Department of Food Science and Technology, Jinan University, Guangzhou 510632, China.
Department of Food Science and Agricultural Chemistry, Macdonald Campus of McGill University, Montréal, QC H3A 0G4, Canada.
Int J Mol Sci. 2024 Mar 19;25(6):3458. doi: 10.3390/ijms25063458.
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), -aminobenzoyl-L-glutamic acid (pABG), 5-formyltetrahydrofolic acid (5-CHOTHF), 5-methyltetrahydrofolic acid (5-CHTHF), 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), -ade-L-homocysteine (SAH), and -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-CHTHF (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-CHTHF (74.3 ng/g), pABG (20.6 ng/g), and 5-CHTHF (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.
一碳叶酸代谢物和一碳相关氨基酸在人体生理学中发挥着重要作用，其在生物样本中的检测至关重要。然而，由于其在不同物种的各种生物样本中稳定性差、浓度低且存在形式多样，因此对其进行定量分析极具挑战性。本研究旨在开发一种简单、快速、灵敏的超高效液相色谱-串联质谱法（UHPLC-MS/MS），用于同时定量检测各种一碳叶酸代谢物（叶酸（FA）、四氢叶酸（THF）、-氨基苯甲酰-L-谷氨酸（pABG）、5-甲酰四氢叶酸（5-CHOTHF）、5-甲基四氢叶酸（5-CHTHF）、10-甲酰叶酸（10-CHOFA）、5,10-亚甲基-5,6,7,8-四氢叶酸（5,10-CH-THF）和 4-α-羟基-5-甲基四氢叶酸（hmTHF））和一碳相关氨基酸（同型半胱氨酸（Hcy）、蛋氨酸（Met）、-S-腺苷-L-同型半胱氨酸（SAH）和 -S-腺苷-L-蛋氨酸（SAM））。该方法通过选择性、拖尾、检测限、定量限、线性、精密度、准确度、回收率和基质效应的确定来进行标准化和验证。采用蛋白酶-淀粉酶处理胚胎、去结合时间、甲醇沉淀和叶酸回收率的等电点沉淀等多种因素优化了提取方法。采用 UHPLC-MS/MS 技术在各种生物样本中检测到十种一碳叶酸代谢物和四种一碳相关氨基酸。人血浆、血清和全血（WB）中叶酸的测定值在正常供体的浓度范围内。小鼠血浆中各分析物的含量如下：pABG（864.0 nmol/L）、5-CHTHF（202.2 nmol/L）、hmTHF（122.2 nmol/L）、Met（8.63 μmol/L）和 SAH（0.06 μmol/L）。小鼠胚胎中各分析物的含量如下：SAM（1.09 μg/g）、SAH（0.13 μg/g）、Met（16.5 μg/g）、5,10-CHTHF（74.3 ng/g）、pABG（20.6 ng/g）和 5-CHTHF（185.4 ng/g）。本研究建立并验证了一种简单、快速的生物样本前处理方法和 UHPLC-MS/MS 方法，用于同时测定不同生物样本中一碳相关叶酸代谢物和一碳相关氨基酸。
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