Comparison of high performance liquid chromatography, radio immunoassay and electrochemiluminescence immunoassay for quantification of serum 25 hydroxy vitamin D

Clin Biochem. 2011 Jul;44(10-11):864-8. doi: 10.1016/j.clinbiochem.2011.04.020. Epub 2011 May 4.


Objective: To compare the performance of radioimmunoassay (RIA) with high-performance liquid chromatography (HPLC) and electrochemiluminescence (ECLIA) for the quantification of vitamin D (25OHD).

Methods: HPLC method for the determination of 25OHD in human biological samples was developed and compared in terms of accuracy and precision with a commercially available RIA assay. Performance of RIA assay with ECLIA technology for 25OHD analysis was further compared.

Results: Median 25OHD levels with HPLC vs. RIA were 50.1nmol/L (IQ=17.7-199.4nmol/L) and 51.1nmol/L (IQ=12.5-187.2nmol/L) respectively, whereas median 25OHD concentration with RIA vs. ECLIA was 32.4nmol/L (9.98-199.7nmol/L) and 29.9nmol/L (4.9-214.6nmol/L), respectively. Comparison data for HPLC vs. RIA showed RIA=-1.13+1.01 (HPLC) (RMSE=11.2nmol/L) and for RIA vs. ECLIA revealed, ECLIA=3.21+0.9 (RIA) (RMSE9.6nmol/L).

Conclusion: Acceptable correlation was observed among HPLC and RIA and also with RIA and ECLIA in quantification of 25OHD.

Publication types

  • Comparative Study

MeSH terms

  • Chromatography, High Pressure Liquid / methods*
  • Electrochemical Techniques / methods*
  • Humans
  • Luminescent Measurements / methods*
  • Radioimmunoassay / methods*
  • Vitamin D / analogs & derivatives*
  • Vitamin D / blood


  • Vitamin D
  • 25-hydroxyvitamin D