Determination of Boc5 in Rat Plasma by HPLC-MS/MS and Its Application to a Pharmacokinetic Study

A simple, robust, and sensitive high-performance liquid chromatography tandem mass spectrometry method was developed and validated for determination of a new nonpeptidic glucose-like peptide-1 receptor (GLP-1R) agonist 1,3-bis(4-((p-butoxycarbonyl)amino)benzamido)-2,4-bis(m-methoxy-p-(thiophene-2-methanoyl)phenyl)-cyclobutane-1,3-dicarboxylic acid (Boc5) in rat plasma using wogonin as internal standard (IS). After a simple protein precipitation with methanol, chromatographic separation was achieved on an Atlantis T3 (100 mm x 4.6 mm, 3.5 mu m) analytical column eluted by mobile phase consisting of methanol, acetonitrile, and 20 mM ammonium formate addition to 2% formic acid (39:39:22, v/v/v) at flow rate of 0.6 mL/min for 7 min. The quantitation analysis was performed using multiple reaction monitoring at the transitions of m/z 1,077.2 -> 784.4 for Boc5 and m/z 285.1 -> 270.4 for IS in positive ion mode with electrospray ionization source on an AB SCIEX API 4000 QTRAP mass spectrometer. Good linearity was achieved over the concentration range of 2-2,000 ng/mL with lower limit of quantification at 2 ng/mL. Intra- and interday precisions were less than 7.3 %, and accuracy ranged from -3.6 to 3.0 %. The mean recovery of Boc5 was 93.4-100.2 %, and the matrix effect of Boc5 was 88.0-93.8 %. The validated method was then successfully applied to a pharmacokinetic study of Boc5 in rats.