Simultaneous determination of 6R-leucovorin, 6S-leucovorin and 5-methyltetrahydrofolate in human plasma using solid phase extraction and chiral liquid chromatography–tandem mass spectrometry

A selective and rapid method was developed and validated for determination of 6R-leucovorin (LV), 6S-leucovorin and 5-methyltetrahydrofolate (5-MeTHF) in human plasma using stereoselective liquid chromatography–tandem mass spectrometry. All analytes and the internal standard were extracted from plasma by solid phase extraction using Oasis® HLB C18 cartridges. A macrocyclic glycopeptide teicoplanin column was used for chiral separation of LV and 5-MeTHF isomers with NH4TFA or NH4OAc in methanol as mobile phase. Detection was performed on an API 4000 tandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode. The calibration curves were linear in the range of 0.050–20.0 μg/mL for 6R-LV and 6S-LV, and 0.025–10.0 μg/mL for 5-MeTHF. The intra- and inter-assay precision was 3.6–13.2%, 3.4–12.9% and 5.3–9.3% for 6R-LV, 6S-LV and 5-MeTHF, respectively. The accuracy was 99.4–102.4%, 95.3–96.8% and 93.0–110% for 6R-LV, 6S-LV and 5-MeTHF, respectively. The lower limit of quantification (LLOQ) was 0.050 μg/mL for each LV isomer and 0.025 μg/mL for 5-MeTHF. The method was successfully applied to a comparative pharmacokinetic study between leucovorin calcium and levoleucovorin calcium in 10 volunteers. No significant differences between levoleucovorin and leucovorin in pharmacokinetic parameters of 6S-LV and 5-MeTHF were found in volunteers.