Simultaneous determination of vitamin D metabolites 25(OH)D3 and 1α,25(OH)2D3 in human plasma using liquid chromatography tandem mass spectrometry

Background

Although measurement of 25(OH)D₃ is a routine analytical method to determine plasma vitamin D status, 1α,25(OH)₂D₃ is the biologically active form. Hence, simultaneous measurement of 25(OH)D₃ and 1α,25(OH)₂D₃ could provide better insight into vitamin D status and pharmacokinetics. However, 1α,25(OH)₂D₃ has a low plasma concentration, making its quantification challenging for most analytical techniques. Here, we demonstrate use of liquid chromatography tandem mass spectrometry (LC-MSMS) for the development of a simple and rapid method for the simultaneous quantification of 25(OH)D₃ and 1α,25(OH)₂D₃.

Methods

Samples were purified from 250 µL human plasma. Chromatography was performed on an analytical column, under gradient conditions using a mobile phase consisting of methanol-lithium acetate. The mass detector was operated in positive multiple reaction monitoring mode. The established method was validated according to the guidance issued by ICH and FDA. Furthermore, a clinical study was performed using this method to detect the plasma concentrations of 1α,25(OH)₂D₃ after oral administration of calcitriol.

Results and conclusion

The method was acceptably linear over the concentration ranges of 20–1200 pg/mL for 1α,25(OH)₂D₃ and 1–60 ng/mL for 25(OH)D₃, respectively, with correlation coefficients of r² > 0.993. Both the inter-assay and intra-assay precision was < 15%, and the analytical recoveries were within 100% ± 10%, with no significant matrix effect or carryover. Thereby, we, provide a facile method for the simultaneous detection of vitamin D metabolites in plasma.