The development and use of isotope dilution mass spectrometry methods for the quantification of target proteins in certified reference materials

Published

July 29, 2026

Author(s)

Gavin O'Connor, Christian Arsene, Claudia Swart, Bernd Guttler, Luise Luckau, Milena Quaglia, David Bunk, Karen Phinney, Liesbet Deprez, Guy Auclair, Qinde Liu, Liqing Wu, Vincent Delatour, Amandine Boeuf

Abstract

Originally developed for small molecules in the late 1990s and early 2000s, isotope dilution mass spectrometry (IDMS) also became the "go to" method of calibration for those wishing to characterize the mass fraction or amount of substance content of proteins in matrix based certified reference materials (CRMs). Whilst instrumental factors can impact on the accuracy of IDMS methods, the use of double IDMS, higher order approaches, sample/calibration blend bracketing and "exact matching" have done a lot to improve the measured isotope ratio precision and reduce the impact of mass bias on the accuracy of the results provided. This review shows the development of IDMS for the quantification of proteins from being a method only used for value assignment of primary calibrators using isotopically labelled peptides as internal standards to a method for quantification of proteins in complex biological matrices such as serum and blood using isotopically labelled recombinant intact proteins as internal standards. It will discuss the challenges and limitations of currently used organic IDMS peptide-based approaches regarding the definition of the measurands, equilibration of sample and internal standard, digestion efficiency, stability and suitability of the internal standards. This paper reviews the steps taken to assess the accuracy of the peptide- and protein-based IDMS methods used for the characterization of CRMs and the requirement to ensure the comparability in laboratory diagnostics down to the patient level. Furthermore, it will give an outlook on future challenges such as including structural and protein activity information in the results.

Citation

Analytical and Bioanalytical Chemistry

Pub Type

Journals

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https://doi.org/10.1007/s00216-026-06689-7

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Keywords

Isotope dilution mass spectrometry, internal standard, certified reference material, characterization, measurement comparability, traceability

Citation

O'Connor, G. , Arsene, C. , Swart, C. , Guttler, B. , Luckau, L. , Quaglia, M. , Bunk, D. , Phinney, K. , Deprez, L. , Auclair, G. , Liu, Q. , Wu, L. , Delatour, V. and Boeuf, A. (2026), The development and use of isotope dilution mass spectrometry methods for the quantification of target proteins in certified reference materials, Analytical and Bioanalytical Chemistry, [online], https://doi.org/10.1007/s00216-026-06689-7, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=962330 (Accessed September 1, 2026)

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