Simultaneous quantitation of folates, flavins and B6 metabolites in human plasma by LC-MS/MS assay: Applications in colorectal cancer

Abstract

An analytical method using electrospray ionization and high- performance liquid chromatography/tandem mass spectrometry (LC/ESI-MS/MS) was developed to quantify the vitamin B metabolites found in the folate one-carbon metabolism, using 50 μL of human plasma. Analytes in plasma were extracted using protein precipitation after being stabilized in 1% ascorbic acid. The analytes were separated using a Kinetex 2.6 μm Pentafluorophenyl (2.1 × 30 mm) column utilizing a gradient mobile phase system of 0.1% formic acid in water and 100% acetonitrile in a 13.2 min run. The MS detector run using a positive multiple reaction monitoring with parameters optimized for each analyte's ion pair. The assay was selective and linear for all analytes at defined dynamic ranges. The recoveries were generally above 80% except for the folate metabolites whose recoveries dipped possibly due to the drying process. The inter-day precision (%coefficient of variation) and accuracy (%calculated concentration of the nominal concentrations) for six replicates of all quality control samples were ≤14% and within 12.2%, respectively. The lower limit of quantification ranged from 0.2 to 3.9 nM. No significant instability was observed after repeated freezing and thawing or in processed samples. The LC-MS/MS assay was found applicable for sensitive, accurate and precise quantitation of vitamin B metabolites in plasma of healthy volunteers and colorectal cancer patients.

Keywords: Colorectal cancer; Folates; LC–MS/MS; Metabolites; Plasma; Vitamin B.

Fig. 1.

The FOCM cycle which drives DNA methylation, synthesis and repair. DHFR, dihydrofolate reductase; 5-MTHF, 5-methyltetrahydrofolate; 5,10-MeTHF, 5,10-methylenetetrahydrofolate; CBS, Cystathionine-β-synthase; MAT, Methionine adenosyl transferase; MTR, methionine synthase; MTRR, methionine synthase reductase; MTHFR, methylenetetrahydrofolate reductase; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; SHMT, Serine hydroxymethyltransferase; THF, tetrahydrofolate; TYMS, thymidylate synthase; dUMP, deoxyuridine monophosphate; dTMP, deoxythymidine monophosphate.

Fig. 2.

Comparison of analyte extraction using protein precipitation (PP) and SPE for low, medium and high QCs.

Fig. 3.

Stability of FOCM analytes in various stabilizing agents. 5MTHF, 5- methyltetrahydrofolate; B₂, riboflavin; B₆, pyridoxine; FA, folic acid; TCEP, (tris(2-carboxyethyl)phosphine); THF, tetrahydrofolate.

Fig. 4.

Representative chromatograms of the vitamin B analytes at concentration of 1/5 dilution of working solution. 5MTHF, 5- methyltetrahydrofolate; B₂, riboflavin; B₆, pyridoxine; DHF, dihydrofolate; FA, folic acid; FMN, Flavin mononucleotide; MTX, methotrexate; PA, 4-pyridoxic acid; PL, pyridoxal; PM, pyridoxamine; THF, tetrahydrofolate.

Fig. 5.

Stability of analytes after 18 h of storage in refrigerated autosampler.