A novel and reliable method for tetrahydrobiopterin quantification: Benzoyl chloride derivatization coupled with liquid chromatography-tandem mass spectrometry analysis
A novel and reliable method for tetrahydrobiopterin quantification: Benzoyl chloride derivatization coupled with liquid chromatography-tandem mass spectrometry analysis
复制标题
一种新颖可靠的四氢生物蝶呤定量方法：苯甲酰氯衍生化结合液相色谱-串联质谱分析
DOI：
10.1016/j.freeradbiomed.2018.02.035
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发表时间：
2018
影响因子：
7.4
通讯作者：
Li Yan
中科院分区：
文献类型：
--
作者：
Yuan Teng Fei;Huang Han Qi;Gao Ling;Wang Shao Ting;Li Yan
四氢生物蝶呤（BH 4）是一氧化氮合酶、酰基甘油单加氧酶和芳香族氨基酸羟化酶的重要辅助因子。长期以来，它在体内氧化还原途径中的重要作用引起了人们的广泛关注。然而，由于BH 4的可氧化性和亚化学计量的性质，分析从来没有真正实现足够的灵敏度和适用性。本工作首次采用苯甲酰氯（BC）对活性仲胺的五位衍生化来稳定BH_4。利用产物（BH 4-BC）良好的化学稳定性和良好的质谱灵敏度，采用液相色谱-串联质谱（LC-MS/MS）技术实现了血浆中内源性BH 4的超灵敏、可靠定量。该方法以BH_4-BC-d_5为稳定同位素内标。定量限（LOQ）可达0.02 ng mL−1。最后，通过对38名健康志愿者血浆BH_4水平的调查，发现BH_4水平与年龄呈显著负相关。与现有方法相比，本方法在敏感性、特异性和实用性方面具有明显的上级优势。可以预期，这项工作将在很大程度上促进未来的研究在BH 4相关领域。
Tetrahydrobiopterin (BH4) is a crucial cofactor for nitric oxide synthase, acylglycerol mono-oxygenase and aromatic amino acids hydroxylases. Its significant function for redox pathways in vivo attracted much attention for long. However, because of the oxidizable and substoichiometric nature, analysis of BH4has never been truly achieved with adequate sensitivity and applicability. In the present work, we pioneeringly stabilized BH4by derivatizing the active secondary amine on five-position with benzoyl chloride (BC). Benefiting from the favorable chemical stability and excellent mass spectrometric sensitivity of the product (BH4-BC), ultra-sensitive and reliable quantification of endogenous BH4in plasma was achieved using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. In such methodology, BH4-BC-d5was introduced as stable isotopic internal standard. And the limit of quantification (LOQ) could reach 0.02 ng mL−1. In the end, after investigation of plasma BH4in healthy volunteers (n = 38), we found that the levels of BH4were significantly and negatively correlated to age. Comparing with all the other existed strategies, the present method was obviously superior in sensitivity, specificity and practical applicability. It could be expected that this work could largely promote the future studies in BH4-related fields.