High-sensitivity quantitation of a biopharmaceutical Nanobody® in plasma by single-cartridge multi-dimensional solid-phase extraction and UPLC-MS/MS

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. For the quantitation of a 28-kDa biopharmaceutical protein, a single-cartridge, multi-dimensional (ion-exchange and reversed-phase) SPE procedure to selectively enrich a signature peptide from a plasma digest was developed, validated and applied. Results: With this procedure, sufficient selectivity was introduced to allow quantitation in 50 µL of plasma down to 10.0 ng/mL (~0.3 nM). An in-house prepared 18O-labeled form of the signature peptide was successfully used as internal standard. The developed procedure was validated for both human and rabbit plasma. Plasma samples from a pre-clinical trial were analyzed with the developed LC-MS/MS method and compared to a conventional ligand binding assay. The results of the LC-MS/MS assay and the ligand binding assay were in good agreement (r2 >0.85) Conclusions: The combination of ion-exchange and (high-pH) reversed-phase SPE principles allowed the sensitive and selective LC-MS/MS quantitation of the Nanobody in digested plasma without the use of antibodies for sample clean-up. When appropriate precautions are taken, the preparation of an 18O-labeled peptide for use as internal standard is a practical and economical alternative to custom synthesis.