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Primary calibrators for peptides

The provision of Primary Calibration Reference Services in the emerging area of health markers such as peptides/proteins has been identified as a core technical competency for National Measurement Institutes (NMIs) [1]. Primary Calibration Reference Services refers to a technical capability for composition assignment, usually as the mass fraction content, of pure substances or solutions thereof [2]. The CCQM strategy for 2013-2023 foresees a limited number of key comparisons to enable NMIs providing measurement services in peptide/protein analysis to test and demonstrate their capabilities in this area.

The quantification of the primary structure purity of a larger molecule is the first step in establishing a primary calibrator material, where the quantity of interest is the mass fraction of the large molecule.

The purity of peptides can be assessed by use of the mass balance approach. However, the mass balance approach could require unviably large quantities of peptide material. Peptide impurity corrected amino acid (PICAA) analysis is a simpler alternative. It requires quantification of constituent amino acids (AAs) following hydrolysis of the material and correction for AAs originating from impurities. Traceability of the AA analysis results is to pure AA certified reference materials (CRMs). NMI capabilities to determine the purity of the amino acid valine, were recently assessed in the CCQM-K55.c key comparison. In addition, AA analysis and peptide hydrolysis capabilities for the mass concentration assignment of peptide solutions are evaluated in the series of CCQM-P55 comparisons. The application of other approaches for the assessment of peptide purity that require only minor quantities of peptide material is conceivable, for example elemental analysis (CHN/O) with a correction for nitrogen originating from impurities or quantitative nuclear magnetic resonance (qNMR) with correction for peptide impurities.


BIPM method development studies for peptide purity comparisons

The BIPM has developed and cross-validated different approaches for the purity value assignment of peptide primary calibrators, for angiotensin I (hypertension biomarker) in collaboration with the National Institute of Standards and Technology (NIST) and for the more complex human insulin (diabetes biomarker). Good agreement of results was found for the application of the full mass balance, PICAA, CHN/O and qNMR approaches for both model compounds. Liquid chromatography coupled to (high-resolution) mass spectrometry was found to be a key analytical technique for the characterization of impurities. The study results are currently in the stage of publication [3, 4]. In addition, the BIPM has value-assigned five AA materials (F, I, L, P and V) for purity by use of the mass balance approach in order to achieve traceable results for the application of the PICAA approach based on LC IDMS.

Primary sequence of angiotensin I: DRVYIHPFHL

  1. Marriott J., O'Connor G., Parkes H., Final Report - Study of Measurement Service and Comparison Needs for an International Measurement Infrastructure for the Biosciences and Biotechnology: Input for the BIPM Work Programme, Rapport BIPM-2011/02, 2011

  2. Westwood S., Choteau T., Daireaux A., Josephs R.D., Wielgosz R.I., Mass balance method for the SI value assignment of the purity of organic compounds, Anal. Chem., 2013, 85, 3118-3126

  3. Stoppacher N., Josephs R.D., Daireaux A., Westwood S., Wielgosz R.I., Impurity identification and determination for the peptide hormone angiotensin I by liquid chromatography - high resolution tandem mass spectrometry and the metrological impact on value assignments by amino acid analysis, Anal. Bioanal. Chem., 2013, 405, 8039-8051

  4. Stoppacher N., Josephs R.D., Daireaux A., Choteau T., Westwood S.W., Wielgosz R.I., Accurate quantification of impurities in pure peptide material – angiotensin I: Comparison of calibration requirements and method performance characteristics of liquid chromatography coupled to hybrid tandem mass spectrometry and linear ion trap-high resolution mass spectrometry, Rapid Comm. Mass Spectrom., 2015, 29, 1651-1660