Son Seok Young, Rhee Hong Soon, Lee Min Woo, Park Jong Moon
a Department of Chemical Engineering , POSTECH , Pohang , Korea.
Biosci Biotechnol Biochem. 2014;78(7):1103-11. doi: 10.1080/09168451.2014.917264.
Effective HPLC-DAD and HPLC-ESI-MS/MS methods have been developed for the analysis of eight benzo[c]phenanthridine alkaloids (sanguinarine, chelirubine, macarpine, chelerythrine, dihydrosanguinarine, dihydrochelirubine, dihydromacarpine and dihydrochelerythrine), which are important metabolites in Eschscholtzia californica cell culture. By adopting a ternary gradient pump system, the dihydro-form alkaloids hardly separable from each other could be successfully separated, and all the target alkaloids could be simultaneously quantified with the LOD values of 0.01-0.79 μg/mL and the LOQ values of 0.03-3.59 μg/mL. This HPLC-DAD method was further confirmed by HPLC-ESI-MS/MS system in multiple reaction monitoring mode. Each separated HPLC peak was identified as the target alkaloid, showing its relevant ionized molecule and selected fragment ion. By applying the established method, alkaloid production during the E. californica cell culture could be successfully monitored and some valuable information on its metabolism could be deduced.
已开发出高效液相色谱-二极管阵列检测法（HPLC-DAD）和高效液相色谱-电喷雾串联质谱法（HPLC-ESI-MS/MS）用于分析8种苯并[c]菲啶生物碱（血根碱、白屈菜红碱、原阿片碱、白屈菜碱、二氢血根碱、二氢白屈菜红碱、二氢原阿片碱和二氢白屈菜碱），这些生物碱是加州罂粟细胞培养中的重要代谢产物。通过采用三元梯度泵系统，成功分离了彼此难以分离的二氢型生物碱，所有目标生物碱均可同时定量，检测限为0.01 - 0.79μg/mL，定量限为0.03 - 3.59μg/mL。该HPLC-DAD方法在多反应监测模式下通过HPLC-ESI-MS/MS系统进一步得到确证。每个分离的HPLC峰均被鉴定为目标生物碱，并显示其相关的离子化分子和选定的碎片离子。通过应用所建立的方法，可成功监测加州罂粟细胞培养过程中的生物碱产生情况，并可推断出有关其代谢的一些有价值的信息。
