Simultaneous Determination of One-Carbon Folate Metabolites and One-Carbon-Related Amino Acids in Biological Samples Using a UHPLC-MS/MS Method

Abstract

One-carbon folate metabolites and one-carbon-related amino acids play an important role in human physiology, and their detection in biological samples is essential. However, poor stability as well as low concentrations and occurrence in different species in various biological samples make their quantification very challenging. The aim of this study was to develop a simple, fast, and sensitive ultra-high-performance liquid chromatography MS/MS (UHPLC-MS/MS) method for the simultaneous quantification of various one-carbon folate metabolites (folic acid (FA), tetrahydrofolic acid (THF), p-aminobenzoyl-L-glutamic acid (pABG), 5-formyltetrahydrofolic acid (5-CHOTHF), 5-methyltetrahydrofolic acid (5-CH₃THF), 10-formylfolic acid (10-CHOFA), 5,10-methenyl-5,6,7,8-tetrahydrofolic acid (5,10-CH⁺-THF), and 4-α-hydroxy-5-methyltetrahydrofolate (hmTHF)) and one-carbon-related amino acids (homocysteine (Hcy), methionine (Met), S-ade-L-homocysteine (SAH), and S-ade-L-methionine (SAM)). The method was standardized and validated by determining the selectivity, carryover, limits of detection, limits of quantitation, linearity, precision, accuracy, recovery, and matrix effects. The extraction methods were optimized with respect to several factors: protease-amylase treatment on embryos, deconjugation time, methanol precipitation, and proteins' isoelectric point precipitation on the folate recovery. Ten one-carbon folate metabolites and four one-carbon-related amino acids were detected using the UHPLC-MS/MS technique in various biological samples. The measured values of folate in human plasma, serum, and whole blood (WB) lay within the concentration range for normal donors. The contents of each analyte in mouse plasma were as follows: pABG (864.0 nmol/L), 5-CH₃THF (202.2 nmol/L), hmTHF (122.2 nmol/L), Met (8.63 μmol/L), and SAH (0.06 μmol/L). The concentration of each analyte in mouse embryos were as follows: SAM (1.09 μg/g), SAH (0.13 μg/g), Met (16.5 μg/g), 5,10-CH⁺THF (74.3 ng/g), pABG (20.6 ng/g), and 5-CH₃THF (185.4 ng/g). A simple and rapid sample preparation and UHPLC-MS/MS method was developed and validated for the simultaneous determination of the one-carbon-related folate metabolites and one-carbon-related amino acids in different biological samples.

Keywords: UHPLC–MS/MS; amino acids; biological samples; folate.

Figure 1

Chemical structures of folate species.

Figure 2

Summary diagram of folate one-carbon metabolism. MS: methionine synthase; CBS: cystathionine β-synthase; MTHFR: methylene-tetrahydrofolate reductase; Ox: oxidation; MA: methionine adenosyltransferase.

Figure 3

A typical UHPLC–MS/MS chromatogram showing one-carbon-related folate metabolites in human plasma: (A) Folate metabolite standards. (B) Folate metabolite in human plasma samples. (C) Amino acids related to folate metabolism standards. (D) Amino acids related to folate metabolism in humans.

Figure 4

Correlation analysis of folate metabolite concentrations in rat plasma. (A) Analysis of correlation between hmTHF and pABG concentrations. (B) Analysis of correlation between hmTHF+pABG and SAH concentrations.