with the use of a triple quadrupole tandem mass spectrometer equipped with an atmospheric pressure photo ionization source that enhances the ionization of D versus earlier chemical methods (atmospheric pressure chemical ionization or APCI and electrospray ionization [ESI]) [37]. This method was used to measure D2, D3, 25(OH)D2, and 25(OH)D3 in the same sample with limits of quantitation (LOQ) for each D of 2 ng/mL and for each 25(OH)D of 1 ng/mL [38].
25-Hydroxyvitamin D
The accurate measurement of 25(OH)D for the assessment of vitamin D status has been the major goal for most commercial laboratories measuring vitamin D metabolites for a good reason. Levels of 25(OH)D in the blood are higher than those of any other vitamin D metabolite, and most of the 25(OH)D in the body is found in the blood stream with limited distribution into less accessible depots like fat (unlike vitamin D). Its level in blood is the best indicator of vitamin D nutritional status because of its relatively long half life in the blood stream and first-order kinetics in which the rate of 25(OH)D production is dependent on vitamin D levels. Testing for 25(OH)D levels has soared over the past several years [39] driven by the appreciation that vitamin D deficiency may be contributing to a number of disease states [1]. This has generated the need for high throughput assays done in specialized laboratories. However, the disparity of results from one laboratory to the next, or one method to another has been a problem. But now many laboratories are part of the Vitamin D External Quality Assessment Scheme in which these laboratories report their results using defined standards from the National Institute of Standards and Technology (NIST). These standards currently include known concentrations of 25(OH)D2, 25(OH)D3, 3-epi 25(OH)D3, 24S,25(OH)2D3, and 24R,25(OH)2D3 [34]. There are 3 general types of assays currently in use today: competitive protein binding assay (CPBA) and immunoassays, LC-UV and LC-MS. LC-MS is becoming the gold standard and is gradually replacing the CPBA and immunoassays, although immunoassays remain the dominant method in use today [40]. However, each method has its advantages and disadvantages.
CPBA. This was the first method developed for 25(OH)D measurements, and was published in 1971 [41]. It used DBP as the binder and 3H-25(OH)D as tracer. Although the method was largely abandoned when efforts to streamline the assay by eliminating the extraction and purification procedures proved unsatisfactory, Roche Diagnostics has introduced an automated CPBA. The sample is incubated with ruthenium red labeled DBP to which 25(OH)D conjugated with biotin is then added to bind the free DBP. Streptavidin coated beads are added to bind the 25(OH)D biotin conjugate, the beads captured magnetically, and chemiluminescence induced. The concentration of 25(OH)D in the sample is inversely proportional to the chemiluminescence signal.
Immunoassays. These fall into 3 main categories.
Radioimmunoassays were introduced in the 1980s [42, 43]. Acetonitrile separation of 25(OH)D from DBP simplified sample preparation. 125I-25(OH)D serves as the tracer. DiaSorin continues to offer this assay using their goat polyclonal antibody, and it correlates well with LC-MS methods. However, this antibody like others crossreacts with 24,25(OH)2D, 25,26(OH)2D, and 25(OH)D3–26,23 lactone, which unless these other metabolites are separated out (generally not done) could increase the apparent level of 25(OH)D measured. This antibody is claimed to recognize both 25(OH)D3 and 25(OH)D2 equally, but that is not always the case for other immunoassays [44].
Enzyme linked immunosorbent assays (ELISA) introduced commercially by IDS use a sheep polyclonal antibody to coat micro-titre wells, which then are incubated with a sample in which 25(OH)D has been dissociated from DBP in competition with biotin labeled 25(OH)D. After washing the wells are incubated with streptavidin conjugated with horseradish peroxidase that enables cleavage of tetramethylbenzidine to produce a chromogenic product. The amount of 25(OH)D in the sample is inversely proportional to the color formed. The IDS antibody is less efficient in detecting 25(OH)D2 than in identifying 25(OH)D3[45].
Chemiluminescent assays were first commercialized by DiaSorin, although a number of such assays are now on the market. In these assays, the antibody is bound to a solid-phase material and sample 25(OH)D competes for binding with 25(OH)D conjugated to a chemiluminescent label. After washing the light signal is induced and quantitated. The amount of light produced is inversely related to the amount of 25(OH)D in the sample.
Immunoassays share a number of advantages and problems relative to LC-MS. As will be discussed, immunoassays generally do not detect the C3-beta epimer of 25(OH)D. However, as mentioned earlier, the different antibodies used have variable ability to measure 25(OH)D2 relative to 25(OH)D3. The expedited extraction procedures do not separate 25(OH)D from other metabolites such as 24,25(OH)2D, which can be found at levels that are 10–15% of 25(OH)D [44]. Increased levels of DBP in the sample can reduce recovery [46]. These assays can show substantial bias when compared to LC-MS (i.e., they do not parallel the results obtained from LC-MS measurements over their range) [47]. This is of particular importance at the lower 25(OH)D levels when a decision to treat or not to treat depends on accurate results. However, as noted above the role of the Vitamin D External Quality Assessment Scheme program with certified NIST standards is improving the variability between laboratories and methods [48].
LC-UV. The use of HPLC for the separation of the vitamin D metabolites was developed in the 1970s [49]. Although the original columns used were silica based, reverse phase C18 columns are more widely used today. Detection is done primarily using UV at 265 nm although electrochemical detection can also be employed. This method readily separates the D3 and D2 metabolites and can separate the C3-beta epimers from their C3-alpha epimers. It is quite accurate, correlating well with LC-MS, but for metabolites circulating at concentrations well below that of 25(OH)D, unacceptably high sample volumes are required. Moreover, samples with high lipid content can alter the elution pattern, requiring careful sample preparation to avoid erroneous results [50]. Because of the large sample requirements and slow sample throughput, LC-UV is being replaced in many laboratories by LC-MS.
LC-MS. As noted earlier, LC-MS is becoming the gold standard for 25(OH)D assays, and is being developed for measurement of other vitamin D metabolites. In this discussion, I use the term LC-MS to refer to HPLC for initial separation of the vitamin D metabolites followed by tandem mass spectrometry. Tandem mass spectrometry, involving several stages of MS, although more expensive and complex than single stage mass spectrometry, is substantially more sensitive with less matrix interference (i.e., fewer interfering substances in the injected sample) [51]. MS does not distinguish the C3-beta epimer from the C3-alpha epimer of 25(OH)D, so requires a preceding chromatographic step that separates these epimers. Pentafluorophenylpropyl columns are often used for this purpose in current methods. The contribution of the C3-beta epimer to
