一碳叶酸代谢产物和一碳相关氨基酸在人体生理中起着重要作用,在生物样本中检测它们是必不可少的。然而,稳定性差以及在各种生物样品中不同物种中的低浓度和发生使其定量非常具有挑战性。这项研究的目的是开发一种简单的,快,和灵敏的超高效液相色谱MS/MS(UHPLC-MS/MS)方法,用于同时定量各种单碳叶酸代谢物(叶酸(FA),四氢叶酸(THF),对氨基苯甲酰基-L-谷氨酸(pABG),5-甲酰基四氢叶酸(5-CHOTHF),5-甲基四氢叶酸(5-CH3THF),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-ade-L-高半胱氨酸(SAH),和S-ade-L-甲硫氨酸(SAM)。该方法进行了标准化和验证,通过测定选择性,结转,检测限,定量极限,线性度精度,准确度,recovery,和矩阵效应。从几个方面对提取方法进行了优化:蛋白酶-淀粉酶处理胚胎,解共轭时间,甲醇沉淀,和蛋白质的等电点沉淀对叶酸的恢复。使用UHPLC-MS/MS技术在各种生物样品中检测到10种一碳叶酸代谢物和4种一碳相关氨基酸。人血浆中叶酸的测定值,血清,和全血(WB)位于正常供体的浓度范围内。小鼠血浆中各分析物含量如下:pABG(864.0nmol/L),5-CH3THF(202.2nmol/L),hmTHF(122.2nmol/L),Met(8.63μmol/L),和SAH(0.06μmol/L)。小鼠胚胎中每种分析物的浓度如下:SAM(1.09μg/g),SAH(0.13μg/g),Met(16.5μg/g),5,10-CH+THF(74.3ng/g),PABG(20.6ng/g),和5-CH3THF(185.4ng/g)。建立了一种简单,快速的样品制备和UHPLC-MS/MS方法,并验证了该方法可同时测定不同生物样品中的一碳相关叶酸代谢物和一碳相关氨基酸。
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.