Enhancement of the LC/MS analysis of fatty acids through derivatization and stable isotope coding
Enhancement of the LC/MS analysis of fatty acids through derivatization and stable isotope coding
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DOI：
10.1021/ac070311t
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发表时间：
2007-07-15
影响因子：
7.4
通讯作者：
Regnier, Fred E.
中科院分区：
文献类型：
--
作者：
Yang, Wen-Chu;Adamec, Jiri;Regnier, Fred E.
本文着重于开发一种通过衍生化鉴定和定量脂肪酸的增强LC/ESI-MS方法。脂肪酸用2-溴-1-甲基碘化吡啶和3-甲醇-1-甲基碘化吡啶衍生，形成3-酰氧基甲基-1-甲基碘化吡啶（AMMP）。该过程将季胺连接到分析物上，并使ESI-MS能够以正离子模式与常见的LC移动的相进行电离。此外，检测灵敏度通常是2500倍，高于使用未衍生化脂肪酸的负离子化模式。对于C10至C24的标准脂肪酸，检测限约为1.0-4.0 nM（或10 pg/进样），线性范围约为2个数量级。AMMP衍生物具有独特的串联质谱，其特征在于m/z 107.0、124.0和178.0处的常见离子。单个脂肪酸也有独特的指纹区域，可以识别它们的碳骨架数，双键数和双键位置。衍生化方法还允许编码分析物作为识别衍生物和增强定量的手段。H-2-编码通过用氘代3-甲醇-1-甲基-d（3）-碘化吡啶鎓衍生化来实现。以两种方式使用H-2编码的衍生化试剂3-酰氧基甲基-1-甲基-d（3）-碘化吡啶鎓。一种是对样品的相等部分进行差异标记，使得在重组并通过ESI-MS分析后，所有脂肪酸都表现为分离约3 amu的离子的双峰簇。这极大地促进了复杂混合物中脂肪酸的鉴定。稳定同位素编码的另一个用途是比较定量。对照和实验样品分别用CPM的非氘代和氘代同位素异构体进行差异标记。混合两种样品后，通过ESI-MS对其进行分析。通过同位素异构脂肪酸的同位素比确定实验样品中脂肪酸相对于对照的丰度。通过将差异标记的脂肪酸标准品加入到含有未知量脂肪酸的实验样品中来实现绝对定量。在人血清样品的分析中检查了该方法的实用性。
This paper focuses on the development of an enhanced LC/ESI-MS method for the identification and quantification of fatty acids through derivatization. Fatty acids were derivatized with 2-bromo-1-methylpyridinium iodide and 3-carbinol-1-methylpyridinium iodide, forming 3-acyloxymethyl-1-methylpyridinium iodide (AMMP). This process attaches a quaternary amine to analytes and enabled ESI-MS in the positive mode of ionization with common LC mobile phases. Moreover, detection sensitivity was generally 2500-fold higher than in the negative mode of ionization used with underivatized fatty acids. The limits of detection were roughly 1.0-4.0 nM (or 10 pg/injection) for standard fatty acids from C10 to C24 and spanned similar to 2 orders of magnitude in linearity. AMMP derivatives had unique tandem mass spectra characterized by common ions at m/z 107.0, 124.0, and 178.0. Individual fatty acids also had unique fingerprint regions that allowed identification of their carbon skeleton number, number of double bonds, and double bond position. The derivatization method also allowed coding of analytes as a means of recognizing derivatives and enhancing quantification. H-2-Coding was achieved through derivatization with deuterated 3-carbinol-1-methyl-d(3)-pyridinium iodide. The H-2-coded derivatization reagent, 3-acyloxymethyl-1-methyl-d(3)-pyridinium iodide, was used in two ways. One was to differentially label equal fractions of a sample such that after being recombined and analyzed by ESI-MS all fatty acids appeared as doublet clusters of ions separated by roughly 3 amu. This greatly facilitated identification of fatty acids in complex mixtures. Another use of stable isotope coding was in comparative quantification. Control and experimental samples were differentially labeled with nondeuterated and deuterated isotopomers of CPM, respectively. After mixing the two samples, they were analyzed by ESI-MS. The abundance of a fatty acid in an experimental sample relative to the control was established by the isotope ratio of the isotopomeric fatty acids. Absolute quantification was achieved by adding differentially labeled fatty acid standards to experimental samples containing unknown quantities of fatty acids. Utility of the method was examined in the analysis of human serum samples.