Quantification of endostar in rat plasma by LC–MS/MS and its application in a pharmacokinetic study

► An LC–MS/MS method for the quantification of endostar in rat was developed and validated. ► This is the first LC–MS/MS quantitative method for endostar in biological matrices. ► The method was applied to the pharmacokinetic studies of endostar in rats. LC–MS/MS is a promising analytical platform for the quantification of recombinant therapeutic proteins in biological fluids for pharmacokinetic (PK) studies. Herein, an absolute quantification method based on LC–MS/MS technique was developed to quantify endostar, which is modified from the recombinant human endostatin by adding a nine-amino acid sequence (MGGSHHHHH) at the N-terminal. A reproducible three-step analytical procedure was adopted: (1) Ni2+ Sepharose was used to selectively extract endostar; (2) the signature peptide “TEAPSATGQASSLLGGR” (m/z 802.32+–651.82+) of endostar and a synthetic peptide “TEAPSATGQVSSLLGGR” (m/z 816.92+–666.42+) as internal standard (IS) were selected and analyzed in the multiple reaction monitoring (MRM) mode; (3) the proposed method was validated and applied to the pharmacokinetic study of endostar. The lower limit of quantification (LLOQ) for quantifying endostar was 50ng/ml and this method is linear over 50–10,000ng/ml. The accuracy was between 85% and 115%, and the intra-batch and inter-batch analytic precision and accuracy were below 15%. This LC–MS/MS approach was validated for the application to the pharmacokinetic study of endostar in rats.