Candidate Reference Measurement Procedure for the Determination of 24R,25-Dihydroxyvitamin D3 in Human Serum using Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry
Susan Tai, Michael A. Nelson
The two major forms of vitamin D, vitamin D3 and vitamin D2, are metabolized in the liver through hydroxylation to 25-hydroxyvitamin D species, and then further hydroxylated in the kidney to various dihydroxyvitamin D species. 24R,25-Dihydroxyvitamin D3 (24R,25(OH)2D3) is a major catabolite of 25-hydroxyvitamin D metabolism and is an important vitamin D metabolite used as a catabolism marker and indicator of kidney disease. NIST has recently developed and critically evaluated a reference measurement procedure for the determination of 24R,25(OH)2D3 in human serum using isotope-dilution LC-MS/MS. The 24R,25(OH)2D3 and its deuterated labeled internal standard 24R,25(OH)2D3-d6 were extracted from a serum matrix using liquid-liquid extraction prior to LC-MS/MS analysis. Chromatographic separation was performed using a fused-core C18 column. Atmosphere pressure chemical ionization (APCI) in the positive ion mode and multiple reaction monitoring (MRM) were used for LC-MS/MS. The accuracy of the measurement of 24R,25(OH)2D3 was evaluated by recovery studies of measuring 24R,25(OH)2D3 in gravimetrically prepared spiked samples of human serum with known 24R,25(OH)2D3 levels. The recoveries of the added 24R,25(OH)2D3 averaged 99.0 % ± 0.8 %. The absolute recoveries (extraction efficiencies) of this method averaged 95 % ± 2 %. Excellent repeatability with within-set coefficients of variations (CVs) of 0.2 % - 1.0 % and between-set CVs of 0.5 % - 1.1 % were demonstrated. The limit of quantitation at a signal-to-noise ratio of approximately 10 was 0.2 ng/g. Potential isomeric interferences from other endogenous species and from impurity components of the reference standard were investigated. LC baseline resolution of 24R,25(OH)2D3 from these isomers was achieved. This method was used for value assignment of 24R,25(OH)2D3 in SRMs of Vitamin D Metabolites in Human Serum, which can serve as an accuracy base for the routine methods used in clinical laboratories.
and Nelson, M.
Candidate Reference Measurement Procedure for the Determination of 24R,25-Dihydroxyvitamin D3 in Human Serum using Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry, Analytical Chemistry, [online], https://doi.org/10.1021/acs.analchem.5b01861
(Accessed December 8, 2023)