LC–MS/MS Method for Detection of a Highly Selective KCa3.1 Blocker, TRAM-34, in Rat Plasma

Abstract

1-[(2-Chlorophenyl)diphenylmethyl]pyrazole (TRAM-34) is a highly selective KCa3.1 channel blocker. TRAM-34 was commonly used to study the role of KCa3.1 in the pathogenesis of disease in vivo, but there was no validated analytical method. Here, we describe the first validated LC–MS/MS analytical method for TRAM-34. Solid-phase extraction (SPE) was performed to extract TRAM-34 from the rat plasma. Chromatographic separation was achieved on the phenyl column. A triple quadrupole mass spectrometer was operated in positive-mode electrospray ionization. There were two multiple-reaction monitoring (MRM) transitions for TRAM-34: m/z 277.2 → 165.1 (for quantification) and m/z 277.2 → 241.2 (for qualification). Bifonazole was used as an internal standard. The lower limit of quantification (LLOQ) achieved was 1 ng mL−1 and the run time was 7.5 min. The linear range was from 1 to 1,000 ng mL−1. The pharmacokinetics profile was acquired for rats following an intraperitoneal injection of TRAM-34, with the following pharmacokinetics parameters found: Cmax 17.03 ± 1.34 ng mL−1; Tmax 8.67 ± 3.06 h; and T1/2 13.45 ± 2.72 h. In addition, a suspected metabolite of TRAM-34 was found using this LC–MS/MS method. Given the results of the detailed validation process and its application to TRAM-34 pharmacokinetics, it is clear that a fast, selective, precise, and reproducible TRAM-34 LC–MS/MS analytical method was successfully established.