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

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• 作者：Susan S.-C. Tai ; Michael A. Nelson
• 刊名：Analytical Chemistry
• 出版年：2015
• 出版时间：August 4, 2015
• 年：2015
• 卷：87
• 期：15
• 页码：7964-7970
• 全文大小：241K
• ISSN：1520-6882

文摘

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鈥揗S/MS. The (24R),25(OH)₂D3 and added deuterated labeled internal standard (24R),25(OH)₂D3-d₆ were extracted from serum matrix using liquid鈥搇iquid extraction prior to LC鈥揗S/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鈥揗S/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 鈭?%. The limit of quantitation at a signal-to-noise ratio of 鈭?0 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.