Demin P, Reynaud D, Pace-Asciak C R
Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Anal Biochem. 1995 Apr 10;226(2):252-5. doi: 10.1006/abio.1995.1222.
Facile methods for the detection of intact hepoxilins, monohydroxy-epoxide derivatives of arachidonic acid formed through the 12-lipoxygenase pathway, are unavailable because (i) an absence in these compounds of an appropriate chromophore for sensitive detection by uv exists, (ii) these compounds are sensitive to the acidic workup leading to varying degrees of decomposition, and (iii) they decompose to the derivatization procedures required for their analysis by gas chromatography mass spectrometry. Herein we apply a method which introduces a fluorescent ester chromophore to the carboxylic group of the hepoxilins under conditions which do not require acidification leading to stabilization of the derivative which is extracted into an organic solvent in situ. This procedure quantitatively derivatizes hepoxilins in a biological sample, permitting the detection of hepoxilins after a TLC purification with a limit of 50 pg/sample. This method permits the profiling of 12-HETE, hepoxilins A3 and B3, as well as the corresponding epoxide hydrolase products, trioxilins A3 and B3, in a biological sample by reverse-phase HPLC with fluorescent detection. We also report on the fluorescent and mass spectral properties of these derivatives using a liquid chromatography mass spectrometry LCMS interface with thermospray ionization.
用于检测完整肝氧杂环素（通过12-脂氧合酶途径形成的花生四烯酸单羟基环氧化物衍生物）的简便方法尚不存在，原因如下：（i）这些化合物缺乏可用于紫外光灵敏检测的合适发色团；（ii）这些化合物对酸性后处理敏感，会导致不同程度的分解；（iii）它们会分解，无法进行气相色谱质谱分析所需的衍生化程序。在此，我们应用一种方法，在不需要酸化的条件下，将荧光酯发色团引入肝氧杂环素的羧基，从而使衍生物稳定化，并原位萃取到有机溶剂中。该程序可对生物样品中的肝氧杂环素进行定量衍生化，经薄层色谱纯化后，可检测低至50 pg/样品的肝氧杂环素。此方法可通过反相高效液相色谱荧光检测，对生物样品中的12-羟基二十碳四烯酸（12-HETE）、肝氧杂环素A3和B3以及相应的环氧水解酶产物三氧杂环素A3和B3进行分析。我们还使用热喷雾电离的液相色谱质谱联用（LCMS）接口，报告了这些衍生物的荧光和质谱特性。
