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

Abstract

Volumetric absorptive microsamplers (VAMS) have been developed recently as a promising tool for clinical blood sampling. Compared to dried blood spot samples analyzed by accurate stable isotope dilution assays (SIDAs), the new technique could provide further substantial miniaturizing of folate assays by eliminating hematocrit effects and uneven analyte distribution within the sample. Herein, we present a miniaturized SIDA coupled with LC-MS/MS measurement of 5-methyltetrahydrofolic acid as main folate vitamer in whole blood (WB) using [13C5]-5-methyltetrahydrofolic acid as internal standard. Elution and extraction of only 10.8 μL-dried WB were carried out by centrifugation followed by enzymatic treatment for polyglutamate deconjugation. Matrix separation was achieved by heating and centrifugation. To verify applicability, WB folate status of 11 volunteers was screened. Limits of detection and limits of quantitation were 9 and 26 nmol·L−1, respectively, which is sufficiently low for screening folate status. Recoveries were 97 (±5.8), 99 (±2.8), and 96 (±6.1)% for 800, 400, and 200 nmol L−1 5-methyltetrahydrofolic acid, respectively. Precision of the LC-MS/MS instrument and inter-assay precision trials revealed CVs of 8.1 and 3.5% (294 nmol L−1), respectively, thus confirming reproducible and precise quantitation. Compared to fresh WB, no significant degradation of 5-methyltetrahydrofolate was observed after 2.5 h of drying at room temperature. VAMS 5-CH3-H4folate was stable for at least 3 weeks at −20°C. In our pilot study, accurate and diagnostically conclusive determination of folate status was verified. Nevertheless, blood sampling should be performed by trained individuals to avoid substantial errors concerning the absorbed volume. Endogenous folate in rat serum and chicken pancreas caused a significant background especially at low blood 5-CH3-H4folate levels and, thus, for polyglutamate deconjugation, these background folates or alternative mixtures need to be removed. The superior feasibility of a minimized blood collection with VAMS allows further progress regarding time- and cost-effective methodologies in newborn or population screenings for 5-methyltetrahydrofolate status. Further steps toward minimization could include an automated assay coupled with UPLC-MS/MS.

Antidepressant Therapeutic Drug Monitoring by Minimally Invasive Techniques in Eating Disorders Patients: Preliminary Results from a Pilot Study with Focus on Vortioxetine

“…Several Proof-Of-Concept studies can be found in literature, demonstrating the usefulness of VAMS devices for various compounds and purposes, including the analysis of caffeine, paraxantine, steroid compounds, 5-methyltetrahydrofolic acid, hydroxyurea, natural and synthetic cannabinoids, cocaine and metabolites, oxycodone and metabolites, asenapine enantiomers and iron isotopes [35,[42][43][44][45][46][47][48]. To date, there are no studies on reliability of VAMS technique for the analysis of antidepressant concentrations, neither on blood nor on OF.…”

Dried blood microsample-assisted determination of vitamins: Recent developments and challenges

“…VAMS devices contain a polymeric tip that absorbs a fixed volume of whole blood, independent of the Hct (40,41). In 2017, Kopp and Rychlik described the determination of 5methyltetrahydrofolic acid (5-MTHF) in VAMS samples (42). Even though analysis of whole DBS and VAMS samples eliminates the Hct-based area bias, the extraction efficiency of analytes from dried samples may still be impacted by the Hct.…”

“…Folate concentrations substantially decreased when samples were stored at higher temperature (24-27°C) and greater humidity (53-68%). Kopp and Rychlik evaluated the short-term stability of 5-MTHF in both DBS and VAMS samples (42,66). 5-MTHF in VAMS samples was stable during 2.5h drying at RT.…”

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

“…23 More recently, Kopp and Rychlik evaluated the stability of the main folate vitamer 5-methyltetrahydrofolate (5MTHF) in DBS and VAMS samples using liquid chromatography tandem mass spectrometry (LC-MS/ MS). 24,25 However, only stability at −20 °C and the effect of drying on VAMS samples was included and no evaluations were done at room or higher temperatures. Thus, based on the available data, it remains unclear whether the observed stability is adequate to perform e.g., home-sampling studies.…”