Preliminary application of liquid chromatography–electrospray-ionization mass spectrometry to the detection of 5-methyltetrahydrofolic acid monoglutamate in human plasma

Liquid chromatography (LC) in direct combination with mass spectrometry (MS) has been shown to be a good analytical technique for the selective separation and detection of labile folate monoglutamates. Reversed-phase LC and electrospray-ionization MS conditions were developed and optimized for the separation and detection of 5-methyltetrahydrofolic acid, 5-formyl tetrahydrofolic acid, tetrahydrofolic acid, dihydrofolic acid and folic acid in aqueous samples. Representative and reproducible positive ion mass spectra were generated for each folate under mild MS conditions. The selective MS detection and identification of endogenous 5-methyltetrahydrofolic acid in human plasma was accomplished through the development of a straightforward C₁₈-based solid-phase extraction procedure. This procedure allows for the qualitative assessment of 5-methyltetrahydrofolic acid in plasma. Based upon an isotope-dilution internal standard calibration study with standards, the LC–MS limit of quantitation for 5M-THF was estimated to be 0.39 ng/ml.