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

Shan Xu; Rui Ni; Lihong Lv; Rui Chen; Yao Chen; Fengjiao Huang; Zhiru Xu

doi:10.1016/j.jmsacl.2022.04.001

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

J Mass Spectrom Adv Clin Lab. 2022 Apr 23:24:65-79. doi: 10.1016/j.jmsacl.2022.04.001. eCollection 2022 Apr.

Authors

Shan Xu¹, Rui Ni², Lihong Lv³, Rui Chen³, Yao Chen³, Fengjiao Huang⁴, Zhiru Xu³

Affiliations

¹ The Affiliated Cancer Hospital of Zhengzhou University & Henan Cancer Hospital, Zhengzhou 450008, China.
² National Pharmaceutical Engineering Research Center, 200437 Shanghai, China.
³ State Key Lab. of New Drug and Pharmaceutical Process/Shanghai Professional and Technical Service Center for Biological Material Druggability Evaluation, Shanghai Institute of Pharmaceutical Industry, 200437 Shanghai, China.
⁴ Division of Endocrinology, Department of Internal Medicine, The First Affiliated Hospital of Zhengzhou University, 450052 Zhengzhou, China.

Abstract

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.

Keywords: 1α; 1α,25(OH)2D3, 1α,25-dihydroxy vitamin D3; 25(OH)2D3; 25(OH)D3; 25(OH)D3, 25-hydroxyvitamin D3; BSA, bovine serum albumin; ESI, electrospray ionization; FDA, Food and Drug Administration; ICH, International Council on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use; IS, internal standard; LC-MS/MS; LC-MS/MS, liquid chromatography tandem mass spectrometry; LLE, liquid liquid extraction; MRM, multiple reaction monitoring; PPT, protein precipitation; Pharmacokinetics; Plasma; SPE, solid phase extraction; VD, vitamin D; m/z, mass-to-charge ratios.