在线固相萃取结合液相色谱-线性离子阱多级质谱法同时检测生物样品中7种乌头类生物碱

摘要

建立了在线固相萃取（on-line SPE）结合液相色谱-线性离子阱多级质谱（LC-LIT／MSn）同时测定全血、尿液和肝组织样品中7种乌头类生物碱的分析方法，并根据7种乌头类生物碱的多级质谱碎片对裂解规律进行了推测和总结。用乙腈沉淀样品中的蛋白质，于稀释和离心后直接进样。经 Waters Oasis® HLB 在线 SPE 柱富集纯化，以0.1％（ v／v）甲酸／乙酸铵溶液-0.1％（ v／v）甲酸／甲醇溶液为流动相，以 Accucore C18为分析柱进行梯度洗脱；在电喷雾电离（ ESI）正离子模式下测定；扫描方式为连续反应监测（ CRM）。在考察的质量浓度范围内，7种乌头类生物碱的标准曲线符合二阶方程（权重因子1／x），相关系数为0.9991～0.9999；在全血和尿液中的方法检出限为0.02～0.60 ng／mL，在肝组织中的方法检出限为0.02～0.40 ng／g；加标回收率为91.1％～104.7％，日内精密度和日间精密度分别为0.2％～10.7％、1.0％～13.7％（ n＝6）。该方法简单准确，灵敏度高，能够满足生物样品中7种乌头类生物碱的快速分析。%An analytical method has been developed for the simultaneous determination of seven aconitum alkaloids in biological samples by liquid chromatography-linear ion trap multistage mass spectrometry(LC-LIT/MSn)coupled with on-line solid phase extraction(on-line SPE). Mass fragmentation regularities of the seven aconitum alkaloids are discussed and summarized according to the analysis of their MS n fragments. The samples were treated with acetonitrile for protein precipitation,followed by dilution and centrifugation. The resulting solution was injec-ted into the LC system directly and processed by Waters Oasis ® HLB on-line SPE column for enrichment and purification. Chromatographic separation was performed on an Accucore C18 column using mobile phases of 0. 01 mol/L ammonium acetate aqueous solution and methanol (both containing 0. 1%(v/v)formic acid)for gradient elution. The analytes were detected in consecutive reaction monitoring( CRM)mode through positive electrospray ionization( ESI﹢). The calibration curves of the analytes within the investigated mass concentration ranges were fitted by second-order equations weighted 1/x,with r2 ranging from 0. 999 1 to 0. 999 9. The method detection limits were 0. 02-0. 60 ng/mL for whole blood and urine,0. 02-0. 40 ng/g for liver tissue. The recoveries at three spiked levels were within 91. 1%-104. 7% for all the ana-lytes,with 0. 2%-10. 7% of intra-day RSDs and 1. 0%-13. 7% of inter-day RSDs( n=6 ). The established method is simple,accurate,and sensitive for rapid determination of the seven aco-nitum alkaloids in biological samples.