Department of Clinical Chemistry and Laboratory Medicine, Saarland University Hospital, Homburg, Saarland, Germany.
Anal Bioanal Chem. 2012 Aug;404(3):895-902. doi: 10.1007/s00216-012-6180-7. Epub 2012 Jun 23.
Folates act as essential coenzymes in many biological pathways, including the synthesis and methylation of DNA. Low folate concentration in serum and whole blood (WB) is associated with several disease conditions. We describe a stable-isotope-dilution ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the quantification of (6S)-5-CH(3)-H(4)folate (where H(4)folate is tetrahydrofolate) and non-CH(3)-H(4)folate [sum of HCO-H(4)folate, (6R)-5,10-CH(+)-H(4)folate, (6R)-5,10-CH(2)-H(4)folate, (6S)-H(4)folate, dihydrofolate, and folic acid] in WB. The assay includes a solid-phase extraction procedure after the hemolysis and deconjugation. The method was linear over the concentration range from 0.2 to 200 nmol/L. The limits of detection were 0.40 nmol/L or lower for the folate forms. The interassay coefficients of variation were 7.4% for (6S)-5-CH(3)-H(4)folate and 15.4% for non-CH(3)-H(4)folate. For the folate forms, the recoveries were between 97.1% and 102.7%. Sample preparation caused the generation of artificial folic acid in WB and serum in a dose-dependent manner, which can lead to misinterpretation of the results. The use of antioxidants could not prevent the formation of folic acid. The median fasting WB folate concentrations from 42 nonsupplemented and nonfortified adults were 576 nmol/L (6S)-5-CH(3)-H(4)folate and 73.6 nmol/L non-CH(3)-H(4)folate, and 1,206 nmol/L (6S)-5-CH(3)-H(4)folate and 155 nmol/L non-CH(3)-H(4)folate for 35 adults who had taken 500 μg of folic acid, 50 mg of vitamin B(6), and 500 μg of vitamin B(12) per day orally for 6 months. In conclusion, the UPLC-MS/MS method is fast and has a good sensitivity and selectivity for WB folates. We observed a dose-dependent oxidation of (6S)-H(4)folate, which resulted in the formation of artificial folic acid in serum and WB. To minimize this effect, we recommend a fast sample preparation.
叶酸在许多生物途径中作为必需的辅酶发挥作用，包括 DNA 的合成和甲基化。血清和全血（WB）中叶酸浓度低与几种疾病状况有关。我们描述了一种稳定同位素稀释超高效液相色谱串联质谱（UPLC-MS/MS）方法，用于定量（6S）-5-CH（3）-H（4）叶酸（其中 H（4）叶酸是四氢叶酸）和非-CH（3）-H（4）叶酸[HCO-H（4）叶酸、（6R）-5,10-CH（+）-H（4）叶酸、（6R）-5,10-CH（2）-H（4）叶酸、（6S）-H（4）叶酸、二氢叶酸和叶酸]在 WB 中的含量。该测定包括在溶血和去共轭后进行固相萃取程序。该方法在 0.2 至 200 nmol/L 的浓度范围内呈线性。各种叶酸形式的检测限均为 0.40 nmol/L 或更低。（6S）-5-CH（3）-H（4）叶酸的批内变异系数为 7.4%，非-CH（3）-H（4）叶酸的批内变异系数为 15.4%。对于各种叶酸形式，回收率在 97.1%至 102.7%之间。样品制备会导致 WB 和血清中的人工叶酸以剂量依赖的方式生成，这可能导致结果的误读。抗氧化剂的使用并不能防止叶酸的形成。42 名未补充和未强化的成年人的空腹 WB 叶酸浓度中位数为 576 nmol/L（6S）-5-CH（3）-H（4）叶酸和 73.6 nmol/L 非-CH（3）-H（4）叶酸，以及 1,206 nmol/L（6S）-5-CH（3）-H（4）叶酸和 155 nmol/L 非-CH（3）-H（4）叶酸，这是 35 名成年人在 6 个月内每天口服 500μg 叶酸、50mg 维生素 B（6）和 500μg 维生素 B（12）的结果。总之，UPLC-MS/MS 方法快速且对 WB 叶酸具有良好的灵敏度和选择性。我们观察到（6S）-H（4）叶酸的剂量依赖性氧化，导致血清和 WB 中人工叶酸的形成。为了最大程度地减少这种影响，我们建议快速进行样品制备。
