UPLC–MS/MS for the Determination of Azilsartan in Beagle Dog Plasma and Its Application in a Pharmacokinetics Study

The purpose of the study is to develop an ultra performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) to determinate the concentration of azilsartan in the dog plasma. After precipitated by methanol, the plasma sample containing azilsartan and diazepam (internal standard, IS) was determined by UPLC–MS/MS. The mobile phase consisted of acetonitrile-water was pumped at a flow rate of 0.3 ml/min in gradient elution. Kinetex 2.6 μ XB-C18 column (50 × 2.1 mm, 100 Å; Phenomenex, USA) were used for LC separations. The column temperature was 30 °C and the injection volume was 5 μl. The electrospray ionization (ESI) and multiple reaction monitoring (MRM) were applied at the transitions of m/z 457 → 279 (azilsartan) and m/z 285 → 193 (diazepam), respectively. The developed method was identified a good linearity over a concentration range of 2.5–5000 ng/ml. The lower limit of quantitation (LLOQ) was 2.5 ng/ml. The intra-day and inter-day precision (relative standard deviation, RSD%) were less than 10% and accuracy (relative error, RE %) was less than 5% at three quality control levels. The extraction recovery of azilsartan at three quality control levels were 82.41 ± 0.68%, 98.66 ± 11.00%, 102.43 ± 0.82%. And the recovery for IS (100 ng/ml) was 91.75 ± 0.54%. A validated UPLC–MS/MS method was firstly developed for the quantification of azilsartan in dog plasma and it was applied to the pharmacokinetics study.