Quantitation of 5-Methyltetrahydrofolic Acid in Dried Blood Spots and Dried Plasma Spots by Stable Isotope Dilution Assays

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.

Assessing Volumetric Absorptive Microsampling Coupled with Stable Isotope Dilution Assay and Liquid Chromatography–Tandem Mass Spectrometry as Potential Diagnostic Tool for Whole Blood 5-Methyltetrahydrofolic Acid

“…VAMS—10.8 μL and whole blood (WB)—10.0 μL were extracted according to the procedure for VAMS; DBS—20.0 μL and WB—20.0 μL were extracted according to Kopp and Rychlik (18). Mean ± SD ( n  = 3).…”

“…However, little effort has been made to minimize WB assays (16–18) because plasma folate has been preferred as the marker of choice to target folate status and deficiency instead of erythrocyte folate, which reflects folate status in the same manner (13, 19). Two principal drawbacks were the complex matrix of erythrocytes, which requires more extraction steps and a longer time, resulting in higher costs (13).…”

Development of stable isotope dilution assays for the quantitation of intra- and extracellular folate patterns of Bifidobacterium adolescentis

“…Methods for extra-and intracellular folate extraction were developed according to [21] and [11], respectively. Sample purification was based on our previous studies [16,17] and further optimized. Bacteria suspension was centrifuged at 5000 g for 10 min at 4°C.…”

“…10 µL of the extracts were injected for folate analysis. Eluate was diverted to waste for the first 10 min and scanning of the SRM transitions (Table A. Analyte concentrations were determined using the HPLC-DAD method published before [17].…”

“…Most recently, SIDAs of folates have been developed in the field of clinical chemistry or food analysis for the quantitation of monoglutamate distribution in erythrocytes [16], dried blood spots [17], and whole blood [18] or polyglutamate distributions in vegetables [19].…”