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Identification and accurate quantification of structurally related peptide impurities in synthetic human C-peptide by liquid chromatography–high resolution mass spectrometry

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Abstract

Peptides are an increasingly important group of biomarkers and pharmaceuticals. The accurate purity characterization of peptide calibrators is critical for the development of reference measurement systems for laboratory medicine and quality control of pharmaceuticals. The peptides used for these purposes are increasingly produced through peptide synthesis. Various approaches (for example mass balance, amino acid analysis, qNMR, and nitrogen determination) can be applied to accurately value assign the purity of peptide calibrators. However, all purity assessment approaches require a correction for structurally related peptide impurities in order to avoid biases. Liquid chromatography coupled to high resolution mass spectrometry (LC-hrMS) has become the key technique for the identification and accurate quantification of structurally related peptide impurities in intact peptide calibrator materials. In this study, LC-hrMS-based methods were developed and validated in-house for the identification and quantification of structurally related peptide impurities in a synthetic human C-peptide (hCP) material, which served as a study material for an international comparison looking at the competencies of laboratories to perform peptide purity mass fraction assignments. More than 65 impurities were identified, confirmed, and accurately quantified by using LC-hrMS. The total mass fraction of all structurally related peptide impurities in the hCP study material was estimated to be 83.3 mg/g with an associated expanded uncertainty of 3.0 mg/g (k = 2). The calibration hierarchy concept used for the quantification of individual impurities is described in detail.

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Abbreviations

CCQM:

Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology

CID:

Collision induced dissociation

ESI:

Electrospray ionization

HCD:

High energy collision dissociation

hCP:

Human C-peptide

hrMS:

High resolution mass spectrometry

IDMS:

Isotope dilution tandem mass spectrometry

LC:

Liquid chromatography

MS:

Mass spectrometry

PAWG:

Protein Analysis Working Group

PICAA:

Peptide impurity corrected amino acid analysis

qNMR:

Quantitative nuclear magnetic resonance spectrometry

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Funding

The authors would like to thank the Ministry of Science and Technology of China for funding support (No. 2014DFA31810).

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Authors and Affiliations

  1. Département de Chimie, Bureau International des Poids et Mesures (BIPM), Pavillon de Breteuil, 92312, Sèvres Cedex, France

    Ming Li, Ralf D. Josephs, Adeline Daireaux, Tiphaine Choteau, Steven Westwood & Robert I. Wielgosz

  2. Division of Chemical Metrology & Analytical Science, National Institute of Metrology, Beijing, 100029, People’s Republic of China

    Ming Li & Hongmei Li

Authors
  1. Ming Li
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  2. Ralf D. Josephs
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  3. Adeline Daireaux
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  4. Tiphaine Choteau
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  5. Steven Westwood
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  6. Robert I. Wielgosz
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  7. Hongmei Li
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Corresponding author

Correspondence to Ralf D. Josephs.

Ethics declarations

The experiments were conducted with synthetic hCP materials provided by the National Institute of Metrology of China commercially sourced from GenScript (China).

Conflict of interest

The authors declare that they have no conflict of interest.

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Li, M., Josephs, R.D., Daireaux, A. et al. Identification and accurate quantification of structurally related peptide impurities in synthetic human C-peptide by liquid chromatography–high resolution mass spectrometry. Anal Bioanal Chem 410, 5059–5070 (2018). https://doi.org/10.1007/s00216-018-1155-y

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  • DOI: https://doi.org/10.1007/s00216-018-1155-y

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