Assay of whole blood (6S)-5-CH₃-H₄folate using ultra performance liquid chromatography tandem mass spectrometry

Abstract

Folates act as essential coenzymes in many biological pathways, including the synthesis and methylation of DNA. Low folate concentration in serum and whole blood (WB) is associated with several disease conditions. We describe a stable-isotope-dilution ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the quantification of (6S)-5-CH₃-H₄folate (where H₄folate is tetrahydrofolate) and non-CH₃-H₄folate [sum of HCO-H₄folate, (6R)-5,10-CH⁺-H₄folate, (6R)-5,10-CH₂-H₄folate, (6S)-H₄folate, dihydrofolate, and folic acid] in WB. The assay includes a solid-phase extraction procedure after the hemolysis and deconjugation. The method was linear over the concentration range from 0.2 to 200 nmol/L. The limits of detection were 0.40 nmol/L or lower for the folate forms. The interassay coefficients of variation were 7.4 % for (6S)-5-CH₃-H₄folate and 15.4 % for non-CH₃-H₄folate. For the folate forms, the recoveries were between 97.1 % and 102.7 %. Sample preparation caused the generation of artificial folic acid in WB and serum in a dose-dependent manner, which can lead to misinterpretation of the results. The use of antioxidants could not prevent the formation of folic acid. The median fasting WB folate concentrations from 42 nonsupplemented and nonfortified adults were 576 nmol/L (6S)-5-CH₃-H₄folate and 73.6 nmol/L non-CH₃-H₄folate, and 1,206 nmol/L (6S)-5-CH₃-H₄folate and 155 nmol/L non-CH₃-H₄folate for 35 adults who had taken 500 μg of folic acid, 50 mg of vitamin B₆, and 500 μg of vitamin B₁₂ per day orally for 6 months. In conclusion, the UPLC-MS/MS method is fast and has a good sensitivity and selectivity for WB folates. We observed a dose-dependent oxidation of (6S)-H₄folate, which resulted in the formation of artificial folic acid in serum and WB. To minimize this effect, we recommend a fast sample preparation.