School of Life Sciences, Liaoning University, Shenyang 110036, China.
School of Engineering, University of Warwick, Coventry CV4 7AL, UK.
Molecules. 2021 Dec 24;27(1):92. doi: 10.3390/molecules27010092.
AWRK6 was a synthesized peptide developed based on the natural occurring peptide dybowskin-2CDYa, which was discovered in frog skin in our previous study. Here, a quantitative determination method for AWRK6 analysis in rat plasma by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was established and validated following U.S. FDA guidelines. A combination of plasma precipitation and liquid-liquid extraction was applied for the extraction. For pharmacokinetics study, the rats were administrated with AWRK6 via intraperitoneal and intravenous injection. The prepared plasma samples were separated on an ODS column and analyzed by tandem MS using precursor-to-product ion pairs of /: 533.4→84.2 for AWRK6 and /: 401.9→101.1 for internal standard Polymyxin B sulfate in multiple reaction monitoring mode. AWRK6 concentrations in rat plasma peaked at about 1.2 h after intraperitoneal injections at 2.35, 4.7 and 9.4 mg/kg bodyweight. The terminal half-life was around 2.8 h. The absolute bioavailability of AWRK6 was 50% after 3 doses via injection, and the apparent volume of distribution was 4.884 ± 1.736 L. The obtained determination method and pharmacokinetics profiles of AWRK6 provides a basis for further development, and forms a benchmark reference for peptide quantification.
AWRK6 是一种基于天然存在的肽 dybowskin-2CDYa 合成的肽，我们之前的研究在青蛙皮肤中发现了它。在此，根据美国 FDA 指南，建立并验证了一种用于大鼠血浆中 AWRK6 分析的液相色谱-串联质谱（LC-MS/MS）定量测定方法。采用血浆沉淀和液液萃取相结合的方法进行提取。进行药代动力学研究时，通过腹腔和静脉注射将 AWRK6 给予大鼠。在 ODS 柱上分离制备的血浆样品，并通过串联质谱在多重反应监测模式下使用 /: 533.4→84.2（用于 AWRK6）和 /: 401.9→101.1（用于内标硫酸多粘菌素 B）的前体到产物离子对进行分析。大鼠腹腔注射 2.35、4.7 和 9.4 mg/kg 体重后，AWRK6 的血浆浓度在约 1.2 小时时达到峰值。半衰期约为 2.8 小时。经过 3 次注射后，AWRK6 的绝对生物利用度为 50%，表观分布容积为 4.884±1.736 L。获得的 AWRK6 测定方法和药代动力学特征为进一步开发提供了基础，并为肽定量提供了基准参考。
