稳定同位素内标高效液相色谱串联质谱检测果蝇体内γ-氨基丁酸  

Determination ofγ-aminobuyric acid in Drosophila by high performance liquid chromatography-tandem mass spectrometry using stable isotopelabelled internal standards

作　　者：孙博群 张紫燕[1,3,4] 唐超 蔡澎[1,3,4] SUN Bo-qun;ZHANG Zi-yan;TANG Chao;CAI Peng(Key Lab of Urban Environment and Health,Institute of Urban Environment,Chinese Academy of Sciences,Xiamen,Fujian 361021,China;University of Chinese Academy of Sciences,Beijing 100049,China;Xiamen Key Laboratory of Physical Environment,Xiamen,Fujian 361021,China;Shanghai Institute of Nutrition and Health,Chinese Academy of Sciences,Shanghai 200031,China)

机构地区：[1]中国科学院城市环境研究所环境与健康重点实验室,福建厦门361021 [2]中国科学院大学,北京100049 [3]厦门市物理环境重点实验室,福建厦门361021 [4]中国科学院上海营养与健康研究所,上海200031

出　　处：《军事医学》2022年第7期540-545,共6页Military Medical Sciences

基　　金：中国科学院重点部署项目(KGFZD-135-160-024)。

摘　　要：目的建立稳定同位素内标高效液相色谱串联质谱法(HPLC-MS/MS)定量检测果蝇体内γ-氨基丁酸(GABA)。方法制备果蝇匀浆,以水为提取剂,甲醇为沉淀剂,加入同位素内标GABA-d6,离心取上清过滤膜用于HPLC-MS/MS检测分析。选用岛津Shim-pack GIST C18-AQ(1.9μm,2.1 mm×50 mm)色谱柱,以0.1%甲酸水-甲醇(30∶70,V/V,0.2 ml/min)为流动相等度洗脱;采用电喷雾离子源(ESI),在正离子扫描下,选择多反应监测(MRM)模式对GABA(m/z 104→87)及稳定同位素内标GABA-d6(m/z 110→93)进行检测。利用机械振荡对果蝇进行夜间睡眠剥夺,检测睡眠补偿期果蝇体内GABA含量。结果通过同位素内标法校正了基质效应,GABA在1.0~400.0 ng/ml范围内线性关系良好,相关系数(R2)≥0.99,定量下限(LOQ)为1.0 ng/ml。在低、中、高3个加标水平下的加标回收率为92.41%~110.05%,相对标准偏差(RSD)为0.84%~4.47%。与对照组相比,睡眠补偿期果蝇每小时睡眠时间显著增加,体内GABA含量显著升高。结论该方法灵敏度高,准确度和精密度较好,能准确、快速、简便地定量检测果蝇体内GABA含量。Objective To establish an HPLC-MS/MS with the stable isotope-labelled internal standard method for quantitative determination of gamma-aminobuyric acid(GABA)in Drosophila.Methods Flies were homogenized by a tissue homogenizer and extracted with water.MeOH was used for precipitation while GABA-d6 was added as the isotopelabelled internal standard and centrifuged at 12000 r/min for 10 min.The clear supernatant was determined by HPLCMS/MS after being filtered through a 0.22μm filter membrane.Chromatographic separation was achieved using a Shim-pack GIST C18-AQ(1.9μm,2.1 mm×50 mm)column at 40℃with the mobile phase composed of 0.1%formic acid-water and MeOH(30∶70,V/V,0.2 ml/min).Mass spectrum conditions included ESI(+)that was performed in the MRM mode using target ions m/z 104→87(GABA)and m/z 110→93(GABA-d6).GABA in flies was determined during sleep compensation after a night of sleep deprivation.Results The matrix effect was corrected by the isotope internal standard method.The calibration curve was linear over the range of 1-400 ng/ml.The LOQ of GABA in flies was 1.0 ng/ml.The extracted recovery was above 90%and the RSD was below 5%.Compared with the control groups,the duration of sleep per hour and the level of GABA were significantly increased during sleep compensation.Conclusion This analytical method is sensitive,accurate and precise,so it can be used for quantitative determination of GABA in Drosophila.

关 键 词：高效液相串联质谱 稳定同位素内标 果蝇 Γ-氨基丁酸 睡眠 

分 类 号：O657.63[理学—分析化学]