Candidate Reference Measurement Procedure for the Determination of (24R),25-Dihydroxyvitamin D3 in Human Serum Using Isotope-Dilution Liquid Chromatography–Tandem Mass Spectrometry

• Published in: Analytical chemistry (Washington) 2015-08, Vol.87 (15), p.7964-7970
• Main Authors: Tai, Susan S.-C, Nelson, Michael A
• Format: Article
• Language: English
• Subjects: 24,25-Dihydroxyvitamin D 3 - blood; Accuracy; Analytical chemistry; Blood Chemical Analysis - instrumentation; Blood Chemical Analysis - methods; Chromatography; Chromatography, Liquid; Human; Humans; Isotopes; Kidney diseases; Liquid-liquid extraction; Mass spectrometry; Metabolites; Reference Standards; Reproducibility of Results; Serums; Tandem Mass Spectrometry; Vitamin D; Vitamins
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.5b01861

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. The National Institute of Standards and Technology has recently developed 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 added deuterated labeled internal standard (24R),25­(OH)2D3-d 6 were extracted from serum matrix using liquid–liquid extraction prior to LC–MS/MS analysis. Chromatographic separation was performed using a fused-core C18 column. Atmospheric pressure chemical ionization in the positive ion mode and multiple reaction monitoring 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% SD), and the extraction efficiencies averaged 95% (2% SD). Excellent repeatability was demonstrated with CVs of ∼1%. The limit of quantitation at a signal-to-noise ratio of ∼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 within 35 min. This method was used for value assignment of (24R),25­(OH)2D3 in Standard Reference Materials of Vitamin D Metabolites in Human Serum, which can serve as an accuracy base for routine methods used in clinical laboratories.