Analytical and Bioanalytical Chemistry

, Volume 407, Issue 11, pp 3137–3146 | Cite as

Development of C-reactive protein certified reference material NMIJ CRM 6201-b: optimization of a hydrolysis process to improve the accuracy of amino acid analysis

  • Megumi KatoEmail author
  • Tomoya Kinumi
  • Mariko Yoshioka
  • Mari Goto
  • Shin-ichiro Fujii
  • Akiko Takatsu
Research Paper
Part of the following topical collections:
  1. Reference Materials for Chemical Analysis


To standardize C-reactive protein (CRP) assays, the National Metrology Institute of Japan (NMIJ) has developed a C-reactive protein solution certified reference material, CRM 6201-b, which is intended for use as a primary reference material to enable the SI-traceable measurement of CRP. This study describes the development process of CRM 6201-b. As a candidate material of the CRM, recombinant human CRP solution was selected because of its higher purity and homogeneity than the purified material from human serum. Gel filtration chromatography was used to examine the homogeneity and stability of the present CRM. The total protein concentration of CRP in the present CRM was determined by amino acid analysis coupled to isotope-dilution mass spectrometry (IDMS-AAA). To improve the accuracy of IDMS-AAA, we optimized the hydrolysis process by examining the effect of parameters such as the volume of protein samples taken for hydrolysis, the procedure of sample preparation prior to the hydrolysis, hydrolysis temperature, and hydrolysis time. Under optimized conditions, we conducted two independent approaches in which the following independent hydrolysis and liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) were combined: one was vapor-phase acid hydrolysis (130 °C, 24 h) and hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) method, and the other was microwave-assisted liquid-phase acid hydrolysis (150 °C, 3 h) and pre-column derivatization liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The quantitative values of the two different amino acid analyses were in agreement within their uncertainties. The certified value was the weighted mean of the results of the two methods. Uncertainties from the value-assignment method, between-method variance, homogeneity, long-term stability, and short-term stability were taken into account in evaluating the uncertainty for a certified value. The certified value and the expanded uncertainty (k = 2) of CRM 6201-b are (40.0 ± 1.6) μmol kg–1.


C-reactive protein Certified reference material Amino acid analysis Isotope dilution mass spectrometry Hydrolysis optimization 

Supplementary material

216_2014_8190_MOESM1_ESM.pdf (185 kb)
ESM 1 (PDF 184 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Megumi Kato
    • 1
    Email author
  • Tomoya Kinumi
    • 1
  • Mariko Yoshioka
    • 1
  • Mari Goto
    • 1
  • Shin-ichiro Fujii
    • 1
  • Akiko Takatsu
    • 1
  1. 1.Organic Analytical Chemistry Division, Bio-Medical Standards SectionNational Metrology Institute of Japan, National Institute of Advanced Industrial Science and TechnologyTsukubaJapan

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