Abstract
Because of minimal data available on folate analysis in dried matrix spots (DMSs), we combined the advantages of stable isotope dilution assays followed by LC-MS/MS analysis with DMS sampling to develop a reliable method for the quantitation of plasma 5-methyltetrahydrofolic acid in dried blood spots (DBSs) and dried plasma spots (DPSs) as well as for the quantitation of whole blood 5-methyltetrahydrofolic acid in DBSs. We focused on two diagnostically conclusive parameters exhibited by the plasma and whole blood 5-methyltetrahydrofolic acid levels that reflect both temporary and long-term folate status. The method is performed using the [2H4]-labeled isotopologue of the vitamin as the internal standard, and three steps are required for the extraction procedure. Elution of the punched out matrix spots was performed using stabilization buffer including Triton X-100 in a standardized ultrasonication treatment followed by enzymatic digestion (whole blood only) and solid-phase extraction with SAX cartridges. This method is sensitive enough to quantify 27 nmol/L whole blood 5-methyltetrahydrofolic acid in DBSs and 6.3 and 4.4 nmol/L plasma 5-methyltetrahydrofolic acid in DBSs and DPSs, respectively. The unprecedented accurate quantification of plasma 5-methyltetrahydrofolic acid in DBSs was achieved by thermal treatment prior to ultrasonication, inhibiting plasma conjugase activity. Mass screenings are more feasible and easier to facilitate for this method in terms of sample collection and storage compared with conventional clinical sampling for the assessment of folate status.
Highlights
Folates, folylpolyglutamates, are coenzymes for methyl, formyl, and other singlecarbon functional group transfer, and are involved in DNA component, protein, and neurotransmitter synthesis [1]
Compared with conventional methods, sensitive Stable isotope dilution assays (SIDAs) based on LC–MS/MS are good tools when combined with the small sample amounts in dried matrix spots (DMS) owing to their low limits of detection (LODs) and quantification (LOQs)
The DMS sampling technique offers an opportunity for cost reduction as no medical surveillance is necessary for this less invasive blood sampling method, which could even be performed at home [18]
Summary
Folylpolyglutamates, are coenzymes for methyl-, formyl-, and other singlecarbon functional group transfer, and are involved in DNA component, protein, and neurotransmitter synthesis [1]. A key metabolic pathway for folates is the methylation cycle, wherein 5-methyltetrahydrofolate (5-CH3-H4folate) is one of the main donors of single-carbon groups, in homocysteine to methionine remethylation [1] These essential functions underline the importance of adequate folate supply. Dried blood spot (DBS) sampling has gained increasing importance in connection with LC–MS since its development in 1963 by Guthrie and Suzi for newborn screenings [16]. Along with these neonatal screenings, metabolic profiling and drug monitoring are other methods based on dried blood or plasma spots [17]. Erythrocytes and enzymes remain stable after drying at room temperature, whereas blood sample freezing leads to analyte and blood constituent degradation [20]
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