Highly sensitive analytical method for the accurate determination of 5-methyltetrahydrofolic acid monoglutamate in various volumes of human plasma using isotope dilution ultra-high performance liquid chromatography-mass spectrometry

One predominant and bioactive folate vitamer circulating in the blood is 5-methyltetrahydrofolate (5-Me-THF). In this study, a method for the accurate determination of 5-Me-THF in human plasma samples of various volumes was established using isotope dilution ultra-high performance liquid chromatography-mass spectrometry (ID-UPLC-MS). For this purpose, 500 μL of homogeneous human plasma was initially employed, and the 5-Me-THF and the ¹³C₅-5-Me-THF standard solutions prepared using 1% ascorbic acid in water gave the calibration solution and spiking sample. The desired amount of ¹³C₅-5-Me-THF standard solution was spiked into the sample followed by sample pretreatment. The method was validated for its repeatability, reproducibility, recovery, and limits of detection and quantification. Subsequently, it was applied to smaller volumes of human plasma samples (i.e., 50 and 10 μL), the results of which corresponded well with those obtained using 500 μL. The feasibility of the method was further confirmed using 10 μL of a standard reference material, SRM 3949, which is a human serum sample containing three different levels of 5-Me-THF. The established ID-UPLC-MS method was successfully applied to various volumes of human plasma or serum ranging from 500 to 10 μL, which exhibited particularly good sensitivity in addition to reliable results for the quantification of 5-Me-THF. Our method therefore expands on the ability to obtain accurate quantitative results for 5-Me-THF using small volumes of blood.