Comparison of high performance liquid Chromatography, Radioimmunoassay and electrochemiluminescence Immunoassay for quantification of serum 25 hydroxy vitamin D

Document Type



Pathology and Microbiology


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 50.1 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).

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


Clinical Biochemistry