A novel LC-MS/MS approach to the pharmacokinetic study of free and bound aflibercept simultaneously

Abstract

To comprehensively evaluate the pharmacokinetic (PK) characteristics of aflibercept, we established a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method to determine the concentration of vascular endothelial growth factor (VEGF)-A-bound aflibercept and free aflibercept. A specific sample preparation method of nano-surface and molecular-orientation limited (nSMOL) proteolysis was performed to extract both free and bound aflibercept from plasma. The tryptic peptides unique to aflibercept and VEGF-A were selected to quantify the amounts of total aflibercept and aflibercept–VEGF complex, respectively. The method was validated by evaluating its selectivity, linearity, precision, accuracy, extraction recovery, matrix effect, and stability. It was then successfully used to quantify total and bound aflibercept concentrations in cynomolgus monkey plasma, while indirectly obtaining the concentration of free aflibercept by subtraction. The PK results of this LC-MS/MS method are comparable to the traditional enzyme-linked immunosorbent assay (ELISA) results. It is thus a reliable and complementary method for the PK evaluation of aflibercept.

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Acknowledgments

The authors would like to thank Shimadzu Co., Ltd., for their technical help.

Funding

This work was supported by grants from the National Natural Science Foundation of China (No.81773679) and the project of Graduate Innovation Foundation of Yantai University, GIFYTU (No.YDZD1917).

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Correspondence to Fei Yu.

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The authors declare that they have no conflict of interest.

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The study had been approved by the Institutional Animal Care and Use Committee with approval number IACUC-A2017033-K001-01.

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Kong, L., Liu, F., Huo, L. et al. A novel LC-MS/MS approach to the pharmacokinetic study of free and bound aflibercept simultaneously. Anal Bioanal Chem 412, 1003–1010 (2020). https://doi.org/10.1007/s00216-019-02316-w

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Keywords

  • Aflibercept
  • VEGF
  • nSMOL
  • Pharmacokinetics
  • LC-MS/MS