Abstract
ObjectivesThe 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.BackgroundVitamin 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.MethodsTo 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.ResultsThe 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).ConclusionThe 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.
Highlights
Vitamin D is a fat-soluble steroid hormone precursor that is mainly produced in the skin by exposure to sunlight
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)
Vitamin D, a fat-soluble steroid hormone produced in the skin by exposure to sunlight or supplied via dietary sources, plays a vital role in skeletal metabolism and calcium homeostasis throughout life (Prentice et al 2008)
Summary
Vitamin D is a fat-soluble steroid hormone precursor that is mainly produced in the skin by exposure to sunlight. It is supplied in the diet and plays a pivotal role in calcium homeostasis and skeletal metabolism throughout life. Vitamin D, a fat-soluble steroid hormone produced in the skin by exposure to sunlight or supplied via dietary sources, plays a vital role in skeletal metabolism and calcium homeostasis throughout life (Prentice et al 2008). Active 25(OH)D is released into the circulation and connected to vitamin D binding protein (DBP) (Ascherio et al 2010) The activity of this enzyme is regulated by numerous factors including parathyroid hormone levels and serum calcium and phosphorus (Holick 2007). Another form is ergocalciferol (vitamin D2), which is derived from plants and used as a supplement
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