Adduct formation in quantitative bioanalysis: Effect of ionization conditions on paclitaxel

AbstractQuantitative analysis of target compounds with liquid chromatography atmospheric pressure ionization mass spectrometry is sometimes hampered by adduct formation. In these situations, cationization with alkali metal ions instead of proton addition is often observed in the positive ion mode. This work studies the process of adduct formation and investigates potential strategies to control this phenomenon. Paclitaxel, a pharmaceutical chemotherapeutic agent, was used as a model compound. Electrospray (ESI), atmospheric pressure chemical ionization (APCI) and sonic spray ionization (SSI) are evaluated and compared. The work was performed on two different instruments, allowing the evaluation of different ionization behavior for different source design for electrospray, if any. Different mobile phase additives were compared, including acetic acid, formic acid, ammonium formate, and a range of primary amines. Continuous infusion was used for a fast screening, to detect optimal conditions. These were then further investigated in detail by LC-MS. The results indicate that electrospray is the more sensitive interface for this compound on the investigated apparatus. Unacceptable quantitative data were acquired without additives in the mobile phase. Generally, additives increased the reproducibility significantly. A response of mainly one ion was achieved with dodecylamine/acetic acid and acetic acid/sodium acetate. The data also point out the importance of evaluating adduct formation for compounds prone to this phenomenon during method development, especially in view of accurate quantitation

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