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.

Keywords: 5-methyltetrahydrofolic acid; Mitra™; dried blood; folate status; volumetric absorptive microsampler.

Figure 1

Blood collection with volumetric absorptive microsamplers (VAMS) after index finger puncture. Approximately 10 μL of whole blood are absorbed by the microsampling device. After drying at room temperature, VAMS can be stored at −20°C with desiccant or removed and suspended for analysis.

Figure 2

Extraction of volumetric absorptive microsamplers with (1) and without additional enzymes (2), without solid phase extraction/enzymes (3). Mean ± SD (n = 3). *Significant difference (p = 0.05).

Figure 3

Liquid chromatography–tandem mass spectrometry chromatogram of the enzyme mixture (75 μL rat serum and 500 μL chicken pancreas suspension).

Figure 4

Liquid chromatography–tandem mass spectrometry chromatogram in the multiple-reaction monitoring mode for volumetric absorptive microsamplers [10.8 μL whole blood sample (329 nmol L⁻¹ 5-CH₃-H₄folate)].

Figure 5

Matrix effects during LC-MS/MS measurement of extracted volumetric absorptive microsamplers. DAD (diode array detector), M (interfering compounds and inorganic salts from whole blood).

Figure 6

Liquid chromatography–tandem mass spectrometry chromatogram of a water blank.

Figure 7

Liquid chromatography–tandem mass spectrometry chromatogram of calibrator 14 (Table 2).

Figure 8

(A) Effect of the drying process (2.5 h) on analyte stability in volumetric absorptive microsamplers (VAMS) compared to a whole blood (WB) sample. (B) Stability at −20°C. (C) Stability in extracted samples during LC-MS/MS analysis. Finger blood from one blood donor analyzed as VAMS containing 10.8 μL of dried WB: freshly drawn WB after forefinger punction. Peak areas of [¹³C₅]-5-CH₃-H₄folate obtained from multiple injections of blood sample extracts to which the same amount of [¹³C₅]-5-CH₃-H₄folate has been added. (A,B) Mean ± SD (n = 3).

Figure 9

Comparison of the volumetric absorptive microsamplers (VAMS) and the dried blood spot (DBS) assay. 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). Significant difference (*p = 0.05).

Figure 10

Screening of whole blood 5-CH₃-H₄folate by means of volumetric absorptive microsamplers. Mean ± SD (n = 3), except value for volunteer 2 (n = 2).