Quantification of JM25-1 in rat plasma by HPLC-MS/MS: Application to a pharmacokinetic study

Abstract 2-diethylamino-N-2,5-dimethylphenyl acetamide (JM25-1), a novel anti-inflammatory agent with epithelial barrier protective function, requires comprehensive pharmacokinetic evaluation to support clinical translation. A quantitative analysis method was developed to characterize the pharmacokinetics of JM25-1 in Wistar rats after oral and intraperitoneal ( i.p ) administration. Plasma, obtained via centrifugation and methanol protein precipitation, was analyzed using HPLC-MS/MS. Chromatographic separation was achieved on a Kinetex column embedded with C 18 stationary phase, using a binary-gradient mobile phases consisting of water containing 0.1% formic acid (phase A) and methanol containing 0.1% formic acid (phase B). The flow rate was set at 0.6 mL min −1 with a total run time of 4 min. Mass spectrometric detection was performed by monitoring the m/z transitions of 235.1/86.2 and 235.1/58.1 for JM25-1, 241.2/86.2 and 241.2/58.1 for Lidocaine-d6 (used as internal standard) in the electrospray ionization positive mode. The method exhibited excellent linearity over concentration ranges of 1–200 ng mL −1 ( r 2 = 0.9998). All validation parameters, including specificity, extraction recovery (89.65–98.44%), matrix effect (absolute: 97.22–99.82%; relative: