Clinical Diagnostics: Analysis of Vitamin D in Human Serum
Validated HPLC-MS/MS method for quantification of vitamin D metabolites in human serum for clinical diagnostics.
Clinical Diagnostics: Analysis of Vitamin D in Human Serum by HPLC Article ID: AN-005 Category: Application Notes Subcategory: Clinical Diagnostics Author: Manus AI Published Date: 2026-02-07 --- Introduction Vitamin D deficiency is a global health problem, linked to a range of conditions including osteoporosis, rickets, and other metabolic bone diseases. The clinical assessment of a patient's vitamin D status is typically performed by measuring the concentration of its major circulating metabolite, 25-hydroxyvitamin D [25(OH)D], in human serum. While immunoassays are widely used, High-Performance Liquid Chromatography (HPLC) is considered the gold standard method because it can separately quantify the two major forms, 25-hydroxyvitamin D₃ (from sun exposure and diet) and 25-hydroxyvitamin D₂ (from supplements). This application note details a reliable RP-HPLC method with UV detection for the quantification of 25-hydroxyvitamin D₃ [25(OH)D₃] in human serum. This method is suitable for clinical diagnostic laboratories in various regions, including the UAE and other parts of the Middle East, where sunshine is abundant but deficiency can still be prevalent. Experimental Conditions | Parameter | Condition | | :--- | :--- | | Instrument | Standard HPLC system with UV Detector | | Column | C18, 3.5 µm, 4.6 x 100 mm | | Mobile Phase | Methanol : Water (90:10, v/v) | | Flow Rate | 1.0 mL/min | | Column Temperature | 40 °C | | Detection Wavelength | 265 nm | | Injection Volume | 100 µL | Sample Preparation Serum is a complex matrix, requiring a multi-step sample preparation procedure to remove proteins and other interferences and to extract the fat-soluble vitamin D metabolites. 1. Protein Precipitation and Liquid-Liquid Extraction (LLE) 1. To 500 µL of human serum in a glass tu