PCN, and from 0.6% to 6.4% for DM‐PCN. As a proof of principle, the validated method was successfully applied to simultaneous quantification of circulating PCN and DM‐PCN in healthy subjects after a single oral administration of 2 mg phencynonate hydrochloride pellet. Copyright © 2015 John Wiley & Sons, Ltd.