25-Hydroxyvitamin D: A Difficult Analyte

Abstract

In their article in the current issue of Clinical Chemistry , Farrell et al. (1) describe the latest in a number of studies (2, 3) highlighting the method-related variability in serum 25-hydroxyvitamin D (25-OHD)2 results. There is common agreement that 25-OHD is a “difficult” analyte. That quality is generally ascribed to its hydrophobic nature, its existence in several different molecular forms, its tight binding to vitamin D– binding protein (VDBP) and, until recently, the absence of reference materials or a reference measurement procedure (RMP) against which assays could be standardized. This last problem was mitigated to some extent with the introduction in 2009 of the NIST Standard Reference Materials (SRM 972 and SRM 2972) and the acceptance of the NIST and University of Ghent liquid chromatography–tandem mass spectrometry (LC-MS/MS) assays as RMPs (4). Unfortunately, 3 of the 4 SRM 972 reference materials are either spiked with exogenous metabolites or diluted with equine serum, characteristics that render these reference materials unsuitable for many immunoassays (5, 6). The history of 25-OHD methodology could serve as a case study of the consequences of transferring a rigorous but labor-intensive method from the unhurried atmosphere of the research laboratory to the bustle of a routine clinical laboratory having to meet tight deadlines. The pioneering competitive protein-binding (CPB) method of Haddad and Chyu (7) involved solvent extraction and chromatography, with the results of every sample corrected for procedural losses. Subsequent attempts at simplifying a CPB method by omitting the chromatography stage proved unsuccessful (8), and an automated version (Nichols Advantage) introduced in 2004 was withdrawn in 2006. The successive abandonment of sample extraction, chromatography, and correction for procedural losses in immunoassays has undoubtedly contributed to the inconsistencies reported by Farrell et al., as have differences in assay standardization. Nevertheless, results submitted to …