Simultaneous determination of four PDE5 inhibitors and metabolites in rat plasma by UPLC-MS/MS

Herein, we developed a sensitive UPLC-MS/MS method for the simultaneous determination of four PDE5 inhibitor and twelve metabolites in rat plasma. A total of 16 chemicals were separated on gradient elution with mobile phase A (0.1 % formate acetonitrile) and mobile phase B (0.1 % formate and 2 mmol/L ammonium formate aqueous solution) on an ACQUITY UPLC HSS T3 column (100 mm×2.1 mm, 1.8 μm) after protein precipitation, followed by the detection on the positive ion mode of electrospray ion source (ESI) via dynamic multi-reaction monitoring mode (MRM). The method provided good linearity over the range of 0.25-20 ng/mL for all compounds with correlation coefficients R greater than 0.99. The recoveries were higher than 75.5 %, the matrix factors were 2.1 %-20.4 %. The precision, accuracy and stability were also within acceptable criteria. A rat model was established to investigate the metabolic profile of Sildenafil, Vardenafil, Acetildenafil and Tadalafil. Blood samples from the experimental group were analyzed using ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). Notably, desmethyltadalafil and methyl-desmethyltadalafil were not detected in the plasma. However, their glucuronide conjugates, desmethyltadalafil glucuronide and methyl-desmethyltadalafil glucuronide, were identified and characterized via tandem mass spectrometry (MS/MS). At present, cases of poisoning or even death due to overdose of phosphodiesterase type 5 (PDE5) inhibitors are often encountered in the practice of forensic science. Few studies on PDE5 inhibitors have been reported in forensic science toxicology identification, and the existing detection methods cannot meet the need for simultaneous and rapid analysis of multiple trace PDE5 inhibitors in vivo. Therefore, it is of great importance to establish and optimize the detection methods for PDE5 inhibitors drugs in biological samples. The significance of this method is to provide a reference for the determination of PDE5 inhibitors poisoning cases.