Peptide mapping is a critical tool in biopharmaceutical analysis for the characterization of protein therapeutics. These separations can be challenging due to the presence of peptides with a wide range of chemical properties, including acidic and phosphorylated peptides. When performing peptide mapping analysis via high-performance liquid chromatography (HPLC), phosphorylated compounds can exhibit non-specific adsorption to the metal surfaces of the instrument. The negatively charged phosphate groups can adsorb to the positively charged metal surfaces due to Lewis acid-base interactions, which then reduces recovery and hinders peak shape for these analytes. Common strategies to mitigate these interactions include the use of mobile phase additives, chemical passivation, or sample priming. However, these solutions can be time consuming and unreliable. For this reason, a variety of technologies have been developed to reduce non-specific adsorption.

 In this study, samples containing a wide range of peptides, including those that exhibit non-specific adsorption, are examined across legacy stainless steel HPLC systems and compared to modern biocompatible and bioinert HPLC systems. The analysis was performed using typical reversed phase method conditions and trifluoroacetic acid as an ion-pairing agent in the mobile phase. The performance of the systems was evaluated with comparisons based on chromatographic criteria, including area recovery, repeatability, sensitivity, and peak asymmetry/tailing.

 Recent developments in HPLC design allow for improvements in analysis of metal sensitive compounds while maintaining performance for all analytes. In this study, the analysis of biomolecules using a system designed to reduce non-specific adsorption allows for improved sensitivity and reproducibility of phosphorylated peptides as compared to traditional HPLC systems.