Development of a sensitive HPLC-MS/MS method for 25-hydroxyvitamin D2 and D3 measurement in capillary blood

Abstract

Background: Measurement of 25-hydroxyvitamin D [25(OH)D)] levels is important. The current method requires a relatively large volume of serum. To minimize the amount of serum needed, we established a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method to measure 25(OH)D in capillary serum. Methods: Venous blood and fingertip blood were collected from 90 participants. Volumes of 100 µL of venous serum and 20 µL of capillary serum were collected. The serum samples were pretreated by protein removal, extraction and concentration, and an HPLC-MS/MS method based on chromatographic separation and multi reactive ion monitoring was conducted. The intra- and inter-batch variation coefficients were less than 10% for both 25-hydroxyvitamin D3[25(OH)D3] and 25-hydroxyvitamin D2[25(OH)D2)]. For venous specimens, the accuracies were 3.87% and 4.91%, respectively. For capillary specimens, the accuracies were 1.65% and 5.32%, respectively. Results: The limit of detection (LOD) of 25(OH)D3 was 0.01 ng/mL, and the LOD of 25(OH)D2 was 0.05 ng/mL. The results showed that the mean concentration of 25(OH)D in venous blood was 22.56 ± 9.50 ng/mL, while the mean concentration of 25(OH)D in capillary blood was 18.14 ± 7.86 ng/mL. Furthermore, the adjusted capillary blood 25(OH)D level was 22.99 ± 10.24 ng/mL by the correction formula in our study. Similarly, the mean concentration of 25(OH)D3 in capillary blood was 17.98 ± 7.98 ng/mL. The adjusted capillary blood 25(OH)D3 level was 22.85 ± 10.42 ng/mL. No difference in the content of 25(OH)D or 25(OH)D3 was found between venous serum and corrected capillary serum. The correlation coefficients between venous and corrected capillary concentrations of 25(OH)D and 25(OH)D3 were 0.7941 and 0.8103, respectively, and the areas under the receiver operating characteristic curve were 0.9367 and 0.9565, respectively. Conclusions: This capillary blood method requires minimal sample preparation and is suitable for routine use in the 25(OH)D detection.