Determination of folates in human plasma using hydrophilic interaction chromatography-tandem mass spectrometry

Folic acid is an essential nutrient, and folate deficiency is associated wi th a variety of disorders including neural tube defects (during pregnancy) and heart disease. A fast, sensitive, and robust HPLC-tandem mass spectrome try (LC-MS-MS) method was developed for the quantification of free folic ac id, tetrahydrofolate, 5'-methyltetrahydrofolate, and 5'-formyltetrahydrofol ate in human plasma. Sample preparation required only acetonitrile precipit ation of proteins followed by filtration instead of solid-phase extraction or solvent- solvent extraction as in other methods. The rapid and streamlin ed sample handling procedure minimized degradation of the highly unstable f olate species. Hydrophilic interaction chromatography was used for addition al sample cleanup on-line, and baseline separation and detection of all fou r folate species was achieved in less than 30 min. The folate species were detected using negative ion electrospray-tandem mass spectrometry with mult iple reaction monitoring of the diagnostic fragment ions of each deprotonat ed molecule. The predominately organic (hydrophobic) solvent system combine d with the microbore flow rate (50 muL/min) used for the chromatography res ulted in enhanced electrospray signal response compared to reversed-phase H PLC using a wider bore column. The recovery of all folate species (from spi ked plasma) was > 97% over a concentration range from 300 pg/L to 12 mg/L w ith intraday precision (RSD, n = 5) of 3.7-6.5%. Stability studies were car ried out for spiked samples in order to define storage and handling conditi ons. The folic acid limit of quantification (LOQ) in human plasma was 80 pm ol/L +/- 10%, and the limit of detection (LOD) was 37.5 pmol/L. The LOQ and LOD for tetrahydrofolate, 5'-methyltetrahydrofolate, and 5 -formyltetrahyd rofolate were 1250, 400, and 360 pmol/L of plasma and 425, 165, and 140 pmo l/L of plasma, respectively.