SPE and LC-MS/MS Method for the Determination of 25-Hydroxyvitamin D2 and 25-Hydroxyvitamin D3 from Human Plasma

A liquid chromatography tandem mass spectrometry method for 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 from human plasma has been developed. Sample preparation was fast and efficient using a Thermo ScientificTM SOLATM hydrophobic reversed phase (HRP) plate. Analysis was carried out using a Thermo ScientificTM SyncronisTM C18 1.7 μm, 50 × 2.1 mm column to give a fast separation with a cycle time of 2 minutes while maintaining excellent peak shape. The reproducibility of the method was measured at three concentrations for each compound and was less than 4.2% (n=6 at each level). Excellent recoveries of 94.4% (25-hydroxyvitamin D2) and 96.3% (25-hydroxyvitamin D3) were also achieved. The dynamic range was linear between 5 and 1000 ng/mL with r2 values of 0.9994 and 0.9958 for 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, respectively. method for assessing a subjects total vitamin D status due to the higher circulating concentrations (nanogram levels) and greater serum half-life. Suggested optimum concentration ranges within the body for 25-OH-D differs between organizations. The Endocrine Society states that a sufficient range is 30–100 ng/mL and a deficient range is 0–20 ng/mL [1]; whereas, the US Food and Nutrition board states that >20 ng/mL is sufficient and 0−11 ng/mL is classified as vitamin D deficient [2]. The dynamic range of this assay, 5 to 1000 ng/mL, allows quantification of 25-OH-D levels within a sample to show the vitamin D status of the subject.