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<p>Traceability in laboratory medicine requires use of well-characterized standards, yet accurate quantitation of intact biological macromolecules remains challenging. Peptide and protein materials are therefore most commonly first hydrolyzed to their constituent amino acids (AA), which are then quantitated by an isotope dilution (ID) approach employing AA standards. As such, the National Research Council Canada has produced two amino acid certified reference materials (CRMs) to facilitate peptide and protein quantitation traceable to the <i>Système international d’unités</i> (SI): ALEU-1 (certified for <span>l</span>-leucine purity) and APHE-1 (<span>l</span>-phenylalanine). Analyte mass fractions were determined via quantitative proton nuclear magnetic resonance (<sup>1</sup>H-qNMR) spectroscopy using SI-traceable internal standards. Structurally related impurities, i.e., alternative amino acids, were quantitated by an ID-standard addition approach via liquid chromatography–tandem mass spectrometry (LC-MS/MS), and relevant NMR signals were corrected accordingly. Additionally, small amounts of the <span>d</span>-enantiomer were detected in each CRM by chiral labeling followed by LC-MS/MS. Vial-to-vial homogeneity was established using qNMR measurements, while a combination of qNMR and LC with ultraviolet–visible spectrophotometric detection was used to evaluate transportation and long-term storage stabilities. The purity of <span>l</span>-leucine in ALEU-1 was 0.9935 ± 0.0054&#xa0;g/g (<i>k</i> = 2, 95&#xa0;% CI), and APHE-1 contained 0.9964 ± 0.0064&#xa0;g/g (<i>k</i> = 2, 95&#xa0;% CI) <span>l</span>-phenylalanine. The amino acid CRMs have been used successfully for peptide and protein quantitation in international comparison exercises.</p>

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Impurity-corrected 1H-qNMR value assignment of amino acid certified reference materials for SI-traceable protein quantitation

  • 期刊论文

  • 发表日期：

    2026年6月24日

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  • Marie-Pier Thibeault,
  • Jeremy E. Melanson,
  • Bradley B. Stocks

摘要

Traceability in laboratory medicine requires use of well-characterized standards, yet accurate quantitation of intact biological macromolecules remains challenging. Peptide and protein materials are therefore most commonly first hydrolyzed to their constituent amino acids (AA), which are then quantitated by an isotope dilution (ID) approach employing AA standards. As such, the National Research Council Canada has produced two amino acid certified reference materials (CRMs) to facilitate peptide and protein quantitation traceable to the Système international d’unités (SI): ALEU-1 (certified for l-leucine purity) and APHE-1 (l-phenylalanine). Analyte mass fractions were determined via quantitative proton nuclear magnetic resonance (1H-qNMR) spectroscopy using SI-traceable internal standards. Structurally related impurities, i.e., alternative amino acids, were quantitated by an ID-standard addition approach via liquid chromatography–tandem mass spectrometry (LC-MS/MS), and relevant NMR signals were corrected accordingly. Additionally, small amounts of the d-enantiomer were detected in each CRM by chiral labeling followed by LC-MS/MS. Vial-to-vial homogeneity was established using qNMR measurements, while a combination of qNMR and LC with ultraviolet–visible spectrophotometric detection was used to evaluate transportation and long-term storage stabilities. The purity of l-leucine in ALEU-1 was 0.9935 ± 0.0054 g/g (k = 2, 95 % CI), and APHE-1 contained 0.9964 ± 0.0064 g/g (k = 2, 95 % CI) l-phenylalanine. The amino acid CRMs have been used successfully for peptide and protein quantitation in international comparison exercises.