Development of stable isotope dilution assays for the quantitation of intra- and extracellular folate patterns of Bifidobacterium adolescentis

- Unique method for accurate quantitation of bacterially synthesized folate vitamers.
- A stable isotope dilution assay using a deuterated internal standard was applied.
- Consistent results between monoglutamate and native polyglutamate folate analysis.
- B. adolescentis produced 5-methyl-, 5-formyltetrahydrofolate and tetrahydrofolate.
- Total 5-methyltetrahydrofolate can be ascribed entirely to the tetraglutamate.

Folate-producing bifidobacteria have been studied extensively but appropriate methods for detailed quantitation of intra- and extracellular pteroylmono- and pteroylpolyglutamate patterns are lacking. Therefore, B. adolescentis DSM 20083T was cultivated in folate-free medium (FFM) for 24 h to develop and validate stable isotope dilution assays (SIDAs) coupled with LC-MS/MS for the determination of 5-formyltetrahydrofolic acid (5-HCO-H4folate), 10-formylfolic acid (10-HCO-PteGlu), tetrahydrofolic acid (H4folate), folic acid (PteGlu) and 5-methyltetrahydrofolic acid (5-CH3-H4folate) including its di-, tri-, and tetraglutamic vitamers (5-CH3-H4PteGlu2-4). The respective monoglutamylated isotopologues labelled with deuterium were used as internal standards for quantitation. Limits of detection and quantitation (LOD/LOQ) were sufficiently low to quantify 48.2 nmol  L−1 5-CH3-H4folate (5.7/17 nmol L−1) and 71.0 nmol L−1 5-HCO-H4folate (10/30 nmol L−1) as major folate vitamers extracellularly and 124 nmol L−1 5-CH3-H4folate (3.4/10 nmol L−1), 213 nmol L−1 5-HCO-H4folate (4.8/14 nmol L−1), and 61.4 nmol L−1 H4folate (2.3/7.0 nmol L−1) intracellularly after deconjugation. The major portion of native 5-CH3-H4folate vitamer was ascribed to its tetraglutamate ( > 95%). Concentrations of mono-, di-, tri-, and pentaglutamylated folates were below LOD or LOQ. Intra-assay precision coefficients of variation (CVs) ranged from 7% (at a concentration of 53.9 nmol L−1 for 5-CH3-H4PteGlu4), 15% (25.5 nmol L−1 5-CH3-H4folate) to 18% (78.5 nmol L−1 5-HCO-H4folate), extracellularly, and from 6% (60.7 nmol L−1 5-CH3-H4PteGlu4), 7% (202 nmol L−1 5-HCO-H4folate), 10% (67.1 nmol L−1 H4folate) to 11% (127 nmol L−1 5-CH3-H4folate), intracellularly. Inter-assay precision CVs ranged from 2% (54.7 nmol L−1 5-CH3-H4PteGlu4), 3% (71 nmol L−1 5-HCO-H4folate) to 11% (48.2 nmol L−1 5-CH3-H4folate), extracellularly, and from 1% (61.4 nmol L−1 H4folate), 5% (213 nmol L−1 5-HCO-H4folate), 6% (63.5 nmol L−1 5-CH3-H4PteGlu4) to 10% (124 nmol L−1 5-CH3-H4folate), intracellularly, thus showing excellent reproducibility. Recoveries for all analytes under study ranged between 81 and 113%. These newly developed methods enable reproducible, precise and sensitive quantitation of eight bacterially synthesized folate vitamers in two totally different matrices, including both monoglutamates and polyglutamates. Furthermore, we here present the first assay using solely monoglutamylated [2H4]-5-CH3-H4folate to quantify native polyglutamate patterns of this vitamer in bacteria which might replace time-consuming determination of monoglutamates in the future.