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Structurally related peptide impurity identification and accurate quantification for synthetic oxytocin by liquid chromatography–high-resolution mass spectrometry

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Abstract

Oxytocin (OXT) is an important peptide that is mainly used as a therapeutic drug to induce labor or strengthen uterine contractions, or to control bleeding after childbirth. OXT has also been reported as a biomarker linked to emotion, and as a potential biomarker for cancer diagnosis. The accurate purity characterization of OXT calibrators is critical for quality control of pharmaceuticals and the development of reference measurement systems for this analyte in laboratory medicine. OXT possesses the particular analytical measurement challenge of a disulfide bond. Accurate value assignment of the purity of oxytocin calibrators can be carried out by applying the mass balance approach or alternative approaches such as amino acid analysis, quantitative nuclear magnetic resonance spectrometry, and nitrogen determination. In order to avoid biases, all these approaches require a correction for structurally related peptide impurities. Structurally related peptide impurities present in a synthetic OXT material have been identified and quantified by a newly developed and in-house-validated liquid chromatography–high-resolution mass spectrometry (LC-hrMS) method. This method was adopted for the measurement of the study material used for an international comparison evaluating the competencies of laboratories to perform peptide characterization. Eighteen structurally related impurities were identified, confirmed, and accurately quantified in the OXT study material by using LC-hrMS. The study material contained a total mass fraction of 31.1 mg/g structurally related OXT impurities with an associated expanded uncertainty of 1.7 mg/g.

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Abbreviations

CCQM:

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

CID:

Collision-induced dissociation

Csa:

Cysteinesulfinic acid

CRM:

Certified reference material

Cya:

Cysteic acid

DOPA:

Dihydroxyphenylalanine

EP:

European Pharmacopoeia

ESI:

Electrospray ionization

HCD:

High-energy collision dissociation

OXT:

Oxytocin

hrMS:

High-resolution mass spectrometry

LC:

Liquid chromatography

MS:

Mass spectrometry

NIBSC:

National Institute for Biological Standards and Control

PAWG:

Protein Analysis Working Group

PICAA:

Peptide impurity–corrected amino acid analysis

qNMR:

Quantitative nuclear magnetic resonance spectroscopy

USP:

United States Pharmacopeia

WHO:

World Health Organization

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Acknowledgments

The authors would like to thank the National Key R&D Program of China for funding support (No. 2017YFF0205403).

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Authors and Affiliations

  1. Département de la 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, Gustavo Martos & Robert I. Wielgosz

  2. Division of Chemical Metrology & Analytical Science, National Institute of Metrology, Beijing, 100029, 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. Gustavo Martos
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  7. Robert I. Wielgosz
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  8. Hongmei Li
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Corresponding author

Correspondence to Ralf D. Josephs.

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

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The authors declare that they have no conflict of interest.

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Li, M., Josephs, R.D., Daireaux, A. et al. Structurally related peptide impurity identification and accurate quantification for synthetic oxytocin by liquid chromatography–high-resolution mass spectrometry. Anal Bioanal Chem 413, 1861–1870 (2021). https://doi.org/10.1007/s00216-021-03154-5

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  • DOI: https://doi.org/10.1007/s00216-021-03154-5

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