Susan Tai (Presenter)
National INstitute of Standards and Technology
Bio: I'm a research chemist at NIST. I have been developed reference measurement procedures for clinical biomarkers including vitamin D metabolites, steroid hormones, and thyroid hormones and value assigned concentrations of these biomarkers on related SRMs.
Authorship: Susan S. Tai, Michael A. Nelson, and James H. Yen
National Institute of Standards and Technology, Gaithersburg, Maryland, United States
| Short Abstract Accurate and precise quantitative evaluations of 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) are important for reliable diagnosis and appropriate treatment of diseases. 24R,25(OH)2D3 is an important vitamin D metabolite used as a catabolism marker and indicator of kidney disease. NIST has developed a candidate RMP for the determination of 24R,25(OH)2D3 in human serum using ID-LC/MS/MS. This method of high precision and high accuracy was used to value assign the concentrations of 24R,25(OH)2D3 in 2 SRMs of Vitamin D Metabolites in Human Serum (SRM 972a and candidate SRM 2973), which can serve as an accuracy base for the routine methods used in clinical laboratories. |
Long Abstract
Accurate and precise quantitative evaluations of 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) are important for reliable diagnosis and appropriate treatment of diseases. 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 a reference measurement procedure for the determination of 24R,25(OH)2D3 in human serum using isotope-dilution LC-MS/MS (APCI). 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.
An isotopically labeled analog of 24R,25(OH)2D3, namely 24R,25(OH)2D3-d6, was used as an internal standard for the 24R,25(OH)2D3 analysis. The 24R,25(OH)2D3 and its internal standard were extracted from the serum matrix using liquid-liquid extraction prior to LC-MS/MS analysis. Chromatographic separation was performed using a fused-core C18 column with a mobile phase consisting of methanol and water. 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 measuring 24R,25(OH)2D3 in gravimetrically prepared samples of human serum spiked 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 approximately 1 %. 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 (including 3-epi-24R,25(OH)2D3) and from impurity components of the reference standard (24S,25(OH)2D3) were investigated. LC baseline resolution of 24R,25(OH)2D3 from these isomers was achieved.
This ID LC-MS/MS candidate reference measurement procedure of high precision and high accuracy was used to value assign the concentrations of 24R,25(OH)2D3 in 2 SRMs of Vitamin D Metabolites in Human Serum (SRM 972a and candidate SRM 2973). Excellent repeatability was obtained with CVs ranging from 0.3 % to 1.4 % for a total of five concentration levels of the two SRMs with concentrations ranging from 1.4 ng/g to 3.1 ng/g. These results are being evaluated and values are being assigned on the Certificates of Analysis for SRM 972a and SRM 2973.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | no | |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |