LC-MS/MS-based determination of 25-hydroxyvitamin D in dried blood spots: crucial importance of combining a robust extraction with knowledge of the hematocrit

Background

While many liquid chromatography – tandem mass spectrometry (LC-MS/MS)-based methods exist for the determination of 25-hydroxyvitamin D (25OHD) using dried blood spots (DBS), none of these comprehensively evaluated the robustness of the employed extraction method. The latter is critical as the hematocrit (Hct) as well as DBS ageing may affect analyte recovery and, thus, the accuracy of the obtained DBS-based 25OHD result. Moreover, as the application potential of DBS for the determination of 25OHD mainly lies in the analysis of DBS collected and archived in the context of newborn screening or epidemiological studies, a large variation in Hct and ageing is to be expected.

Results

A Hct- and ageing-independent extraction of 25OHD from a single 6 mm DBS subpunch was obtained in the Hct range of 0.23 to 0.53 L/L, using thermoshaking in 50/50 acetonitrile/water for 1 h at 60 °C. The optimized LC-MS/MS-based method allowed fast (<2 min) separation of 3-epi-25OHD3 from 25OHD3 using an inverse gradient without the need for a dedicated column. The method was successfully validated for the determination of 25OHD3 and 25OHD2 in DBS, whole blood and plasma, with lower limits of quantification of 1.97 and 2.53 ng/mL in DBS and whole blood, and 3.94 and 5.06 ng/mL in plasma for 25OHD3 and 25OHD2, respectively. For both 25OHD3 and 25OHD2, accuracy and imprecision were respectively within -14.7% to 2.3% and within 1.1% to 10.0% for all matrices. Stability studies (up until three months of storage at room temperature) as well as the evaluation of spotted blood volume did not reveal any relevant impact on the quantification of 25OHD in DBS. Finally, a proof-of-concept study indicated that, when the Hct is taken into account, comparable results can be obtained in DBS and whole blood samples and plasma concentrations can be derived from DBS, respectively.

Significance and novelty

The presented manuscript is the first to address in-depth one of the major remaining research gaps in the determination of 25OHD from DBS, namely the evaluation of the robustness of the employed extraction procedure. Using thermoshaking at elevated temperature, we demonstrated Hct- and ageing-independent recovery of 25OHD3 and 25OHD2, confirming the method’s suitability for large-scale applications such as newborn screening and epidemiological studies.