Saarland University Hospital, Department of Clinical Chemistry and Laboratory Medicine, Building 57, 66421 Homburg/Saar, Germany.
J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Jan 1;878(1):68-75. 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 0.17nmol/L (5-formylTHF) and 1.79nmol/L (THF), and the assay was linear up to 100nmol/L (5-methylTHF) and 10nmol/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 18.09nmol/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.
叶酸在许多生物途径中作为必需的辅酶发挥作用。叶酸形式分布的改变可能具有生物学意义，特别是与某些遗传多态性有关。我们开发了一种稳定同位素稀释超高效液相色谱-质谱联用（UPLC-MS/MS）方法，用于定量血清中的叶酸形式 5-甲基四氢叶酸（5-methylTHF）、5-甲酰基 THF、5,10-亚甲基 THF、THF 和叶酸。使用离子交换和混合模式固相萃取后，使用 UPLC-MS/MS 系统分离和检测样品。定量下限在 0.17nmol/L（5-甲酰基 THF）和 1.79nmol/L（THF）之间，检测范围在 0.17nmol/L 到 100nmol/L（5-methylTHF）和 10nmol/L（5-甲酰基 THF、5,10-亚甲基 THF、THF 和叶酸）之间呈线性。5-methylTHF 和 5-甲酰基 THF 的日内 CV 分别为 2.0%和 7.2%。THF 的平均回收率在 82.3%之间，5,10-亚甲基 THF 的平均回收率在 110.8%之间。新方法测量的总叶酸浓度与免疫测定法测量的浓度呈强相关（r=0.939；p<0.001）。32 名看似健康的受试者的平均总叶酸为 18.09nmol/L，其中 87.23%为 5-methylTHF。与免疫测定法相比，同型半胱氨酸浓度与新方法测量的总叶酸相关性更好。我们还证实，在这个未补充的小人群中，MTHFR 多态性对叶酸分布有显著影响。
