Quantification of key folate forms in serum using stable-isotope dilution ultra performance liquid chromatography-tandem mass spectrometry.
Folates act as essential coenzymes in many biological pathways. Alteration in folate form distribution might have biological significance, especially in relation to certain genetic polymorphisms. We developed a stable-isotope dilution ultra performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method for quantification of the folate forms 5-methyltetrahydrofolate (5-methylTHF), 5-formylTHF, 5,10-methenylTHF, THF, and folic acid in serum. After extraction using an ion exchange and mixed mode solid-phase, samples were separated and detected using an UPLC-MS/MS system. The quantification limits were between 0.17nmol/L (5-formylTHF) and 1.79nmol/L (THF), and the assay was linear up to 100nmol/L (5-methylTHF) and 10nmol/L (5-formylTHF, 5,10-methenylTHF, THF, and folic acid). The intraassay CVs for 5-methylTHF and 5-formylTHF were 2.0% and 7.2%, respectively. Mean recoveries were between 82.3% for THF and 110.8% for 5,10-methenylTHF. Concentrations of total folate measured by the new method showed a strong correlation with those measured by an immunologic assay (r=0.939; p<0.001). The mean total folate from 32 apparently healthy subjects was 18.09nmol/L, of which 87.23% was 5-methylTHF. Concentrations of homocysteine showed a better correlation to the total folate measured by the new method compared to that obtained by an immunologic assay. We also confirmed that MTHFR polymorphism has a significant effect on folate distribution in this small population of non-supplemented subjects.
Kirsch
Susanne H
SH
Saarland University Hospital, Department of Clinical Chemistry and Laboratory Medicine, Building 57, 66421 Homburg/Saar, Germany.
Knapp
Jean-Pierre
JP
Herrmann
Wolfgang
W
Obeid
Rima
R
D045103:5,10-Methylenetetrahydrofolate Reductase (FADH2) / Q000235:genetics; D000293:Adolescent; D000328:Adult; D002247:Carbon Isotopes / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004355:Drug Stability; D005260:Female; D005492:Folic Acid / Q000097:blood*; D006710:Homocysteine / Q000097:blood; D006801:Humans; D016014:Linear Models; D008297:Male; D008875:Middle Aged; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013763:Tetrahydrofolates / Q000097:blood*
D002247:Carbon Isotopes; D013763:Tetrahydrofolates; D006710:Homocysteine; D005492:Folic Acid; D045103:5,10-Methylenetetrahydrofolate Reductase (FADH2); C005984:5-methyltetrahydrofolate