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SPECIAL FEATURE :: VITAMIN D


the relative low cost, availability, using small sample volume, rapid turnaround, and ease of use. The first automated vitamin D assay developed was based on Competitive-Protein Binding Assays (CPBA) for the Nichols Advantage analyzer. It has the advantages of being inexpensive, can be performed on small sample size, and is co-specific for 25-OH-D2 and 25-OH-D3.7


This assay underestimated 25-OH-D at


low levels and overestimated it at high levels. Immu- noassay methods were first reported in the 1980s with RIA. This assay formed the basis for a subsequent chemiluminescent detection-based system. The RIA requires a small sample size and the incorporation of iodine-125 as a tracer. It is not subjected to nonspe- cific interference, and in addition to being rapid it is inexpensive and accurate.7 However, it still requires the use of radionuclides, and some RIA assays discriminate between 25-OH- D2 and 25-OH-D3. The challenges with vitamin D immunoassays include demonstrated accuracy and specificity and the recognition of fluctuations of assay performance.


Chemical assays have traditionally been more tech- nically involved but are now able to accommodate a higher number of tests per workday. Chemical meth- ods (HPLC and LC-MS/MS) can report vitamin D2 D3 independently.8


and Ultraviolet quantitation following


HPLC is a very stable, repeatable assay, and provides separate quantitation of 25-OH-D2 and 25-OH-D3. Nevertheless, it requires a larger sample size, needs a


preparation step before chromatography and some- times is subject to interferences with other compounds measured in the ultraviolet spectrum. This assay also requires a high level of technical expertise.8 Isotope dilution LC-MS/MS is currently considered the most desirable method for 25OHD measurement, being able to simultaneously quantitate 25OHD2 and 25OHD3, with summation of the two values resulting in total 25OHD.9


A criticism of LC-MS/MS is that there


is a multitude of ‘‘home-brew’’ or ‘‘in-house’’ methods available using different sample preparation and extrac- tion methods, varying running conditions and buffers, different HPLC systems, and multiple MS detection systems which utilize different transitions for each molecule of interest.9


Blood vs. hair testing Currently, the best method to measure the presence of vitamin D is blood. However, there is a recent study regarding extraction and determination of vitamin D concentration from human hair.10


concentration and could capture a large seasonal dif-


capability to account for the high variability of 25(OH) D3


This method has the a large seasonal dif-


ference, since hair only grows approximately one cm per month. Whereas blood can only account for a snapshot of vitamin D and is not able to provide information of vitamin D year-round. These findings potentially pres- ent a new approach to epidemiological studies relating vitamin D to bone and non-bone related medical condi- tions which have been associated with its deficiency.10


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