Tsikas Dimitrios, Rothmann Sabine, Schneider Jessica Y, Suchy Maria-Theresia, Trettin Arne, Modun Darko, Stuke Nadine, Maassen Norbert, Frölich Jürgen C
Bioanalytical Research Laboratory for NO, Oxidative Stress and Eicosanoids (BIOFORNOX20), Centre of Pharmacology and Toxicology, Hannover Medical School, Hannover, Germany.
Bioanalytical Research Laboratory for NO, Oxidative Stress and Eicosanoids (BIOFORNOX20), Centre of Pharmacology and Toxicology, Hannover Medical School, Hannover, Germany.
J Chromatogr B Analyt Technol Biomed Life Sci. 2016 Apr 15;1019:95-111. doi: 10.1016/j.jchromb.2015.10.009. Epub 2015 Oct 17.
Malondialdehyde (MDA, CH2(CHO)2) is one of the best investigated and most frequently measured biomarkers of lipid peroxidation in biological fluids, a constituent of the so called thiobarbituric acid reactive substances (TBARS). The reaction of thiobarbituric acid with MDA and other carbonyl compounds is the basis for the batch TBARS assay, one of the most commonly and widely used assays of oxidative stress. Yet, the TBARS assay lacks specificity even if combined with HPLC separation prior to visible absorbance or fluorescence detection. In this article, we report highly specific and sensitive stable-isotope dilution GC-MS and GC-MS/MS methods for the quantitative determination of MDA in human plasma (0.1 mL). These methods utilize the acidity (pKa, 4.46) of the two methylene H protons of MDA in aqueous solution, which are as acidic as acetic acid. Endogenous MDA in native plasma and the externally added internal standard [1,3-(2)H2]-MDA (d2-MDA, CH2(CDO)2) are derivatized in aqueous acetone (400 μL) with pentafluorobenzyl (PFB) bromide (10 μL). The reaction products were identified as C(PFB)2(CHO)2 (molecular weight, 432) and C(PFB)2(CDO)2) (molecular weight, 434), respectively. After solvent extraction with toluene (1 mL) quantification is performed by selected-ion monitoring (SIM) in GC-MS and by selected-reaction monitoring (SRM) in GC-MS/MS in the electron-capture negative-ion chemical ionization (ECNICI) mode. In the SIM mode, the anions M-PFB at m/z 251 for MDA and m/z 253 for d2-MDA are detected. In the SRM mode, the mass transitions m/z 251 to m/z 175 for MDA and m/z 253 to m/z 177 for d2-MDA are monitored. The method was thoroughly validated in human plasma. Potential interfering substances including anticoagulants and commercially available monovettes commonly used for blood sampling were tested. The lowest MDA concentrations were measured in serum followed by heparinized and EDTA plasma. The GC-MS and GC-MS/MS methods were found to be specific, precise, accurate and sensitive. Thus, the LOD of the GC-MS/MS method was determined to be 2 amol (2 × 10(-18)mol) MDA. The GC-MS/MS method is exceedingly useful in clinical settings. We report several biomedical applications and discuss the utility of circulating MDA as a biomarker of lipid peroxidation, especially in long-term clinical studies, and its relation to the F2-isoprostane 15(S)-8-iso-prostaglandin F2α and nitric oxide (NO).
丙二醛（MDA，CH2(CHO)2）是生物体液中研究最深入、最常检测的脂质过氧化生物标志物之一，是所谓硫代巴比妥酸反应性物质（TBARS）的一种成分。硫代巴比妥酸与MDA及其他羰基化合物的反应是批量TBARS测定法的基础，该测定法是最常用和广泛应用的氧化应激测定法之一。然而，即使在可见光吸收或荧光检测之前结合高效液相色谱分离，TBARS测定法仍缺乏特异性。在本文中，我们报告了用于定量测定人血浆（0.1 mL）中MDA的高特异性和灵敏的稳定同位素稀释气相色谱 - 质谱联用（GC-MS）和气相色谱 - 串联质谱联用（GC-MS/MS）方法。这些方法利用了MDA在水溶液中两个亚甲基H质子的酸度（pKa，4.46），其酸度与乙酸相当。天然血浆中的内源性MDA和外部添加的内标[1,3-(2)H2]-MDA（d2-MDA，CH2(CDO)2）在400 μL丙酮水溶液中与五氟苄基（PFB）溴（10 μL）进行衍生化反应。反应产物分别鉴定为C(PFB)2(CHO)2（分子量，432）和C(PFB)2(CDO)2（分子量，434）。用甲苯（1 mL）进行溶剂萃取后，通过气相色谱 - 质谱联用中的选择离子监测（SIM）和气相色谱 - 串联质谱联用中的选择反应监测（SRM）在电子捕获负离子化学电离（ECNICI）模式下进行定量。在SIM模式下，检测到MDA的m/z 251处的阴离子M - PFB和d2-MDA的m/z 253处的阴离子。在SRM模式下，监测MDA的m/z 251到m/z 175以及d2-MDA的m/z 253到m/z 177的质量跃迁。该方法在人血浆中进行了全面验证。测试了包括抗凝剂和常用于血液采样的市售单管采血管在内的潜在干扰物质。在血清中测得的MDA浓度最低，其次是肝素化血浆和乙二胺四乙酸（EDTA）血浆。发现气相色谱 - 质谱联用和气相色谱 - 串联质谱联用方法具有特异性、精密度、准确性和灵敏性。因此，气相色谱 - 串联质谱联用方法的检测限确定为2阿摩尔（2×10(-18)摩尔）MDA。气相色谱 - 串联质谱联用方法在临床环境中非常有用。我们报告了几个生物医学应用，并讨论了循环MDA作为脂质过氧化生物标志物的效用，特别是在长期临床研究中，以及它与F2-异前列腺素15(S)-8-异前列腺素F2α和一氧化氮（NO）的关系。
