Rapid quantification of a chemically synthesized peptide GAP162 in rat plasma by liquid chromatography/triple quadrupole tandem mass spectrometry and application to a pharmacokinetic study

Abstract

Liquid chromatography/tandem mass spectrometry (LC-MS/MS) is a promising analytical platform for the quantification of therapeutic peptide in biological fluids for pharmacokinetics (PK) studies. Herein, an absolute quantification method based on liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) technique was developed to quantify GAP162, a new synthetic peptide derived from RasGAP_301–326, which is a promising candidate as antitumor drug. A synthetic peptide P119 was used as internal standard. Solid phase extraction (SPE) of the mixed-mode of ion exchange and reversed-phase was employed for sample preparation. Chromatographic separation was performed on a reversed phase C4 column (30 mm × 2.1 mm, 5 μm) with a mobile phase consisting of acetonitrile–water containing 0.1% formic acid with gradient elution at a flow rate of 0.8 mL min⁻¹ for 2.0 min. Multiple reaction-monitoring (MRM) mode was performed with ion pairs of m/z: 748.2 → 830.2, and 526.7 → 585.5 for GAP162 and internal standard of P119, respectively. Calibration curve was linear over a concentration range of 5–500 ng mL⁻¹ with a correlation coefficient >0.99. The lower limit of detection was at 5 ng mL⁻¹ in rat plasma for GAP162. The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits. The validated method was successfully applied to investigate the pharmacokinetics study of GAP162 after single intravenous administration to male Sprague-Dawley rats at 5 mg kg⁻¹.