Simultaneous measurement of folate cycle intermediates in different biological matrices using liquid...

The folate cycle is an essential metabolic pathway in the cell, involved in nucleotide synthesis, maintenance of the redox balance in the cell, methionine metabolism and re-methylation reactions. Standardised methods for the measurement of folate cycle intermediates in different biological matrices are in great demand. Here we describe a rapid, sensitive, precise and accurate liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method with a wide calibration curve range and a short run time for the simultaneous determination of folate cycle metabolites, including tetrahydrofolic acid (THF), 5‑methyl THF, 5‑formyl THF, 5,10‑methenyl THF, 5,10‑methylene THF, dihydrofolic acid (DHF) and folic acid in different biological matrices. Extraction of folate derivatives from soft and hard tissue samples as well as from adherent cells was achieved using homogenisation in buffer, while extraction from the whole blood and plasma relied on the anion exchange solid-phase extraction (SPE) method. Chromatographic separation was completed using a Waters Atlantis dC₁₈ 2.0 × 100 mm, 3-μ column with a gradient elution using formic acid in water (0.1% v/v) and acetonitrile as the mobile phases. LC gradient started with 95% of the aqueous phase which was gradually changed to 95% of the organic phase during 2.70 min in order to separate the selected metabolites. The analytes were separated with a run time of 5 min at a flow rate of 0.300 mL/min and detected using a Waters Xevo-TQS triple quadrupole mass spectrometer in the multiple reaction monitoring mode (MRM) at positive polarity. The instrument response was linear over a calibration range of 0.5 to 2500 ng/mL (r² > 0.980). The developed bioanalytical method was thoroughly validated in terms of accuracy, precision, linearity, recovery, sensitivity and stability for tissue and blood samples. The matrix effect was compensated by using structurally similar isotope labelled internal standard (IS), ¹³C₅‑methyl THF, for all folate metabolites. However, not all folate metabolites can be accurately quantified using this method due to their high interconversion rates especially at low pH. This applies to 5,10‑methylene THF which interconverts into THF, and 5,10‑methenyl‑THF interconverting into 5‑formyl‑THF. Using this method, we measured folate cycle intermediates in mouse bone marrow cells and plasma, in human whole blood; in mouse muscle, liver, heart and brain samples.