Development of an Online-SPE–LC–MS/MS Method for 26 Hydroxylated Polyunsaturated Fatty Acids as Rapid Targeted Metabolomics Approach for the LOX, CYP, and Autoxidation Pathways of the Arachidonic Acid Cascade
Development of an Online-SPE–LC–MS/MS Method for 26 Hydroxylated Polyunsaturated Fatty Acids as Rapid Targeted Metabolomics Approach for the LOX, CYP, and Autoxidation Pathways of the Arachidonic Acid Cascade
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DOI：
10.1007/s10337-014-2768-8
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发表时间：
2015-03
期刊：
影响因子：
1.7
通讯作者：
A. I. Ostermann;Ina Willenberg;K. Weylandt;N. Schebb
中科院分区：
文献类型：
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作者：
A. I. Ostermann;Ina Willenberg;K. Weylandt;N. Schebb
羟基化脂肪酸（OH-FA）在花生四烯酸（AA）级联的所有分支中由多不饱和脂肪酸（PUFA）形成。OH-FAs作为有效的脂质介质，并作为AA级联途径的活性标志物，特别是脂氧合酶分支。目前的AA级联靶向代谢组学方法涵盖了其他氧脂类中的几种OH-FA，但它们需要较长的运行时间和费力的样品制备。在本研究中，我们开发了一种新的快速LC-MS方法与自动化样品制备的同时定量26 OH-FA在6.5分钟内。加入四种同位素标记的内标物并离心后，直接进样粗生物样品。在Oasis HLB色谱柱上，以3.5 mL min− 1流速，通过在线固相萃取从基质中提取分析物。在RP-18色谱柱上用1.3 µm融合核心颗粒进行LC分离。该方法灵敏度高，检测限为0.5-10 fmol，线性范围宽。对细胞培养基以及人血浆中分析物的批内和批间精密度和准确度进行了表征，发现通常在100 ± 15%范围内。该方法被施加到OH-FA的形成在五个细胞系的AA，二十碳五烯酸（EPA），二十二碳六烯酸（DHA）孵育后的调查。结肠癌细胞系HCA-7和SW-480以及成纤维细胞系Balb/c 3 T3在细胞培养基中显示出显著的OH-FA形成，其中由前体PUFA主要形成15-HETE、18-HEPE、20-HDHA和8-HDHA。
Hydroxylated fatty acids (OH-FAs) are formed in all branches of the arachidonic acid (AA) cascade from polyunsaturated fatty acids (PUFA). OH-FAs act as potent lipid mediators and serve as activity marker for pathways of the AA cascade, particularly the lipoxygenase branch. Current targeted metabolomics methods of the AA cascade cover several OH-FAs among other oxylipins, yet they require long runtimes and laborious sample preparation. In the present study, we developed a new rapid LC–MS method with automated sample preparation for the simultaneous quantification of 26 OH-FAs within 6.5 min. Crude biological samples are directly injected following addition of four isotopically labeled internal standards and centrifugation. The analytes are extracted from the matrix by means of online solid phase extraction on an Oasis HLB column at 3.5 mL min−1flow rate. LC separation was carried out on a RP-18 column with fused core 1.3 µm particles. The method showed a high sensitivity with a limit of detection of 0.5–10 fmol on column and a broad linear range. Intra- and inter-batch precision and accuracy for the analytes were characterized for cell culture medium as well as human plasma and were found to be generally within 100 ± 15 %. The method was applied to the investigation of OH-FA formation in five cell lines following incubation with AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). The colon cancer cell lines HCA-7 and SW-480, as well as the fibroblast line Balb/c 3T3 showed significant formation of OH-FAs in the cell culture medium, with dominant formation of 15-HETE, 18-HEPE, 20-HDHA, and 8-HDHA from the precursor PUFAs.