Quantitation of folates and their catabolites in blood plasma, erythrocytes, and urine by stable isotope dilution assays

New stable isotope dilution assays were developed for the simultaneous quantitation of the folates 5-methyltetrahydrofolic acid, 5-formyltetrahydrofolic acid, tetrahydrofolic acid, 10-formylfolic acid, and folic acid as well as for their catabolites para-aminobenzoylglutamate (pABG) and acetyl-para-aminobenzoylglutamate (ApABG) in clinical samples. The methods were based on cleanup by strong anion exchange followed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) detection. Deuterated analogues of the folates and [13C5]-labeled isotopologues of the catabolites were applied as the internal standards in stable isotope dilution assays. Extraction in 4-morpholineethanesulfonic acid (MES) buffer at pH 5.0 ensured the optimum stability of folates and, in combination with solid-phase extraction (SPE) based on strong anion exchange, resulted in higher recoveries compared with other combinations of extraction buffers and SPE. The method was sensitive enough to detect pABG in plasma generally and unmetabolized folic acid in the plasma of a volunteer after oral dosage of an aqueous folic acid solution. The sum of folate catabolites increased by a factor of 2 in the urine of the latter volunteer, compared with that resulting when only water was dosed.