Bioactivity assay of porcine relaxin based on cAMP accumulation in THP-1 cells quantified by LC-MS/MS.
作者信息
Ren Xiaohong, Wang Caifen, Wu Li, Li Haiyan, Chi Liandi, Sun Lixin, Wang Bin, Cui Hu, Angiolini Luca, Bianchi Alessandro, Caccia Paolo, Xing Shu, Fu Xueqi, Zhang Jiwen
机构信息
Edmond H. Fischer Signal Transduction Laboratory, College of Life Sciences, Jilin University, Changchun 130012, China; Center for Drug Delivery Systems, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Center for Drug Delivery Systems, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China.
出版信息
J Pharm Biomed Anal. 2015;111:320-3. doi: 10.1016/j.jpba.2015.04.016. Epub 2015 Apr 20.
This study describes a reliable bioactivity assay of porcine relaxin (pRLX) based on cyclic adenosine 3',5'-monophosphate (cAMP) accumulation in the human monocyte cell-line quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). As a result, the LC-MS/MS was based on a positive selected reaction monitoring of cAMP with a stable internal standard, 8-Br-cAMP and a protein precipitation procedure by HClO4. The standard curve of cAMP was linear from 5.0 ng mL(-1) to 992.0 ng mL(-1), with lower limits of detection and quantification of 0.5 ng mL(-1) and 5.0 ng mL(-1), respectively. The satisfactory validation data including stability assay were obtained. When measured by the LC-MS/MS, the pRLX sample showed a time- and dose-dependent stimulation of cAMP with the concentration for 50% of the maximal effect (EC50) of 40.6 ng mL(-1). The developed method indicated higher precision and selectivity than the commercial enzyme linked immunosorbent assay kits, which showed EC50 of 66.6 ng mL(-1).
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