Clinical determination of folates: recent analytical strategies and challenges

Abstract

Since the introduction of liquid chromatography tandem mass spectrometry in clinical laboratories, folate analysis has shifted from microbiological or protein binding assays to chromatographic methods. Now it is possible to sensitively and selectively determine several folate species in clinical samples where only a total folate content could be quantified using a microbiological or a binding assay. Although several chromatographic methods have been developed, validated and published, interlaboratory variability limits the comparability of the results. In this review we provide an overview of the latest strategies for sampling, sample treatment and analysis and how these may influence the final analytical result. Amongst the variables covered are the effect of pH, temperature, storage and the use of antioxidants and anticoagulants on analyte stability. In addition, we highlight the importance of correct assay calibration and the use of (labeled) certified reference materials in order to obtain correct and comparable results among different laboratories.

Is the stability of folates in dried blood microsamples sufficient to perform home-sampling studies?

“…This is especially relevant for folates, as they are well-known for their light sensitivity. 15,31 However, our analyses revealed that drying and/ or storage in the presence of light did not relevantly affect the individual folate levels when using VAMS samples. Only for the major folate vitamer 5MTHF and the minor folate vitamer 10FoFA a decrease was observed compared to drying in protected conditions, however the mean recovery relative to t 0 was still above 75% (Fig.…”

“…O'Broin and Gunter hypothesized that this was caused by AA contributing to red blood cell lysis at the time of spotting and by enhancing deconjugation of red blood cell polyglutamates, resulting in an increased level of (less stable) folate monoglutamates. 15,32 This hypothesis implies that when red blood cells dry naturally on filter paper a greater stability would be achieved compared to using AA-treated paper. This is in contrast to serum, where folates are already present as monoglutamates.…”

“…The collected anticoagulated blood was stored at 4 °C prior to preparing dried blood microsamples within 24 h after blood collection. Based on the previously determined stability of folates in whole blood 15 and the fact that the focus of the manuscript is on the comparative evaluation of the stability of the different folates in dried blood microsamples, with dried blood microsamples freshly prepared from liquid blood being considered the ‘baseline’ condition, we considered the possible impact of the time frame between blood collection and dried blood microsample generation (and, hence, the possible impact of differences in oxidative and enzymatic stressors between fresh and 1-day old blood) negligible. VAMS samples were prepared by dipping the tip in whole blood at an angle of 45° for 6 s, as recommended by the manufacturer.…”

“…Apart from their important role within the human body, folates are also well-known for their instability e.g., after blood sampling, which is in essence primarily a chemical phenomenon, as elaborately discussed elsewhere. 15 Also here, the use of microsampling could offer a solution. 6,15,[18][19][20] Indeed, already in the 90's relatively good stability was observed in DBS compared to liquid whole blood using a microbiological assay for total folate determination.…”

“…15 Also here, the use of microsampling could offer a solution. 6,15,[18][19][20] Indeed, already in the 90's relatively good stability was observed in DBS compared to liquid whole blood using a microbiological assay for total folate determination. Of note, caution should still be paid as with increasing temperatures increased degradation was observed.…”