Quantification of recombinant human relaxin-2 (B-29/A-24) in non-pregnant rat plasma using ultra performance liquid chromatography-mass spectrometry

Abstract

A rapid, sensitive and high throughput method using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was first developed for the determination of recombinant human relaxin-2 (B-29/A-24) in non-pregnant rat plasma for characterizing the pharmacokinetics. The method was operated under pseudo-multiple reaction monitoring in the positive electrospray ionization mode. H2 relaxin and internal standard (Levemir) were extracted under acidic conditions by one-step protein precipitation with acetonitrile. Chromatographical separation was obtained on a XBridge BEH300 C4 column with a gradient elution profile consisting of acetonitrile and 0.2% aqueous formic acid. The method was fully validated in terms of linearity, selectivity, precision, accuracy, recovery, matrix effect and stability. The assay was validated over a concentration range of 10.0–1000 ng mL⁻¹ and no interfering peaks were detected at the retention time of H2 relaxin and internal standard in blank rat plasma. Recoveries from spiked controls were >83% for the analytes at all quality control levels and no obvious matrix effects were found. Stability studies indicated that H2 relaxin in rat plasma underwent no significant degradation. In conclusion, this method was successfully applied to determine the concentration of H2 relaxin in plasma collected from Sprague-Dawley rats during the pharmacokinetic study of H2 relaxin.