Vitamin radioligand assay

Vitamin D and its metabolites can be detected on the basis of their native UV absorption (Amax = 264 nm) or after conversion to isotachysterols (Amax = 301 nm). However, because of the inherent poor selectivity and sensitivity of this approach quantitation is mostly performed offline by a radioligand assay. Several metabolites have also been determined in plasma by LC-thermospray MS. 

Resolution of hydroxylated metabolites of vitamin D and their isolation from total lipid extracts (removal of interferences) as part of GC or radioligand assays of these metabolites in human plasma or serum... 

For the quantification of relatively large amounts of vitamin D, e.g. in pharmaceutical preparations, off-line spectrometric procedures can be considered. Spots eluted from the plates can be quantitated by spectrometric or gas-chromatographic procedures (28). Photodensitometry (reflectance spectrometry at 265 nm) and fluorodensitometry (after spraying the plate) are usually sufficiently sensitive to permit the determination of vitamin D in foods (34-36,38). The low concentrations of vitamin D metabolites in human plasma/serum can only be measured off-line by GC (25(OH)D) (40) or radioligand assays (25(OH)D and l,25(OH)2D) (41-43). 

In recent years, focus has shifted from vitamin D to the hydroxylated metabolites of vitamin D. New HPTLC systems display a performance comparable to HPLC for the resolution of individual metabolites. Most important, conventional TLC and HPTLC have been repeatedly advocated as alternatives to HPLC, particularly for laboratories that specialize in radioligand assays but lacking HPLC equipment (41-43,45). These applications suggest a certain future for TLC for the analysis of vitamin D metabolites, primarily because of the nonsophisticated nature of the technique. 

New developments in the TLC of vitamin E have been concerned mainly with refinement of detection and quantitation (e.g. densitometry), rather than with new chromatographic systems, although a few HPTLC procedures as well as a separation of D and L isomers (50) have been reported. This trend toward increasing sophistication hardly makes these new TLC approaches attractive to poorly equipped laboratories as routine techniques for vitamin E determination. As shown above, the opposite may be true for assays of vitamin D metabolites, in which TLC can conveniently replace HPLC as a sample purification method. The explanation for this difference in the position of TLC with respect to both vitamins lies in the availability of suitable techniques for quantitation. In case of vitamin D metabolites, TLC can be easily coupled with a simple and yet highly specific radioligand assay. In contrast, for vitamin E no such off-line determination of equally powerful performance exists, the older colorimetric or gas chromatographic procedures being obsolete. 

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