Determination of 25-hydroxyvitamin D in serum by HPLC and immunoassay.

Vitamin D status is usually assessed by measuring the serum concentration of 25-hydroxyvitamin D [25(OH)D]. Its measurement is important as a clinical indicator of nutritional vitamin D deficiency, which is one of the causes of osteoporosis (1). Vitamin D exists in two forms: cholecalciferol (vitamin D 3) and ergocalciferol (vitamin D 2). Vitamin D 2 is further metabolized to 25(OH)D 2. Vitamin D 3 is formed in the skin from its precursor 7-dehydrocholesterol after ultraviolet irradiation or is absorbed from the diet (2). It is further hydroxylated in the liver to 25(OH)D 3 as the first step of its conversion in the kidney to 1,25-dihydroxyvitamin D 3 , which is the biologically active form. 25(OH)D 3 is the main circulating form of vitamin D. Clinically it is important to measure both forms of 25-hydroxyvitamin D to monitor the effect of vitamin D 2 supplementation on total vitamin D status. The first routine methods for measurement of 25(OH)D concentrations in human plasma were based on competitive protein binding and used vitamin D-binding protein and a tritium-labeled tracer (3). These methods were replaced by a simpler, rapid RIA (4), and a radioiodinated tracer was incorporated into the RIA in 1993 (5). This assay principle is the basis of several commercially available methods. Quantitative HPLC assays have been developed based on ultraviolet detection and normal-phase separation (6), combined use of normal-and reversed-phase separations (7), or reversed-phase separation alone (8). Recently, reversed-phase HPLC methods for 25(OH)D 3 in human plasma have been developed with normal-phase prepurifi-cation of the sample (9) or liquid extraction only (10). Earlier HPLC methods for 25(OH)D 3 in serum were designed mainly for research purposes and were therefore too complicated for routine use. The present method was designed to be easy to use, sensitive, and rapid with simple sample preparation. Separation and quantification of 25(OH)D 3 from 25(OH)D 2 are achieved with an iso-cratic elution. To 0.5 mL of serum, we added 350 ␮L of methanol–2-propanol (80:20 by volume). The tubes were mixed in a Multitube vortex mixer for 30 s. 25(OH)D was extracted by mixing three times (60 s each time) with 2 mL of hexane. The phases were separated by centrifugation, and the upper organic phase was transferred to a conical tube and dried under nitrogen. The residue was dissolved in 100 ␮L of mobile phase. Calibration curves were constructed using four concentrations of 25(OH)D 3 (15–120 nmol/L; …