Evaluation of 25-OH vitamin D by high performance liquid chromatography: validation and comparison with electrochemiluminescence

Objectives

The aim of the present study was to develop a robust and easy to use high performance liquid chromatography (HPLC) to analyze 25(OH)D3 in human serum.

Background

Vitamin D is a fat-soluble steroid hormone precursor that is mainly produced in the skin by exposure to sunlight. It is also supplied in the diet and plays a pivotal role in calcium homeostasis and skeletal metabolism throughout life.

Methods

To assess its analytical performance, we used the RECIPE HPLC Complete Kit and an HPLC-UV instrument. Our HPLC results were compared with a validated electrochemiluminescence method.

Results

The method was linear for the lower limit of quantification from 3 ng/l up to at least 200 ng/l for 25(OH)D3, with the following equation for the regression line: y = 0.172 X + 2.45 (R² = 0.989). Intra-assay precision was determined by extracting and quantifying 10 serum replicates from one patient. The mean was 37.875 ng/ml, the standard deviation was 0.22, and the coefficient of variation was 0.58%. Comparisons of results demonstrated good agreement between HPLC and ECL methods (R² = 0.883).

Conclusion

The HPLC assay demonstrates excellent linearity, acceptable accuracy and precision, and good agreement with a validated ECL method. The simple sample preparation and ease of use make it practical for the routine clinical laboratory.

Keywords: 25-OH vitamin D, Validation, High-performance liquid chromatography, Electrochemiluminescence