Guo Xiaochun, Xie Ping, Chen Jun, Tuo Xun, Deng Xuwei, Li Shangchun, Yu Dezhao, Zeng Cheng
College of Fisheries, Huazhong Agricultural University, Wuhan, Hubei 430070, China.
Donghu Experimental Station of Lake Ecosystems, State Key Laboratory of Freshwater Ecology and Biotechnology of China, Institute of Hydrobiology, Chinese Academy of Sciences, Donghu South Road 7, Wuhan 430072, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Jul 15;963:54-61. doi: 10.1016/j.jchromb.2014.05.046. Epub 2014 Jun 2.
The roles of glutathione (GSH) and cysteine (Cys) in the detoxification of Microcystin-LR (MC-LR) have recently become a popular area of research. However, lacking analysis methods for MC-LR-GSH and MC-LR-Cys (two main GSH pathway metabolites) in mammals, elucidation of the detoxification mechanism and metabolic pathway of MC-LR in mammals is difficult. In this study, a novel method for the simultaneous quantitative analysis of MC-LR, MC-LR-GSH and MC-LR-Cys in rat liver was developed and validated. The analytes were simultaneously extracted from rat liver using 3M sodium chloride solution containing 0.01M EDTA-Na2-5% acetic acid, followed by solid-phase extraction (SPE) on Oasis HLB and silica cartridges and determination by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS/MS). Under the optimized pretreatment conditions and instrument parameters, good recoveries of MC-LR, MC-LR-GSH and MC-LR-Cys were obtained at three concentrations (0.2, 1.0 and 2.5 μg g(-1) dry weight (DW)) with values ranging from 97.7 ± 4.2 to 98.7 ± 5.1%, 70.1 ± 4.8 to 71.1 ± 4.1% and 79.8 ± 3.5 to 81.4 ± 4.0%, respectively. The relative standard deviations (RSDs) of these compounds at 0.2, 1.0 and 2.5 μg g(-1) DW were between 4.3% and 6.9%. The limits of detection (LODs) were 0.005, 0.007 and 0.006 μg g(-1) DW and the limits of quantification (LOQs) were 0.017, 0.023 and 0.020 μg g(-1) DW for MC-LR, MC-LR-GSH and MC-LR-Cys, respectively. Furthermore, this method was successfully applied to both time- and dosage-effect studies of MC-LR, MC-LR-GSH and MC-LR-Cys in vivo.
谷胱甘肽（GSH）和半胱氨酸（Cys）在微囊藻毒素-LR（MC-LR）解毒过程中的作用，近来已成为热门研究领域。然而，由于缺乏针对哺乳动物体内MC-LR-GSH和MC-LR-Cys（GSH途径的两种主要代谢产物）的分析方法，因此难以阐明MC-LR在哺乳动物体内的解毒机制和代谢途径。在本研究中，开发并验证了一种同时定量分析大鼠肝脏中MC-LR、MC-LR-GSH和MC-LR-Cys的新方法。使用含有0.01M EDTA-Na2-5%乙酸的3M氯化钠溶液从大鼠肝脏中同时提取分析物，随后在Oasis HLB和硅胶柱上进行固相萃取（SPE），并通过液相色谱-电喷雾电离质谱（LC-ESI-MS/MS）进行测定。在优化的预处理条件和仪器参数下，MC-LR、MC-LR-GSH和MC-LR-Cys在三个浓度（0.2、1.0和2.5 μg g(-1)干重（DW））下的回收率良好，分别为97.7±4.2%至98.7±5.1%、70.1±4.8%至71.1±4.1%和79.8±3.5%至81.4±4.0%。这些化合物在0.2、1.0和2.5 μg g(-1) DW时的相对标准偏差（RSD）在4.3%至6.9%之间。MC-LR、MC-LR-GSH和MC-LR-Cys的检测限（LOD）分别为0.005、0.007和0.006 μg g(-1) DW，定量限（LOQ）分别为0.017、0.023和0.020 μg g(-1) DW。此外，该方法已成功应用于MC-LR、MC-LR-GSH和MC-LR-Cys在体内的时间和剂量效应研究。
