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

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)(2)D3) 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)(2)D3 in human serum using isotope-dilution LC-MS/MS. The (24R),25(OH)(2)D3 and added deuterated labeled internal standard (24R),25(OH)(2)D3-d6 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)(2)D3 was evaluated by recovery studies of measuring (24R),25(OH)(2)D3 in gravimetrically prepared spiked samples of human serum with known (24R),25(OH)(2)D3 levels. The recoveries of the added (24R),25(OH)(2)D3 averaged 99.0% (0.8% SD), and the extraction efficiencies averaged 95% (2% SD). Excellent repeatability was demonstrated with CVs of similar to 1%. The limit of quantitation at a signal-to-noise ratio of similar to 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)(2)D3 from these isomers was achieved within 35 min. This method was used for value assignment of (24R),25(OH)(2)D3 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.