Background: The detection of 25-hydroxyvitamin D (25OHD) from dried blood spots (DBS) has been widely studied. However, the existing pretreatment methods suffer from limitations in terms of throughput (usually exceeding 2 h), complexity (involving liquid-liquid extraction or solid-phase extraction), and contamination (including multiple steps of organic solvent evaporation).
Results: We first released 25OHD from DBS samples by 50% acetonitrile solution through ultrasonication. Subsequently, the cold-induced phase separation technique was introduced for in-situ concentration and purification. Afterward, the PTAD derivatization of 25OHD was performed directly, profiting from the high acetonitrile content in the concentrated solution. In the end, the resulting solution was submitted to LC-MS/MS for quantification. The established LC-MS/MS methodology possessed favorable analytical performance, possessing lower limit of quantification of 1 ng/mL pointing to plasma, accuracy of 86.8-110.1% and imprecision of 5.4-16.8%. Method comparison with plasma samples demonstrated that over 93% of the detections met the acceptance limit for cross-validation of ±20%.
Significance and novelty: The novel sample preparation can be finished within 15 min and eliminated the traditional steps of extraction and organic solvent evaporation. Based on this high-throughput, reliable and applicable LC-MS/MS method, the detection of 25OHD in DBS samples can be better achieved for clinical patients and researchers with relevant demands.
Keywords: 25-Hydroxyvitamin D2; 25-Hydroxyvitamin D3; Cold-induced phase separation (CIPS); Dried blood spot (DBS); LC-MS/MS.
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