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Development and validation of a LC-MS/MS assay for quantitation of plasma citrulline for application to animal models of the acute radiation syndrome across multiple species

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Abstract

The potential risk of a radiological catastrophe highlights the need for identifying and validating potential biomarkers that accurately predict radiation-induced organ damage. A key target organ that is acutely sensitive to the effects of irradiation is the gastrointestinal (GI) tract, referred to as the GI acute radiation syndrome (GI-ARS). Recently, citrulline has been identified as a potential circulating biomarker for radiation-induced GI damage. Prior to biologically validating citrulline as a biomarker for radiation-induced GI injury, there is the important task of developing and validating a quantitation assay for citrulline detection within the radiation animal models used for biomarker validation. Herein, we describe the analytical development and validation of citrulline detection using a liquid chromatography tandem mass spectrometry assay that incorporates stable-label isotope internal standards. Analytical validation for specificity, linearity, lower limit of quantitation, accuracy, intra- and interday precision, extraction recovery, matrix effects, and stability was performed under sample collection and storage conditions according to the Guidance for Industry, Bioanalytical Methods Validation issued by the US Food and Drug Administration. In addition, the method was biologically validated using plasma from well-characterized mouse, minipig, and nonhuman primate GI-ARS models. The results demonstrated that circulating citrulline can be confidently quantified from plasma. Additionally, circulating citrulline displayed a time-dependent response for radiological doses covering GI-ARS across multiple species.

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Acknowledgments

This work was funded with Federal funds from the National Institute of Allergy and Infectious Diseases (contract no. HHSN272201000046C). This work is also supported in part by the University of Maryland Baltimore, School of Pharmacy Mass Spectrometry Center (SOP1841-IQB2014). The authors would like to thank all members of the Medical Countermeasures Against Radiological Threats (MCART) consortium for their dedication, support, and guidance in establishing biomarker identification and validation as a priority in the radiation medical counter measure field. Additionally, we would like to acknowledge and thank all members of the Kane laboratory.

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Authors and Affiliations

  1. Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, 20 N. Pine Street, Room 721N, Baltimore, MD, 21201, USA

    Jace W. Jones

  2. Epistem Ltd, 48 Grafton St, Manchester, M13 9XX, UK

    Gregory Tudor & Catherine Booth

  3. Department of Radiation Oncology, School of Medicine, University of Maryland, 655 W. Baltimore Street, Baltimore, MD, 21201, USA

    Alexander Bennett, Ann M. Farese & Thomas J. MacVittie

  4. Radiation Countermeasures Program, Armed Forces Radiobiology Research Institute, Uniformed Services University of the Health Sciences, 8901 Wisconsin Avenue, Building 42, Bethesda, MD, 20889-5603, USA

    Maria Moroni

  5. Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, 20 N. Pine Street, Room 723N, Baltimore, MD, 21201, USA

    Maureen A. Kane

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  1. Jace W. Jones
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  2. Gregory Tudor
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Correspondence to Maureen A. Kane.

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Jones, J.W., Tudor, G., Bennett, A. et al. Development and validation of a LC-MS/MS assay for quantitation of plasma citrulline for application to animal models of the acute radiation syndrome across multiple species. Anal Bioanal Chem 406, 4663–4675 (2014). https://doi.org/10.1007/s00216-014-7870-0

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