CPBA, immunoassay, and LC-MS. LC-UV does not have the sensitivity to measure 1,25(OH)2D so is not included in this discussion.
CPBA. The first assay for 1,25(OH)2D was a CPBA using a chicken intestinal extract containing the VDR and 3H-1,25(OH)2D as tracer [58]. The intestinal extract was subsequently replaced by calf thymus VDR [59]. These assays required substantial sample preparation by HPLC to avoid interfering substances, and have subsequently been replaced by immunoassays.
Immunoassays. The most common immunoassay in use today is the RIA available in kit form from DiaSorin and IDS among others with antibody (e.g., sheep polyclonal) and 125I-1,25(OH)2D as tracer. This assay does not require HPLC preparation of the sample and uses serum samples as standards without an internal standard to monitor recovery [60]. However, the IDS kit has been reported to have less than 100% recovery for 1,25(OH)2D2[61]. ELISA kits are also available from IDS and Immunodiagnostik. The IDS assay uses solid phase immunoextraction in terms of their RIA and colorimetric detection as described for 25(OH)D, but again may underestimate 1,25(OH)2D2. Less is known about the performance of the Immunodiagnostik kit. Recently, a fully automated chemiluminescent assay for 1,25(OH)2D has been introduced (DiaSorin Liason XL) [62]. This method uses the ligand binding domain of VDR as the capture molecule, reaction conditions favoring the binding of 1,25(OH)2D to VDR versus DBP in the sample but retaining the preferential binding of 25(OH)D, 24,25(OH)2D, and 25,26(OH)2D to DBP, and using a monoclonal antibody that selectively detects the VDR conformation induced by ligand binding. This antibody is attached to magnetic beads enabling nonbound materials to be washed away and then followed by a monoclonal antibody conjugated with a chemiluminescent label and specific for an epitope in the ligand binding domain. After washing, the chemiluminescent signal is triggered and quantitated, the strength of which is directly proportional to the amount of 1,25(OH)2D in the sample. This assay was compared to 2 LCMS assays using immuno enrichment as a preliminary step and found to have a correlation coefficient around 0.92–0.94, a slope approximately of 1, a mean bias of 2.4–15.5%, and an intercept of approximately 2–4. In this regard, it outperformed the earlier DiaSorin immunoassay. Its LOQ was reported as 2 pg/mL using 75 microliter samples.
An important problem for all immunoassays for 1,25(OH)2D with the possible exception of the above-described chemiluminescent assay is that the polyclonal antibodies generally employed show some cross reactivity with 25(OH)D and 24,25(OH)2D, which circulate at much higher concentrations in blood than does 1,25(OH)2D [63]. This can be prevented by careful preparation of the sample to separate these metabolites from 1,25(OH)2D, but this is not always done.
LC-MS. The major problem to be overcome using LC-MS to measure 1,25(OH)2D is the limited sensitivity caused by the low circulating levels of 1,25(OH)2D and the poor ionization efficiency. The sensitivity problem can be addressed by using immunoaffinity extraction with an antibody to 1,25(OH)2D prior to HPLC and tandem mass spectrometry. This would eliminate the use of LC-MS to measure 1,25(OH)2D along with other vitamin D metabolites and does not lend itself to making this measurement in small samples. The second method is to use derivatization with polar groups such as PTAD [64] or adduct formation with ammonia [65] or lithium [66]. In these measurements, deuterated 1,25(OH)2D is used as an internal standard to calculate recovery. Combining immunoaffinity extraction with derivitazation has achieved an LOQ for 1,25(OH)2D3 of 3 pM (1.2 pg/mL) and for 1,25(OH)2D2 of 1.5 pM (0.6 pg/mL) [63]. The advantages and limitations of LC-MS over and above the sensitivity issue are similar to those described for the LC-MS measurement of 25(OH)D. However, at this point, there is no universally accepted NIST standard for 1,25(OH)2D by which labs can compare their results, or investigators and clinicians can compare one assay to another. Moreover, like 25(OH)D, the C3-beta epimer of 1,25(OH)2D is found in serum [67], and other dihydroxy vitamin D metabolites such as 23,25(OH)2D, 24,25(OH)2D, 25,26(OH)2D, and 4β,25(OH)2D (the product of CYP3A4 hydroxylation of 25[OH]D) need to be separated from 1,25(OH)2D prior to MS, as all exhibit the same molecular weight and m/z ratios. 4β,25(OH)2D was found in similar concentrations as 1,25(OH)2D in one study [68].
24,25(OH)2D
Although theoretically 24,25(OH)2D could be measured by CPBA using DBP because of its equivalent affinity for DBP compared to 25(OH)D or immunoassay using a 1,25(OH)2D antibody that cross reacts 24,25(OH)2D [69,] given its relatively high concentrations in the blood (0.7–24 nM) [70], modern assays for this metabolite use LC-MS exclusively. Frequently this is done as part of a multimetabolite profile. Like that for 25(OH)D and 1,25(OH)2D the C3-beta epimer of 24,25(OH)2D has been identified [71]. Moreover, 24,25(OH)2D exists as both the 24R,25(OH)2D and 24S,25(OH)2D epimer, but only the R epimer is biologically active [72]. Therefore, careful separation of these epimers as well as other dihydroxylated vitamin D metabolites prior to MS is required to obtain accurate results. As noted, 24,25(OH)2D measurement is often part of a multimetabolite profile. The approaches to such assays have recently been reviewed [40]. Most of these assays used ESI for ionization, triplequadrupole instruments for MS, and nonspecific water loss transitions for monitoring. Derivitazation is often employed to increase sensitivity for the less abundant metabolites in the profile, but PTAD derivatization of 25(OH)D was found to interfere with the separation of C3-beta epi-25(OH)D from 25(OH)D [73]. Other methods of derivitazation have been developed that may circumvent this problem [74], and derivitazation may not be required if larger samples are used [75].
Free Vitamin D Metabolite Measurements
Up to this point, I have focused on measurements of total vitamin D metabolite levels. However, the free levels may be a better marker of vitamin D status. The vitamin D metabolites circulate in blood extensively bound to 2 liver-produced proteins, vitamin DBP and albumin. Approximately 85% of this binding is to DBP, 15% to albumin. Given the high affinity of these metabolites for DBP, and the abundance of DBP in normal individuals, free vitamin D metabolite levels are very low (approximately 0.03% of total for free 25(OH)D and 0.4% of total for free 1,25(OH)2D) [76, 77]. However, in conditions such as liver disease (and likely nephrotic syndrome and protein losing enteropathies), DBP and albumin levels are low. The reverse is true during the 3rd trimester of pregnancy when DBP levels are increased. In these situations, the free level of 25(OH)D