Quantitation of 5-methyltetraydrofolic acid in plasma for determination of folate status and clinical studies by stable isotope dilution assays

Abstract

Folates play a key role in the prevention of neural tube defects in newborns. Thus, it is important to reliably determine the bioavailability of folates from various foods. Accurate analytical methods are essential for quantifying blood-folates, especially in human studies. Here, we present the development and validation of a sensitive method using stable isotope dilution liquid chromatography coupled with mass spectrometry for determining various folates in plasma. Moreover, this study reports the applicability of the developed method to a human pilot study using strawberries as a test food. Validation of the assay revealed the precision, sensitivity, and accuracy of the method in determining the predominant 5-methyltetrahydrofolate in plasma. This method was also applicable for the screening of individual folate status using finger prick blood and for monitoring the post-absorptive plasma-concentration curve. Moreover, the human study revealed a high recovery of strawberry folates with a calculated relative bioavailability of 96.2%. Thus, the developed method enables prospective bioavailability studies. This work also confirmed, via human studies, that strawberries are a rich and natural source of folates that are available for human metabolism.

Fig 1. Study design.

Overview of the timeline and the different blood sampling time points for one of the three interventions. Details are described in the text. 5-CH₃-H₄folate, 5-methyltetrahydrofolate; STD, standardized.

Fig 2

Comparison of extraction chromatograms of 60 μL (A) and 30 μL (B) of plasma; 5-CH₃-H₄folate m/z 460.20→313.20 (black), 460.20 → 180.15 (pink), and 460.20 → 194.25 (blue).

Fig 3. Comparison of the matrix effect of K3 EDTA and lithium-heparin.

Extraction of 60 μL plasma drawn with a K3 EDTA coated tube (A) and lithium-heparin coated tube (B); 5-CH₃-H₄folate m/z 460.20 → 313.20 (black), 460.20 → 180.15 (pink), and [¹³C₅]-5-CH₃-H₄folate m/z 465.30 → 313.20 (blue), 460.30 → 180.15 (brown).

Fig 4. Postresorptive plasma concentration on the folate-free control day, strawberry test day, and tablet test day (subject 1).

Standard deviation [nmol], calculated from a technical triplicate.