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

Lena Jafri, Aga Khan University
Aysha Habib Khan, Aga Khan University
Anwar Ali Siddiqui
Shamim Mushtaq
Romaina Iqbal, Aga Khan University
Farooq Ghani, Aga Khan University
Imran Siddiqui, Aga Khan University


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


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.


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


Acceptable correlation was observed among HPLC and RIA and also with RIA and ECLIA in quantification of 25OHD. (C) 2011 The Canadian Society of Clinical Chemists.