Bioanalysis Zone

High-sensitivity quantitation of a Nanobody® in plasma by single-cartridge multidimensional SPE and ultra-performance LC–MS/MS

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his article is available for members, for free, to celebrate Nico van der Merbel being awarded the BOSCA – Excellence in research.

Background: A major challenge in protein quantitation based on enzymatic digestion of complex biological samples and subsequent LC–MS/MS analysis of a signature peptide is dealing with the high complexity of the matrix after digestion, which can reduce sensitivity considerably. Results: Using single cartridge multidimensional SPE, sufficient selectivity was introduced to allow quantitation in 50 µl of plasma down to 10.0 ng/ml (˜0.3 nM). An inhouse prepared 18O-labeled signature peptide was used as the internal standard. The procedure was validated for human and rabbit plasma. Conclusion: The developed SPE procedure allowed the sensitive and selective LC–MS/MS quantitation of the Nanobody® without the use of antibodies. When appropriate precautions are taken, the 18O-labeled peptide is a practical and economical alternative to custom synthesis.

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