Development of a HPLC-MS/MS method for the pharmacokinetic studies of Pirfenidone in pig plasma

Pirfenidone is a drug active against idiopathic pulmonary fibrosis, but recently its use was evaluated also in the myocardial infarction to reduce the heart damage. The aim of this study was the development and validation of a HPLC-MS/MS method for the quantitative determination of Pirfenidone in pig plasma for preliminary pharmacokinetic studies in order to confirm the activity against heart attack. For this study, a HPLC Varian Prostar coupled with a triple quadrupole Varian 1200L was employed. The development of the HPLC-MS/MS method started with an Energy Resolved Mass Spectrometry (ERMS) experiment on the Pirfenidone for study its fragmentation in order to select the best MRM transitions. Phenacetin, was chosen as internal standard due to its structural and molecular weight similarities. The chromatographic parameters employed to analyse the samples, were finely tuned to minimize the run time, maintaining the sensitivity and the reliability requested for the study. The best performances were obtained by using a Phenomenex Luna PFP 30x2 mm, 3 m of particle size, column with a gradient elution of 10 mM formic acid and 5 mM ammonium formate solution (Solvent A), 5mM formic acid and 10 mM ammonium formate in methanol solution (Solvent B). The elution gradient was started at 90 % solvent A, then decreased to 5 % in 4.0 min, kept for 4.0 min, returned to initial conditions in 0.1 min and maintained for 3.9 min for reconditioning, to a total run time of 12 min. Finally, the mobile phase flow, sample injection and column temperature were kept at 0.25 mL/min, 5 microL and 20°C respectively. The pharmacokinetic studies involved three different Pirfenidone administrations to Pigs (ev, im and os). Then plasma samples in different times (0, 30, 60, 90, 120, 180 and 240 minutes) have been collected. Moreover, in order to establish the chemical stability of Pirfenidone in studied samples, a series of experiments in PBS buffer and pig plasma were carried out. Each sample was prepared by simply protein precipitation and dilution of supernatant, in order to increase productivity. The evaluation of matrix effect was carried out by following the guidelines of Matuszewski et al using Pig plasma. Accuracy and Precision experiments were performed to validate the proposed HPLC-MS/MS method. Concerning the pharmacokinetics studies, the different administrations shown different kinetic profiles and the related PK parameters were calculated. The drug plasma stability experiments demonstrated that the Pirfenidone was stable in all matrices and incubation times. The method didn’t show matrix effect while recovery, precision and accuracy values were 97.2%, 1,9- 4% and 83-94% respectively. The proposed HPLC-MS/MS method has proven to be suitable for the determination of Pirfenidone in the pharmacokinetic studies described above. Moreover, the obtained preliminary data will be useful to support the Pirfenidone study to reduce the heart damages in the myocardial infarction.