Online ISSN : 1348-3315
Print ISSN : 1342-8284
ISSN-L : 1342-8284
Original Papers
Fluorescence Bioanalysis of Bevacizumab Using Pre-Column and Post-Column Derivatization – Liquid Chromatography After Immunoaffinity Magnetic Purification
Kenichiro TODOROKI Tatsuki NAKANOHideki HAYASHIHajime MIZUNOJun Zhe MINToshimasa TOYO’OKA
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2020 Volume 41 Issue 3 Pages 115-122


This report presents two fluorescence labeling methods for therapeutic monoclonal antibody, bevacizumab, to increase its detection sensitivity for fluorescence detection. One method is high-temperature reversed-phase LC (HT-RPLC) following post-column fluorogenic derivatization using o-phthalaldehyde with thiol. Another method is pre-column derivatization using Zenon Alexa Fluor 488 protein-tag following size-exclusion chromatography (SEC). The calibration curves of bevacizumab were 1–50 μg/mL (post-column method) and 0.1–10 μg/mL (pre-column method). Both methods showed good correlation coefficients (r2 > 0.991). The LOD and the LOQ of bevacizumab were, respectively, 0.13 and 0.43 μg/mL (post-column method) and 0.03 and 0.1 μg/mL (pre-column method). The sensitivities were about 2 and 10 times higher than that of native fluorescence detection. The proposed methods were applied to bevacizumab spiked human plasma samples. The bevacizumab in plasma samples was purified selectively with immunoaffinity beads and detected as a single peak using HT-RPLC or SEC with fluorescence detection.

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© 2020 The Society for Chromatographic Sciences
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