Quantification of 25-hydroxyvitamin D2 and D3 in Mitra® devices with volumetric absorptive microsampling technology (VAMS®) by UHPLC-HRMS for regular vitamin D status monitoring

Abstract

The numbers of vitamin D inadequacies has reportedly increased in the general population, especially in the Northern hemisphere. However, routine measurement of 25(OH) vitamin D is usually associated with a substantial effort due to the requirement of a venous blood sample taken by medical professionals. Thus, the objective of this work is to develop and validate an easy and minimal-invasive method, using a microsampling technique for autonomous blood collections by medically untrained individuals. The assay enables a simplified monitoring of the vitamin D-status in both, risk group and normal population throughout the year. For this purpose, a simple methanol extraction without derivatization combined with a UHPLC-HRMS method was developed to quantify 25(OH)D2 and 25(OH)D3 in capillary blood. For sample collection, a 20 μl Mitra® device with VAMS® technology is used. By employing the six-fold deuterium-labelled 25(OH)D3 as internal standard, the validated assay provides accurate (<10%) and precise (<11%) results. With a LOQ of 5 ng/ml, the approach also proved sensitive enough to adequately identify potential vitamin D deficiencies (< 12 ng/ml), and proof-of-concept analyses of authentic VAMS® samples (n = 20) yielded test results in the expected blood concentration range. Implementing VAMS® sampling for vitamin D-status monitoring enables a higher frequency due to a simplified, straightforward, and time-effective sample collection. VAMS® assures accurate sample volumes because of its absorptive capacities and, thus, area bias and homogeneity issues associated with conventional DBS are avoided. Regular monitoring of 25(OH)D status throughout the year supports people in high-risk groups for vitamin D-deficiency by early identifying inadequacies and, thus, preventing adverse health consequences.

Keywords: 25(OH)D; Mass spectrometry; NIST; Orbitrap; Validation.