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 total 25(OH)D measurements (if not separated) is substantial in infants (mean level 18 nM but up to 61% of total) but also can be significant in adults (mean levels 4.3 nM, but up to 47% of total 25(OH)D [52]). Moreover, the concentration (and % of total) of the C3-beta epimer increases with vitamin D supplementation. Following chromatographic separation, the metabolites must be ionized. The vitamin D metabolites are lipophilic and so ionization can be a limit to sensitivity. As mentioned previously in discussing vitamin D measurements, atmospheric pressure photo ionization appears to be more efficient in ionizing these metabolites than APCI or ESI resulting in greater sensitivity with lower limits of detection [38, 53]. Increased sensitivity can also be obtained with the use of readily ionizable derivatives such as 4-phenyl-1,2,4-triazoline-3,5 dione (PTAD). This modification enabled the measurement of 25(OH)D in saliva [54], presumably the free fraction. I will be discussing the measurement of free (i.e., non-protein bound) vitamin D metabolites subsequently, but the free concentration of 25(OH)D is well below 0.1% of total in individuals with normal DBP and albumin levels.
Although LC-MS is a very versatile method, and unlike immunoassays readily measures multiple metabolites in a single sample, it is not without problems some of which I have already discussed (e.g., inability to distinguish epimers). These include ion suppression by interfering substances (so called matrix effects) [55] and mass spectral overlaps with isobaric compounds with comparable m/z ratios (e.g., 7α-hydroxy-4 cholestene-3-one) [56]. The problem with mass spectral overlaps is in part due to the standard use of nonspecific transitions (e.g., loss of H2O) used for multiple reaction monitoring. These problems can be mitigated by the use of an internal standard, namely, deuterated 25(OH)D as a control for ionization efficiency [57], better sample preparation including an LC step to separate the epimers and potential isobars, and high resolution MS (and tandem MS) to distinguish potential spectral overlaps.
1,25(OH)2D
The measurement of 1,25(OH)2D is more challenging than that of 25(OH)D because it circulates in blood at levels that are nearly 1/1,000 that of 25(OH)D. As for the measurement of 25(OH)D, there are