Production of isotopically labeled standards from a uniformly labeled precursor for quantitative volatile metabolomic studies.
Production of isotopically labeled standards from a uniformly labeled precursor for quantitative volatile metabolomic studies.
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DOI：
10.1021/ac300933d
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发表时间：
2012-06-19
影响因子：
7.4
通讯作者：
Sacks, Gavin L.
中科院分区：
文献类型：
--
作者：
Gomez-Cortes, Pilar;Brenna, J. Thomas;Sacks, Gavin L.
通过质谱法进行小分子分析的最佳准确性和精密度通常需要在化学上代表所有感兴趣化合物的同位素标记标准品。然而，从市售纯化合物制备混合标准品通常非常昂贵且耗时，并且许多标记化合物不能以纯形式获得。我们使用统一标记的 [U-13C]-化合物的单一原型来生成 [U-13C]-挥发性标准品，用于后续的实验分析研究。 [U-13C]-α-亚麻酸（C18:3n-3，ALA）被热氧化产生标记的脂质降解挥发物，随后对其进行定性和定量表征。通过顶空固相微萃取-气相色谱-飞行时间质谱 (HS-SPME-GC-TOF-MS) 与未标记挥发物的光谱比较，鉴定了 25 种 [U-13C] 标记的挥发物。使用 250 μL 起始样品，通过反向同位素稀释程序对标记的挥发物进行定量。使用 [U-13C] 标记的标准品，检测限达到了与之前的 HS-SPME 报告相当或更好的水平，即 0.010–1.04 ng/g。使用在 60°C 氧化 0 至 15 d 的商品大豆油 (CSO) 评估 [U-13C]-挥发性标准品的性能。当 CSO 基质被氧化时，正癸烷（混合物中不存在的未标记内标）和 [U-13C]-氧化产物的相对响应变化高达 8 倍，这表明在挥发性分析研究中依赖单一标准会因基质效应的变化而产生不准确的结果。 [U-13C]-标准混合物用于定量氧化 CSO 和低 ALA 大豆油中的 25 种挥发物，平均相对标准偏差为 8.5%。将此方法扩展到其他标记底物（例如 [U-13C]-糖和氨基酸）进行分析研究应该是可行的，并且与使用单一标准品相比可以显着改善定量结果。
Optimal accuracy and precision in small molecule profiling by mass spectrometry generally requires isotopically labeled standards chemically representative of all compounds of interest. However, preparation of mixed standards from commercially available pure compounds is often prohibitively expensive and time consuming, and many labeled compounds are not available in pure form. We used a single prototype uniformly labeled [U-13C]-compound to generate [U-13C]-volatile standards for use in subsequent experimental profiling studies. [U-13C]-α-linolenic acid (C18:3n-3, ALA) was thermally oxidized to produce labeled lipid degradation volatiles which were subsequently characterized qualitatively and quantitatively. Twenty-five [U-13C]-labeled volatiles were identified by headspace solid-phase microextraction-gas chromatography-time of flight-mass spectrometry (HS-SPME-GC-TOF-MS) by comparison of spectra with unlabeled volatiles. Using 250 μL starting sample, labeled volatiles were quantified by a reverse isotope dilution procedure. Using the [U-13C]-labeled standards, limits of detection comparable to or better than previous HS-SPME reports were achieved, 0.010–1.04 ng/g. The performance of the [U-13C]-volatile standards was evaluated using a commodity soybean oil (CSO) oxidized at 60°C from 0 to 15 d. Relative responses of n-decane, an unlabeled internal standard otherwise absent from the mixture, and [U-13C]-oxidation products changed by up to 8-fold as the CSO matrix was oxidized, demonstrating that reliance on a single standard in volatile profiling studies yields inaccurate results due to changing matrix effects. The [U-13C]-standard mixture was used to quantify 25 volatiles in oxidized CSO and low-ALA soybean oil with an average relative standard deviation of 8.5%. Extension of this approach to other labeled substrates, e.g., [U-13C]-sugars and amino acids, for profiling studies should be feasible and can dramatically improve quantitative results compared to use of a single standard.
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