Background: Hemolysis can result in analyte suppression or enhancement and it can affect the extraction efficiency and analyte stability. Triskelion developed an LC-MS method to monitor hemolysis. The concept can be integrated into existing and new quantitative protein LC-MS methods and can be validated according to the most appropriate tier. Results/methodology: In this proof of concept study, the tryptic target LLVVYPWTQR was used to quantify hemoglobin. The peptide target has only few variations considering the most common (laboratory) animals and is thus nearly generic. It was shown that LC-MS is a suitable technique for the quantification of hemoglobin in hemolyzed samples and that the signals are not affected by lipemia. Conclusion: LC-MS exhibited the best performance to monitor hemolysis when the results were compared with UV-VIS and visual inspection, especially when samples were lipemic.
Keywords: LC–MS; biotherapeutics; hemoglobin; hemolysis; integrated monitoring; lipemia; method development; proteins.