Details:

Alternative Title:
Anal Bioanal Chem
Publisher's site:
http://dx.doi.org/10.1007/s00216-015-8733-z
Personal Author:
Mineva, Ekaterina M. ; Schleicher, Rosemary L. ; Chaudhary-Webb, Madhulika ; Maw, Khin L. ; Botelho, Julianne C. ; ... More +
Mineva, Ekaterina M. ; Schleicher, Rosemary L. ; Chaudhary-Webb, Madhulika ; Maw, Khin L. ; Botelho, Julianne C. ; Vesper, Hubert W. ; Pfeiffer, Christine M. Less -
Description:
The inaccuracy of routine serum 25-hydroxyvitamin D measurements hampers the interpretation of data in patient care and public health research. We developed and validated a candidate reference measurement procedure (RMP) for highly accurate quantitation of two clinically important 25-hydroxyvitamin D metabolites in serum, 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3]. The two compounds of interest together with spiked deuterium-labeled internal standards [d 3-25(OH)D2 and d 6-25(OH)D3] were extracted from serum via liquid-liquid extraction. The featured isotope-dilution LC-MS/MS method used reversed-phase chromatography and atmospheric pressure chemical ionization in positive ion mode. A pentafluorophenylpropyl-packed UHPLC column together with isocratic elution allowed for complete baseline resolution of 25(OH)D2 and 25(OH)D3 from their structural C-3 isomers within 12 min. We evaluated method trueness, precision, potential interferences, matrix effects, limits of quantitation, and measurement uncertainty. Calibration materials were, or were traceable to, NIST Standard Reference Materials 2972. Within-day and total imprecision (CV) averaged 1.9 and 2.0% for 25(OH)D3, respectively, and 2.4 and 3.5% for 25(OH)D2, respectively. Mean trueness was 100.3% for 25(OH)D3 and 25(OH)D2. The limits of quantitation/limits of detection were 4.61/1.38 nmol/L for 25(OH)D3 and 1.46/0.13 nmol/L for 25(OH)D2. When we compared our RMP results to an established RMP using 40 serum samples, we found a nonsignificant mean bias of 0.2% for total 25(OH)D. This candidate RMP for 25(OH)D metabolites meets predefined method performance specifications (≤5% total CV and ≤1.7% bias) and provides sufficient sample throughput to meet the needs of the Centers for Disease Control and Prevention Vitamin D Standardization Certification Program. Graphical abstract Bias assessment using NIST standard reference materials. Legend CDC mean mass fractions (ng/g) ± U 95 (6 replicates per mean). NIST-certified mass fractions (ng/g) ± U 95 from the Certificates of Analysis.
Subject:
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25-Hydroxyvitamin D 2
25-Hydroxyvitamin D2
25-Hydroxyvitamin D3
Article
Calcifediol
Chromatography, High Pressure Liquid
Humans
Isotopes
LC-MS/MS
Reference Method
Tandem Mass Spectrometry
Vitamin D

Source:
Anal Bioanal Chem. 407(19):5615-5624.
Pubmed ID:
25967149
Pubmed Central ID:
PMC4823009
Document Type:
Journal Article
Funding:
CC999999/Intramural CDC HHS/United States
Collection(s):
CDC Public Access
Main Document Checksum:
[+]
urn:sha256:c9827fedf22631c1280a2f3da5139908989f49b9dfdcdb1f03fccdbc2fc1549b
File Type:

Supporting Files

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