Comparación entre las técnicas HPLC y ELISA para la detección de vitamina D en suero sanguíneo

Abstract

The present study aims to compare the detection of Vitamin D in human serum using highperformance liquid chromatography (HPLC) and enzyme-linked immunosorbent assays (ELISA). For this purpose, serum was isolated from four volunteers, and the levels of Vitamin D were quantified using HPLC to identify the metabolites 25-(OH) D2 and 25(OH) D3, and a commercial kit was used to quantify the measurable metabolites of Vitamin D through ELISA. The work was structured based on the stated objectives, including the purification of the serum, the development of a control serum called "zero serum" for HPLC analysis, and the measurement of Vitamin D using the two mentioned methods: ELISA assay using the commercial kit Accu Bind ELISA Microwells, and HPLC along with an analysis of linearity, detection limit, quantification limit, precision, and accuracy to ensure reliable results. Six repetitions were performed for each sample. The average results obtained by HPLC for 25(OH) D3 were 31.22 ng/mL, 32.92 ng/mL, and 31.52 ng/mL for samples M1, M2, and M4, respectively, which were considered optimal. However, sample M3 showed a value of 22.9 ng/mL, indicating a deficiency of Vitamin D. On the other hand, the average results from the ELISA assay were 30.67 ng/mL, 32.67 ng/mL, and 29.83 ng/mL for M1, M2, and M4, respectively, while sample M3 had a concentration of 19.50 ng/mL, coinciding with the parameters related to HPLC. Finally, a MannWhitney U test was performed to determine if there were differences between the results of the two techniques. The results showed that HPLC had different outcomes compared to the ELISA assay in determining Vitamin D levels in human serum.