摘要
采用HPLC-MS/MS监控反应,合成了微囊藻毒素RR的谷胱甘肽和半胱氨酸代谢物(MC-RRGSH及MC-RR-Cys)。该产物经Accu BOND C18固相萃取小柱纯化后,纯度大于95%(HPLC检测)。结果表明,合成反应的最佳时间为60 min。在SPE洗脱过程中,采用纯水淋洗,1%甲酸-90%甲醇洗脱时,MCRR-GSH和MC-RR-Cys的平均回收率可达86.4%±1.5%和90.2%±2.8%。运用ESI-HPLC-MS/MS对以上产物进行一级及二级质谱扫描。两种产物的一级质谱电离均以[M+2H]2+为基峰,对应的质荷比(m/z)分别为673.6和580.5。以MC-RR-GSH和MC-RR-Cys一级质谱图中丰度最高的双电荷离子为母离子进行二级质谱扫描,对应的子离子对为m/z 609.0/536.9和519.4/520.8,由上述裂解碎片推断合成的产物为MC-RR-GSH和MC-RR-Cys。以合成产物为标准品,测定出太湖鲤鱼肾脏中MC-RR-GSH,MC-RR-Cys和MC-RR的含量(干重)分别为未检出,1.587 5,0.001 5μg·kg-1。本文合成的高纯度代谢产物,可作为相关研究中非商品化的标准对照品,代谢产物的质谱裂解碎片亦可为环境中微囊藻毒素MC-RR的GSH/Cys代谢途径分析提供参考。
GSH and Cys metabolites of microcystin- RR（ MC- RR） were synthesized chemically by the monitoring of high liquid chromatography- electrospray ionization ion trap mass spectrometry（ HPLC- ESI- ITMS）. Based on the enrichment and purification of Accu BOND C18 solid phase extraction（ SPE）,the purity of these s-conjugates were higher than 95% by HPLC. In this study,the optimum reaction time and SPE cartridges treatment procedure were also studied,and 60 min was obtained as the optimal time for the s-conjugates synthesis. Extraction parameters including proportions of methanol in elution and washing solution were optimized,and the best mean recoveries were obtained by 90% methanol in elution after water washing,and their recoveries were in the ranges of86. 4% ± 1. 5% and 90. 2% ± 2. 8% for GSH conjugates of MC- RR（ MC- RR- GSH） and Cys conjugates of MC- RR（ MC- RR- Cys）,respectively. These metabolites were subsequently confirmed by high phase liquid chromatography- electrospray ionization ion trap mass spectrometry（ HPLC- ESI- ITMS）. The fragment ions of the metabolites of MC- RR were characterized,and the most aboundant ions for MC- RR- GSH and MC- RR- Cys were at m / z 673. 6 and m / z 580. 5,respectively,and these ions were further selected as precursor ion in MS / MS,while the product ions of these parent ions were at m / z 609. 0 /536. 9 and 519. 4 /520. 8,respectively. These purified me-tabolites were successfully applied as standard compounds in the quantification of MC- RR- GSH,MC- RR- Cys and MC- RR in the kidney of commom carp,and the contents（ dry weight） were obtained at the levels of undetected（ ND）,1. 587 5,0. 001 5 μg·kg- 1,respectively. Futhermore,these metabolites could be used as significant reference substances in various studies,and the cleavage rules of MC- RR- GSH and MC- RR- Cys could provide some metabolic mechanism of microcystin- RR.
出处
《分析测试学报》
CAS
CSCD
北大核心
2015年第6期664-669,共6页
Journal of Instrumental Analysis
基金
国家自然科学基金项目(21307164)
中央高校基本科研业务费专项资金资助项目(CZQ11023)
中南民族大学大学生创新创业训练资助项目(KYCX140211
KYCX120208Z)
