Accreditation and Quality Assurance

, Volume 21, Issue 1, pp 57–63 | Cite as

Instability of the DNPH-formaldehyde derivative in the certified reference material BCR-551 stored at −70 °C but not at −20 °C

  • A. Perez
  • G. Vanermen
  • B. Noten
  • T. P. J. LinsingerEmail author
Practitioner's Report


During the post-certification stability monitoring of the certified reference material (CRM) BCR-551 (DNPH derivatives dissolved in acetonitrile), a decreased concentration of one of the analytes of this CRM, the DNPH-formaldehyde derivative, was detected in reference samples (stored at −70 °C), while the concentration of normal “on-sale” samples (stored at −20 °C) remained stable. This behaviour is contrary to the expectation of better stability at lower temperatures. Apparently, the DNPH-formaldehyde derivative reacts with dinitrophenylhydrazine (DNPH) remaining from the synthesis phase to produce two new substances. These substances have been identified as C13H12N8O8 (substance 1) and C20H16N12O12 (substance 2) which, based on their structure, are suggested to be produced consecutively: DNPH + DNPH-formaldehyde derivative → substance 1 and substance 1 + DNPH-formaldehyde derivative → substance 2. Since acetonitrile freezes at −45 °C, reference samples are frozen at −70 °C, while normal samples are still liquid at −20 °C. We believe that this leads to a cryo-concentration of the solutes above the eutectic point and thus to an increased reaction rate in the reference samples. This case demonstrates that care should be taken when extrapolating stability results towards conditions that never have been tested, especially if phase transitions are involved, even at temperature as low as −70 °C. Furthermore, a slower degradation rate at lower temperatures can be overcompensated by a higher concentration due to cryo-concentration above the eutectic temperature.


Cryo-concentration Reference materials BCR-551 DNPH-formaldehyde derivative DNPH derivative Reference stock 

Supplementary material

769_2015_1185_MOESM1_ESM.docx (677 kb)
Supplementary material 1 (DOCX 676 kb)


  1. 1.
    ISO Guide 34 (2009) General requirements for the competence of reference material producers. International Organization for Standardization, GenevaGoogle Scholar
  2. 2.
    Linsinger TPJ, Gawlik BM, Trapmann S, Lamberty A, Emons H (2004) Preservation of sensitive CRM’s and monitoring their stability at JRC-IRMM. Anal Bioanal Chem 378:1168–1174CrossRefGoogle Scholar
  3. 3.
    Levin JO, Lindahl R, Heeremans CEM, Gawlik BM, Lamberty A (2003) The certification of reference materials related to the monitoring of aldehydes in air by derivatization with 2,4-dinitrophenylhydrazine and subsequent determination with HPLC-UV. BCR-546, BCR-547, BCR-548, BCR-549, BCR-550, BCR-551, BCR-552, BCR-553, BCR-554. European CommissionGoogle Scholar
  4. 4.
    Linsinger TPJ, Van Der Veen AMH, Gawlik BM, Pauwels J, Lamberty A (2004) Planning and combining of isochronous stability studies of CRMs. Accred Qual Assur 8(9):464–472CrossRefGoogle Scholar
  5. 5.
    Braun A, Franks K, Kestens V, Roebbens G, Lamberty A, Linsinger T (2011) Certification of equivalent spherical diameters of silica nanoparticles in water. Certified reference material ERM-FD100. European CommissionGoogle Scholar
  6. 6.
    Franks K, Braun A, Charoud-Got J, Couteau O, Kestens V, Lamberty A, Linsinger T, Roebben G (2013) Certification of the equivalent spherical diameters of silica nanoparticles in water. Certified reference material ERM-FD304. European CommissionGoogle Scholar
  7. 7.
    European Commission (2006) Preparation and certification of a reference material of haemoglobincyanide for the standardization of blood haemoglobin measurements. CRM-522. European CommissionGoogle Scholar
  8. 8.
    Zweens J, Frankera H, Zijlstra WG (1979) Decomposition on freezing of reagents used in the ICSH-recommended method for the determination of total haemoglobin in blood; its nature, cause and prevention. Clin Chim Acta 91:337–352CrossRefGoogle Scholar
  9. 9.
    Klein A (1966) Physiochemical requirements regarding reacting solutions for the determination of haemoglobin as haemoglobincyanide. Bibl Haematol 24:150–156Google Scholar
  10. 10.

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • A. Perez
    • 1
  • G. Vanermen
    • 2
  • B. Noten
    • 2
  • T. P. J. Linsinger
    • 1
    Email author
  1. 1.European Commission (EC), Joint Research CentreInstitute for Reference Material and Measurements (JRC-IRMM)GeelBelgium
  2. 2.Vlaamse Instelling voor Technologisch Onderzoek (VITO)MolBelgium

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