Survey of peptide quantification methods and comparison of their reproducibility: A case study using oxytocin

Abstract USP’s peptide reference standards content is typically determined using an HPLC assay against an external standard for which the purity was determined by a mass balance approach. To explore the use of other analytical methods, the USP Biologics Department conducted a multi-laboratory collaborative study. The study determined the inter-laboratory variability for peptide quantitation using the following methods: HPLC assay, quantitative nuclear magnetic resonance (qNMR) spectroscopy, or amino acid analysis (AAA). The three methods were compared with regard to their suitability for quantitation of the nonapeptide oxytocin. In this study, the HPLC assay method using the same peptide bulk material as the standard showed the lowest inter-lab variability. The coefficient of variation (%CV) was calculated without counting the uncertainty associated with the purity assignment of the standard with mass balance. The proton qNMR method is a direct measurement of the peptide against an internal standard, which is not difficult to perform under common laboratory conditions. Because of the simpler operation and shorter analytical time, qNMR as a primary method for peptide reference standard value assignment deserves further exploration.