Iron Redox Speciation Analysis Using Capillary Electrophoresis Coupled to Inductively Coupled Plasma Mass Spectrometry (CE-ICP-MS)
Iron Redox Speciation Analysis Using Capillary Electrophoresis Coupled to Inductively Coupled Plasma Mass Spectrometry (CE-ICP-MS)
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DOI：
10.3389/fchem.2019.00136
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发表时间：
2019-03-14
影响因子：
5.5
通讯作者：
Venkataramani, Vivek
中科院分区：
文献类型：
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作者：
Michalke, Bernhard;Willkommen, Desiree;Venkataramani, Vivek
多种神经退行性疾病均发生神经元铁平衡失调。铁(II)/铁（III）向铁（II）的比值变化与氧化应激、脂质过氧化密切相关，是铁死亡的标志特征。特别是对于体液，如脑脊液（CSF），需要可靠的定量方法进行铁(II)/(III)氧化还原-形态分析，以更好地评估铁（II）介导的脑组织损伤的风险。目前在金属组学领域，分析这两种铁最直接的方法是LC-ICP-MS。然而，这种Fe(II)/(III)形态分析方法存在一些局限性。在这里，我们描述了一种独特的方法，使用毛细管电泳(CE)-ICP-MS进行定量铁(II)/(III)形态分析，可用于细胞裂解物和生物流体样品。与LC相比，CE具有以下优点：(1)毛细管没有固定相，不依赖于固定相的批次一致性；(2)老化或堵塞的毛细血管更换迅速，无性能变化；(3)吹扫步骤有效，时间短；(4)样品分析时间短。最后一种方法采用20mM盐酸作为背景电解液，分离电压为+ 25kv。与lc法相反，没有使用铁与吡啶二羧酸（PDCA）络合，因为它阻碍了分离。电导率- ph -复合叠加提高了峰形和浓度检出限，在13 nL注射量下达到3 μ g/L检出限（3 s）。从LOD-150 μ g/L开始，r(2) [Fe(II)] = 0.9999, r(2) [Fe(III)] = 0.9951呈线性关系。在较高浓度下，Fe（II）曲线明显变平。测量精度为3.5%[铁（II）， 62 μ g/L]或2.2%[铁（III）， 112 μ g/L]，铁（III）和铁（II）的迁移时间精度分别为2%和3%，分别在1:2稀释的人神经母细胞瘤细胞溶解物中测定。采用经验证的LC-ICP-MS法平行测定SH-SY5Y细胞裂解液，并在标准加入后进行回收率实验，以验证浓度测定的准确性。准确度（n = 6）为97.6 +/- 3.7% Fe（III）和105 +/- 6.6% Fe（II）。回收率[(a) + 33 μ g/L或(b) + 500 μ g/L，每个物种]分别为(a):97.2 +/- 13% [Fe(II)], 108 +/- 15% [Fe(III)], 102.5 +/- 7%（种总数）；(b) 99 +/- 4% [Fe(II)], 101 +/- 6% [Fe(III)], 100 +/- 5%（种总数）。脑脊液样品的迁移时移是由于高盐度造成的，但两种铁都是通过标准添加来鉴定的。
Neuronal iron dyshomeostasis occurs in multiple neurodegenerative diseases. Changes in the Fe(II)/Fe(III) ratio toward Fe(II) is closely related to oxidative stress, lipid peroxidation, and represents a hallmark feature of ferroptosis. In particular for body fluids, like cerebrospinal fluid (CSF), reliable quantitative methods for Fe(II)/(III) redox-speciation analysis are needed to better assess the risk of Fe(II)-mediated damage in brain tissue. Currently in the field of metallomics, the most direct method to analyze both iron species is via LC-ICP-MS. However, this Fe(II)/(III) speciation analysis method suffers from several limitations. Here, we describe a unique method using capillary electrophoresis (CE)-ICP-MS for quantitative Fe(II)/(III) speciation analysis that can be applied for cell lysates and biofluid samples. Compared to LC, CE offers various advantages:(1) Capillaries have no stationary phase and do not depend on batch identity of stationary phases; (2) Replacement of aged or blocked capillaries is quick with no performance change; (3) Purge steps are effective and short; (4) Short sample analysis time. The final method employed 20mM HCl as background electrolyte and a separation voltage of +25 kV. In contrary to the LC-method, no complexation of Fe-species with pyridine dicarboxylic acid (PDCA) was applied, since it hampered separation. Peak shapes and concentration detection limits were improved by combined conductivity-pH-stacking achieving 3 mu g/L detection limit (3 s) at 13 nL injection volume. Calibrations from LOD-150 mu g/L were linear [r(2) [Fe(II)] = 0.9999, r(2) [Fe(III)] = 0.9951]. At higher concentrations Fe(II) curve flattened significantly. Measurement precision was 3.5% [Fe(II) at 62 mu g/L] or 2.2% [Fe(III) at 112 mu g/L] and migration time precision was 2% for Fe(III) and 3% for Fe(II), each determined in 1:2 diluted lysates of human neuroblastoma cells. Concentration determination accuracy was checked by parallel measurements of SH-SY5Y cell lysates with validated LC-ICP-MS method and by recovery experiments after standard addition. Accuracy (n = 6) was 97.6 +/- 3.7% Fe(III) and 105 +/- 6.6% Fe(II). Recovery [(a) + 33 mu g/L or (b) + 500 mu g/L, addition per species] was (a):97.2 +/- 13% [Fe(II)], 108 +/- 15% [Fe(III)], 102.5 +/- 7% (sum of species), and (b) 99 +/- 4% [Fe(II)], 101 +/- 6% [Fe(III)], 100 +/- 5% (sum of species). Migration time shifts in CSF samples were due to high salinity, but both Fe-species were identified by standard addition.