Folates labelled samples

Recently, radioassay methods have been refined to measure folates in biological samples. These techniques use radioactively labeled folates and competitive protein binding.80 Johnson et al.81 compare this method with traditional microbiological assay with L. casei. 

The ability to conduct bioavailability studies on foods labeled intrinsically with stable isotopes is particularly important as this type of labeling is the gold standard for metabolic studies, i.e., intrinsic label should mimic the behavior of endogenous nutrients most closely. However, the mass spectrometric measurement of the metabolism of intrinsically stable isotope labeled materials is far more challenging than measuring extrinsically labeled nutrients. Low levels of isotope incorporation must be determined in small samples and it is only quite recently that modern LC-MS and LC-MS/MS methods, as demonstrated by the folate studies described above, have begun to rise to this challenge. 

In quantitative immunoassays, e.g., enzyme-linked immunosorbent assay and radioimmunoassay, a known amount of labeled vitamin is mixed with sample extract in which the vitamin content should be determined. Methods of labeling include radioisotopes (e.g., cobalamine), fluorescence, or luminescence markers (e.g., folate). The mixture is subjected to binding agent, equally forming complexes with both labeled and unlabeled vitamin. This complex is then isolated, and the amount of labeled vitamin present is measured. Sample vitamin concentration can be deduced from the ratio of labeled vitamin added to labeled vitamin measured after isolation. Advantages of immunoassays are short analysis time, and the possibility of automating them on clinical analyzers. 

The m/z values monitored in SRM mode for various folate vitamers are shown in Table 6.1. For quantitative purposes, commercially available Cj-labeled internal standards (Merck-Eprova, Schaffhausen, Switzerland) are added at the start of the sample extraction. 

Sample preparation was carried out under subdued light. The folate samples were treated with a-amylase, protease, and deconjugase and then extracted with phosphate buffer at pH 7.5. Matrix effects were compensated by use of isotopically labelled internal standards. 

Furthermore, an important roadblock for propagation of SIDAs is the price of labelled standards. However, this is not a convincing argument as illustrated by the following example 20 mg of commercially available [ C]-labelled folate may cost around 1500. As less than 200 ng of the labelled standard is required for a SID A, 20 mg of the standard enables the performance of at least 10000 analyses. Hence the material costs for using a labelled standard accounts for 0.10 per sample, which is negligible compared with the cost of labour and equipment. 

Buettner, B., Oehrvik, V., Witthoeft, C.M., and Rychlik, M., 2011. Quantitation of isotope-labelled and unlabelled folates in plasma, ileostomy and food samples. Analytical and Bioanalytical Chemistry. 399 429-439. 

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