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

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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: C max 17.03 ± 1.34 ng mL−1; T max 8.67 ± 3.06 h; and T 1/2 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.

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Acknowledgments

We acknowledge Mr. Pei-Wei Huang, Senior Assistant, School of Pharmacy, National Defense Medical Center, for his technical support.

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Author notes

  1. Li-Heng Pao

    Present address: Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Kweishan, Taoyuan, 333, Taiwan, Republic of China

Authors and Affiliations

  1. Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China

    Jia-Hung Ye

  2. Department of Disaster Management, Taiwan Police College, Shing-Lung Rd, Section 3, Taipei, Taiwan, Republic of China

    Ryh-Nan Pan

  3. Forensic Laboratory, Taiwan Police College, Shing-Lung Rd, Section 3, Taipei, Taiwan, Republic of China

    Cong-Cheng Guo

  4. School of Pharmacy, National Defense Medical Center, Taipei, Taiwan, Republic of China

    Chao-Lin Liu & Li-Heng Pao

  5. Department of Nutrition and Health Sciences, Chang Gung University of Science and Technology, Kweishan, Taoyuan, Taiwan, Republic of China

    Li-Heng Pao

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Ye, JH., Pan, RN., Guo, CC. et al. LC–MS/MS Method for Detection of a Highly Selective KCa3.1 Blocker, TRAM-34, in Rat Plasma. Chromatographia 77, 695–705 (2014). https://doi.org/10.1007/s10337-014-2674-0

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