Quantification of key folate forms in serum using stable-isotope dilution ultra performance liquid chromatography–tandem mass spectrometry

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 878; no. 1; pp. 68 - 75
• Main Authors: Kirsch, Susanne H., Knapp, Jean-Pierre, Herrmann, Wolfgang, Obeid, Rima
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.01.2010; Elsevier
• Subjects: 5,10-Methylenetetrahydrofolate Reductase (FADH2) - genetics; 5-Methyltetrahydrofolate; Adolescent; Adult; Analysis; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Carbon Isotopes - chemistry; Chromatography, High Pressure Liquid - methods; Drug Stability; Female; Folate; Folic acid; Folic Acid - blood; Fundamental and applied biological sciences. Psychology; General pharmacology; Homocysteine; Homocysteine - blood; Humans; Linear Models; Male; Medical sciences; Middle Aged; Pharmacology. Drug treatments; Reproducibility of Results; Sensitivity and Specificity; Tandem mass spectrometry; Tandem Mass Spectrometry - methods; Tetrahydrofolates - blood; LOD; Tandem mass spectrometry; DHF; pABA; TFOL; MRM; MTHFR; SPE; MS/MS; LC–MS/MS; 5-Methyltetrahydrofolate; Folate; Folic acid; LOQ; TIC; THF; UPLC; Homocysteine; GC/MS; HPLC; PCR; tHcy; Biological fluid; Thiol; Vitamin; Pteridine derivatives; Isotope dilution; Sulfur containing aminoacid; α-Aminoacid; Homocystein; Mass spectrometry MS/MS; Quantitative analysis
• ISSN: 1570-0232; 1873-376X; 1873-376X
• DOI: 10.1016/j.jchromb.2009.11.021

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 0.17 nmol/L (5-formylTHF) and 1.79 nmol/L (THF), and the assay was linear up to 100 nmol/L (5-methylTHF) and 10 nmol/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 18.09 nmol/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.