Simultaneous Determination of Vitamin D Metabolites 25(OH)D and 1α,25(OH)D in Human Plasma Using Liquid Chromatography Tandem Mass Spectrometry

Overview

Journal J Mass Spectrom Adv Clin Lab

Specialty General Medicine

Date 2022 May 16

PMID 35572785

Authors

Shan Xu

Rui Ni

Lihong Lv

Rui Chen

Yao Chen

Fengjiao Huang

Zhiru Xu

Affiliations

Soon will be listed here.

Abstract

Background: Although measurement of 25(OH)D is a routine analytical method to determine plasma vitamin D status, 1α,25(OH)D is the biologically active form. Hence, simultaneous measurement of 25(OH)D and 1α,25(OH)D could provide better insight into vitamin D status and pharmacokinetics. However, 1α,25(OH)D 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)D and 1α,25(OH)D.

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)D 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)D and 1-60 ng/mL for 25(OH)D, respectively, with correlation coefficients of r > 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.

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