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Updated: Sep 20, 2025
Biobanking of Human Aqueous and Vitreous Liquid Biopsies for Molecular Analyses
Published on: September 11, 2023
Microvolume Analysis of Aflibercept in Aqueous Humor Using Mass Spectrometry
Natsuka Kimura1, Hidenori Takahashi2, Shinichi Sakamoto2
1Division of Clinical Pharmacology, Department of Pharmacology, Jichi Medical University, Shimotsuke, Tochigi, Japan.
Purpose:
To develop a microvolume analytical method for measurement of the aflibercept concentration in human intraocular fluid and plasma.
Methods:
We analyzed trace amounts of aflibercept in human aqueous humor using Fab-selective proteolysis and nano-surface and molecular-orientation limited (nSMOL) proteolysis, coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Patients with age-related macular degeneration or diabetic macular edema were recruited. Just after an injection of 50 µL of aflibercept, regurgitate from needle holes was collected with a micropipette pressed to the side of the injection hole within 10 seconds. The median amount of regurgitate was 4 µL (range, 1-18 µL).
Results:
In human plasma, the aflibercept concentration ranged between 0.195 and 50 µg/mL when using the quantitative signature peptide IIWDSR (aa. 56-61) present on the vascular endothelial growth factor receptor 1 domain of aflibercept. The method was validated by evaluating its linearity, carryover, selectivity, accuracy and precision, dilution effect, and sample/processing stability. As only a minimal amount of regurgitate through needle holes can be sampled, we performed and verified the aflibercept assay using patient samples after 1:10 dilution with control human plasma, a recognized diluent. The median concentration of aflibercept in the regurgitate was 240 µg/mL (range, 13-4300 µg/mL).
Conclusions:
Our findings indicate that the aflibercept assay using human intraocular fluid can be reliably performed using nSMOL coupled with LC-MS/MS.
Translational Relevance:
This technique for quantifying aflibercept in the regurgitate suggests that the amount of drug lost post-injection can be ignored, even in patients with a relatively large leak after vitreous injection. This new methodology suggests possible therapeutic responses and may be employed as a general analytical method for trapping many biologics, such as vascular endothelial growth factor, in various types of clinical samples, unaffected by proteinaceous or small organic pharmaceuticals.
