粪便内中短链脂肪酸柱前衍生化UHPLC-QTOF-MS/MS分析方法研究  

Determination of medium-and short-chain fatty acids in feces by UHPLC-QTOF-MS/MS with pre-column derivatization

作　　者：丛龙杰 邵玉健 沈红[2] 康东周[3] 徐金娣[2] 周姗姗[1] 李松林[1,2] CONG Long-Jie;SHAO Yu-jianu;SHEN Hong;KANG Dong-zhou;XU Jin-di;ZHOU Shan-shan;LI Song-lin(.Department of Pharmaceutical Analysis,Affiliated Hospital of Integrated Traditional Chinese and Western Medicine to Nanjing University of Chinese Medicine,Nanjing 210028,China;Department of Metabolomics,Jiangsu Province Academy of Traditional Chinese Medicine and Jiangsu Branch of China Academy of Traditional Chinese Medicine,Nanjing 210028,China;Yanbian University,Yanji 133000,China)

机构地区：[1]南京中医药大学附属中西医结合医院中药质量研究室,江苏南京210028 [2]江苏省中医药研究院和中国中医科学院江苏分院中药代谢组研究室,江苏南京210028 [3]延边大学,吉林延吉133000

出　　处：《药学学报》2020年第1期131-138,共8页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金面上项目(81774012)

摘　　要：建立柱前衍生化超高效液相色谱-串联四级杆飞行时间质谱(UHPLC-QTOF-MS/MS)定性、定量表征小鼠粪便内中短链脂肪酸的方法,并用于评价其在抗生素处置前后小鼠粪便样品中的变化。动物实验方案获得江苏省中医药研究院动物实验伦理委员会的批准。以3-硝基苯肼为衍生化试剂,对衍生化反应条件、色谱及质谱条件进行优化,建立了粪便中16个中短链脂肪酸的定性、定量方法。所有分析物在各自浓度范围内线性关系良好(R2>0.99),日内、日间精密度RSD均小于10%,重复性RSD小于6%,加样回收率介于80%~120%之间,样品36 h内稳定性RSD小于7%;联合抗生素处置后小鼠粪便内的中短链脂肪酸种类和含量均发生了明显改变。其中,甲酸、乙酸、丙酸、丁酸、异丁酸、戊酸、乳酸等含量明显降低,而庚酸和丁二酸的含量明显增加。该方法准确、可靠,可作为粪便内中短链脂肪酸的表征方法。A pre-column derivatization and ultra-high performance liquid chromatography quadrupole time-offlight mass spectrometry(UHPLC-QTOF-MS/MS) method was developed for qualitative and quantitative determination of medium-and short-chain fatty acids in mice feces, and was further applied to evaluate variations in the feces of mice before and after antibiotic treatment. This animal experiment had been approved by Animal Experimental Ethics Committee of Jiangsu Province Academy of Traditional Chinese Medicine. By optimizing the derivatization conditions and UHPLC-QTOF-MS/MS parameters a new UHPLC-QTOF-MS/MS method with 3-nitrophenylhydrazine as the derivatization reagent was developed for simultaneous determination of 16 mediumand short-chain fatty acids. Validation studies showed that the linearity of the calibration curves was good(R2>0.99), the RSD of intra-day and inter-day precision was less than 10%, the repeatability RSD was less than 6%, the recovery rate was between 80%-120% at three spiked levels, and the stability RSD was less than 7% within 36 h.The types and amounts of the detected medium-and short-chain fatty acids in feces significantly changed after the mice were treated with antibiotics. The content of formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and lactic acid decreased, whereas that of heptanoic acid and succinic acid increased significantly. All these results suggest that the newly established method is accurate and reliable, and can be used for determination of medium-and short-chain fatty acids in feces.

关 键 词：中短链脂肪酸 UPLC-QTOF-MS/MS 柱前衍生化 粪便 定性定量分析 

分 类 号：R917[医药卫生—药物分析学]