Yang Wen-Chu, Adamec Jiri, Regnier Fred E
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA.
Anal Chem. 2007 Jul 15;79(14):5150-7. doi: 10.1021/ac070311t. Epub 2007 May 11.
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 approximately 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. 2H-Coding was achieved through derivatization with deuterated 3-carbinol-1-methyl-d3-pyridinium iodide. The 2H-coded derivatization reagent, 3-acyloxymethyl-1-methyl-d3-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.
本文重点介绍一种通过衍生化来鉴定和定量脂肪酸的增强型液相色谱/电喷雾电离质谱（LC/ESI-MS）方法。脂肪酸用2-溴-1-甲基碘化吡啶和3-甲醇-1-甲基碘化吡啶进行衍生化，形成3-酰氧基甲基-1-甲基碘化吡啶（AMMP）。该过程将季铵基团连接到分析物上，并能在使用常见液相色谱流动相的正离子模式下进行电喷雾电离质谱分析。此外，检测灵敏度通常比未衍生化脂肪酸在负离子模式下高2500倍。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），线性范围跨越约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处有常见离子。每种脂肪酸也有独特的指纹区域，可用于识别其碳骨架数量、双键数量和双键位置。衍生化方法还允许对分析物进行编码，作为识别衍生物和增强定量的一种手段。通过用氘代的3-甲醇-1-甲基-d3-碘化吡啶进行衍生化实现2H编码。2H编码的衍生化试剂3-酰氧基甲基-1-甲基-d3-碘化吡啶有两种使用方式。一种是对样品的等量部分进行差异标记，使得在重新组合并通过电喷雾电离质谱分析后，所有脂肪酸都呈现为离子双峰簇，间隔约3 amu。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在比较定量中。对照样品和实验样品分别用CPM的非氘代和氘代同位素异构体进行差异标记。将两个样品混合后，通过电喷雾电离质谱进行分析。实验样品中脂肪酸相对于对照的丰度通过同位素异构体脂肪酸的同位素比来确定。通过向含有未知量脂肪酸的实验样品中添加差异标记的脂肪酸标准品实现绝对定量。该方法的实用性在人血清样品分析中得到了检验。
