, Volume 77, Issue 7–8, pp 581–589 | Cite as

A Stability-Indicating HPLC Method for the Determination of Nitrosylcobalamin (NO-Cbl), a Novel Vitamin B12 Analog

  • Michael J. Dunphy
  • Annette M. Sysel
  • Joseph A. Lupica
  • Kristie Griffith
  • Taylor Sherrod
  • Joseph A. Bauer


Nitrosylcobalamin (NO-Cbl), a novel vitamin B12 analog and anti-tumor agent, functions as a biologic ‘Trojan horse’, utilizing the vitamin B12 transcobalamin II transport protein and cell surface receptor to specifically target cancer cells. A stability-indicating HPLC method was developed for the detection of NO-Cbl during forced degradation studies. This method utilized an Ascentis® RP-Amide (150 mm × 4.6 mm, 5 μm) column at 35 °C with a mobile phase (1.0 mL min−1) combining a gradient of methanol and an acetate buffer at pH 6.0. Detection wavelengths of 450 and 254 nm were used to detect corrin and non-corrin-based products, respectively. NO-Cbl, synthesized from hydroxocobalamin and pure nitric oxide gas, was subjected to degradative stress conditions including oxidation, hydrolysis and thermal and radiant energy challenge. The method was validated by assessing linearity, accuracy, precision, detection and quantitation limits and robustness. The method was applied successfully for purity assessment of synthesized NO-Cbl and for the determination of NO-Cbl during kinetic studies in aqueous solution and in solid-state degradation assessments. This HPLC method is suitable for the separation of cobalamins in aqueous and methanolic solutions, for routine detection of NO-Cbl and for purity assessment of synthesized NO-Cbl. Additionally, this method has potential application in identification and monitoring of diseases involving altered nitric oxide homeostasis where vitamin B12 therapy is utilized to scavenge excess nitric oxide, subsequently resulting in the in vivo production of NO-Cbl.


Column liquid chromatography Stability-indicating method Nitrosylcobalamin Vitamin B12 Nitric oxide 



This work was funded in part by federal funds from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services, under SBIR contract number HHSN261201000107C to BNOAT Oncology, Inc.


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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Michael J. Dunphy
    • 1
    • 2
  • Annette M. Sysel
    • 1
  • Joseph A. Lupica
    • 1
    • 2
  • Kristie Griffith
    • 2
  • Taylor Sherrod
    • 2
  • Joseph A. Bauer
    • 1
  1. 1.BNOAT Oncology, Inc.AkronUSA
  2. 2.Walsh UniversityNorth CantonUSA

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