果蝇组织中多不饱和脂肪酸代谢产物--类二十烷酸的高效液相色谱-串联质谱法测定

Determination of the metabolites, eicosanoids derived from polyunsaturated fatty acids in Drosophila tissue with high performance liquid chromatography tandem mass spectrometry

果蝇样品经固相萃取后,采用高效液相色谱-串联质谱法进行样品分析,检测条件用Endeavorsil^(TM)C_(18)色谱柱(100 mm×2.1 mm,1.8μm),以0.1%甲酸水-乙腈为流动相,流速为0.3 m L/min,柱温40℃;采用电喷雾离子源负离子多重反应监测模式,内标标准工作曲线定量,建立了果蝇组织中15种多不饱和脂肪酸代谢产物——类二十烷酸的测定方法。结果显示,在2.5~200 ng/m L范围内呈良好的线性关系(r=0.991),回收率范围在89.3%~108.9%,日内精密度相对标准偏差(relative standard deviation,RSD)在1.2%~12.4%,日间精密度RSD在1.0%~15.0%,检出限0.1~2.6 ng/g,定量限0.3~8.7 ng/g。所建方法简便、准确、选择性高,为研究模式生物果蝇对多不饱和脂肪酸的代谢特性提供了科学依据。

Eicosanoids, which mediate various physiological and pathophysiological processes, are mainly formed from C20 polyunsaturated fatty acids （PUFAs） such as arachidonic acid （AA, 20：4n-6） and eicosapentaenoic acid （EPA, 20： 5n-3） through cyclooxygenases （COX）, lipoxygenases （LOXs） and cytochrome P450 （CYPs） pathways, including prostaglandins （PGs）, thromboxanes （TXs） and the lipoxin/leukotriene family of eicosanoids such as hydroperoxyeicosatetraenoic acids （ HPETEs ）, hydroxyeicosatetraenoic acids （ HETEs ）, and epoxyeicosatrienoic acids （EETs）. Vast knowledge of eieosanoids stems from works in mammals. Lipid signaling that complicates our understanding of fatty acid signaling is highly complex and fine-tuned in mammal species. Fortunately, the small genetic model Drosophila melanogaster is considered to be an ideal model to investigate the flexible nature of eicosanoids signaling pathways. However, it seems that Drosophila possess a special lipid metabolic system which is different （rom mammals. Thus, before studying the physiological mechanism of eicosanoids by using Drosophila, it is necessary to clarify its metabolic characteristics to C20 PUFAs based on the detection of eicosanoids in Drosophila. Therefore, this study is aimed to develop a high performance liquid chromatography tandem mass spectrometry （HPLC-MS/MS） method for the determination of eicosanoids in Drosophila. Fifteen metabolites of AA and EPA produced by human in COX, LOX and CYP pathway were chosen, and each pathway contained 1 or 2 metabolites as delegates to ensure the representative of the method. These eicosanoids included PGF2~, PGE2, PGFao, PGE3, 15（S）-HETE, LTB4, 15（S）-HEPE, 11（12）-EET, 20-HETE, 17（18）- EpETE, 17, 18-DiHETE, 15（S）-HpEPE, 15（S）-HpETE, PGH2 and 5（S）-HpETE, and PGE2 d4, 15（S）- HETE-d8 and 20-HETE-d6 were used as internal standards. Samples were prepared by solid phase extraction and separated on an EndeavorsilTM C18 column （100 mm×2.1 mm,1.8μm）. The analytes were detected by using multiple reaction monitoring （MRM） in a negative electrospray ion mode. The HPLC-MS/MS method to analyze 15 selected eicosanoids in Drosophila qualitatively and quantitatively was established by optimizing the sample pretreatment and the detection conditions. It was found that the recoveries were significantly influenced by the pH of sample adjusted by 1 mol/L sodium acetate buffer containing 5% methanol, and the optimized pH was at 6. The response values of analytes separated on a mobile phase of ultrapure water with 0. 1 % formic acid acetonitrile were higher than these of ultrapure water with 0.1% formic acid-methanol, ultrapure water acetonitrile or ultrapure water methanol. To reduce the matrix interference, the blank Drosophila matrix was used to prepare the standard working solution. Obtained results showed that the calibration curves were of good linearity for the 15 metabolites in the range of 2.5 - 100 ng/mL with the correlation coefficient （r） of 0. 991. The limits of detection and quantitation were about 0. 1 - 2.6 ng/g and 0.3 -8.7 ng/g, respectively. Spiked recovery experiments showed that both recoveries （89.3%- 111.5%） and relative standard deviations （1.0 %0- 15.0% ） met the requirements of analytical methods. In conclusion, our study has established a simple, specific and sensitive method that suitable for the determination of eicosanoids in Drosophila which serves an approach to clarify the metabolic characteristics of Drosophila to C20 PUFAs.