Zhu Quan-Fei, Yan Jing-Wen, Gao Yang, Zhang Jing-Wei, Yuan Bi-Feng, Feng Yu-Qi
Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, PR China.
Department of Oncology, Zhongnan Hospital of Wuhan University, Wuhan 430071, PR China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2017 Sep 1;1061-1062:34-40. doi: 10.1016/j.jchromb.2017.06.045. Epub 2017 Jun 27.
Recently, a new class of endogenous lipids, branched fatty acid esters of hydroxy fatty acids (FAHFAs), was discovered with anti-diabetic and anti-inflammatory effects in mammals. FAHFAs attracted increasing attention because of their critical physiological function. However, accurate quantitation of FAHFAs is still a challenge due to their high structure similarity and low abundance in biological samples. Herein, we developed a highly sensitive method for the determination of 16 FAHFAs (PAHSAs, OAHSAs, SAHSAs and POHSAs) in biological samples by coupling strong anion exchange solid phase extraction (SAX-SPE) with chemical labeling assisted ultra-high performance liquid chromatography/mass spectrometry (SAX-SPE-CL-UHPLC/MS). In the developed method, SAX-SPE was employed to selectively enrich and purify FAHFAs from biological samples. And then a pair of isotope labeling reagents, 2-dimethylaminoethylamine (DMED) and d-DMED were used to label the purified samples and standard FAHFAs, respectively. The labeled samples were mixed and further subjected to UHPLC/MS analysis. Our results demonstrated that the detection sensitivities of FAHFAs increased by 7-72 folds upon DMED labeling and the limits of detections (LODs) of labeled FAHFAs ranged from 0.01 to 0.14pg. Moreover, a good separation of FAHFAs isomers was achieved on C18 column in a UHPLC system and all FAHFAs could be analyzed in 20min with sharp peak shape. The established method provided substantial sensitivity, high specificity, and broad linear dynamic range (3 orders of magnitude). Using this method, we successfully measured the contents and distribution of FAHFAs in rat white adipose, lung, kidney, thymus, liver and heart tissues. The results showed that 7 FAHFAs (13-, 12-, 9-, 5-PAHSA, 13-, 12- and 9-SAHSA) were observed in different tissues of rat. In addition, we successfully detected the above 7 FAHFAs in human serum samples; and among the 7 FAHFAs, 13-, 9-PAHSA, 13- and 12-SAHSA were found remarkably decreased in human breast cancer serum. The proposed method could be successfully applied for the detection of FAHFAs in various biological samples, which will facilitate the understanding of the physiological functions of FAHFAs.
最近，一类新的内源性脂质——羟基脂肪酸支链脂肪酸酯（FAHFAs）被发现，它在哺乳动物中具有抗糖尿病和抗炎作用。由于其关键的生理功能，FAHFAs受到了越来越多的关注。然而，由于FAHFAs在生物样品中的结构相似性高且丰度低，其准确定量仍然是一个挑战。在此，我们开发了一种高灵敏度的方法，通过将强阴离子交换固相萃取（SAX-SPE）与化学标记辅助超高效液相色谱/质谱联用（SAX-SPE-CL-UHPLC/MS）来测定生物样品中的16种FAHFAs（PAHSAs、OAHSAs、SAHSAs和POHSAs）。在该方法中，SAX-SPE用于从生物样品中选择性富集和纯化FAHFAs。然后，使用一对同位素标记试剂，2-二甲基氨基乙胺（DMED）和d-DMED分别标记纯化后的样品和标准FAHFAs。将标记后的样品混合并进一步进行UHPLC/MS分析。我们的结果表明，DMED标记后FAHFAs的检测灵敏度提高了7至72倍，标记后的FAHFAs检测限范围为0.01至0.14 pg。此外，在UHPLC系统中的C18柱上实现了FAHFAs异构体的良好分离，所有FAHFAs均可在20分钟内以尖锐的峰形进行分析。所建立的方法具有高灵敏度、高特异性和宽线性动态范围（3个数量级）。使用该方法，我们成功测量了大鼠白色脂肪、肺、肾、胸腺、肝脏和心脏组织中FAHFAs的含量和分布。结果表明，在大鼠的不同组织中观察到了7种FAHFAs（13-、12-、9-、5-PAHSA、13-、12-和9-SAHSA）。此外，我们在人血清样品中成功检测到了上述7种FAHFAs；在这7种FAHFAs中，发现13-、9-PAHSA、13-和12-SAHSA在人乳腺癌血清中显著降低。所提出的方法可成功应用于各种生物样品中FAHFAs的检测，这将有助于了解FAHFAs的生理功能。
