A candidate reference measurement procedure for quantifying serum concentrations of 25-hydroxyvitamin D.sub.3 and 25-hydroxyvitamin D.sub.2 using isotope-dilution liquid chromatography-tandem mass spectrometry

• Published in: Analytical and bioanalytical chemistry Vol. 407; no. 19; p. 5615
• Main Authors: Mineva, Ekaterina M, Schleicher, Rosemary L, Chaudhary-Webb, Madhulika, Maw, Khin L, Botelho, Julianne C, Vesper, Hubert W, Pfeiffer, Christine M
• Format: Journal Article
• Language: English
• Published: Springer 01.07.2015
• Subjects: Analysis; Chemical properties; Evaluation; Liquid chromatography; Mass spectrometry; Measurement; Vitamin D; United States
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-015-8733-z

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 D.sub.2 [25(OH)D.sub.2] and 25-hydroxyvitamin D.sub.3 [25(OH)D.sub.3]. The two compounds of interest together with spiked deuterium-labeled internal standards [d.sub.3-25(OH)D.sub.2 and d.sub.6-25(OH)D.sub.3] 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)D.sub.2 and 25(OH)D.sub.3 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)D.sub.3, respectively, and 2.4 and 3.5 % for 25(OH)D.sub.2, respectively. Mean trueness was 100.3 % for 25(OH)D.sub.3 and 25(OH)D.sub.2. The limits of quantitation/limits of detection were 4.61/1.38 nmol/L for 25(OH)D.sub.3 and 1.46/0.13 nmol/L for 25(OH)D.sub.2. 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 ([less than or equal to]5 % total CV and [less than or equal to]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. Electronic supplementary material The online version of this article (doi:10.1007/s00216-015-8733-z) contains supplementary material, which is available to authorized users.