HPLC-MS/MS法同时检测生物检材中芬太尼及其代谢物

Simultaneous detection of fentanyl and its metabolites in biological samples by HPLC-MS/MS

目的建立生物检材中芬太尼及其代谢物去甲芬太尼、去丙酰基芬太尼、羟基去甲芬太尼的液相色谱-串联质谱(HPLC-MS/MS)检测方法。方法LD_(50)剂量组待大鼠呼吸心跳停止立即取心脏、肝脏、脾脏、肺脏、肾脏、大脑、胃、肌肉、睾丸和心血。所取血液和组织样品中加入内标芬太尼-D5后经乙腈沉淀蛋白提取,以水(含0.1%甲酸加10mmoL乙酸铵)和甲醇溶液为流动相进行梯度洗脱,通过Eclipse XDB C18(2.1×150mm,3.5μm)色谱柱分离,在电喷雾离子源多反应监测(MRM)模式下进行检测。结果血和肝中芬太尼及其代谢物的线性检测范围为0.02~100ng/mL和0.1~20000ng/g,相关系数(R^(2))大于0.995和0.992,最低检出限(LOD)为0.005~0.01ng/m L和0.005~0.15ng/g,最低定量限(LOQ)为0.02~0.03ng/mL和0.02~0.50ng/g,日内和日间精密度分别小于11.83%、13.01%,准确度分别为90.55%~113.96%、91.33%~114.26%,绝对提取回收率均大于50%,基质效应均在75%~125%范围内。LD_(50)剂量组大鼠各脏器和心血中均可检测到芬太尼及其代谢物,心血中未检测到去丙酰基芬太尼。结论建立同时检测血和组织中芬太尼及其3种代谢物的HPLC-MS/MS方法,可为芬太尼相关案件鉴定和法医毒物学研究提供可靠方法。

Objective To establish a liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)method for the determination of fentanyl and its metabolites norfentanyl,depropionylfentanyl and hydroxynorfentanyl in biological samples.Methods The heart,liver,spleen,lung,kidney,brain,stomach,muscle,testis and blood of rats in LD_(50) dose group were taken immediately after respiratory heartbeat stopped.The blood and tissue samples were added with the internal standard fentanyl-D5,and then extracted by precipitated protein with acetonitrile.The mobile phase was gradient elution with water(containing 0.1%formic acid and 10 mmoL ammonium acetate)and methanol solution.The separation was performed on an Eclipse XDB C18(2.1 x150 mm,3.5μm)column.Electrospray ionization source was used for detection in the multiple reaction monitoring(MRM)mode.Results The linear detection ranges of fentanyl and its metabolites in blood and liver were 0.02~100 ng/m L and 0.1~2000 ng/g,the correlation coefficients(R^(2))were greater than 0.995 and 0.992,the lower limit of detection(LOD)were 0.005~0.01 ng/m L and 0.005~0.15 ng/g,the lower limits of quantification(LOQ)were 0.02~0.03 ng/m L and 0.02~0.50 ng/g,and the intra-day and inter-day precision were less than 11.83%and 13.01%,respectively.The extraction accuracy was 90.55%~113.96%and 91.33%~114.26%,respectively.The absolute recovery was more than 50%,and the matrix effect was in the range of 75%~125%.Fentanyl and its metabolites could be detected in all organs and blood of rats in LD_(50) dose group,but no depropanylfentanyl was detected in blood.Conclusion The HPLCMS/MS method for simultaneous detection of fentanyl and its three metabolites in blood and tissue can provide a reliable method for the identification of fentanyl-related cases and the study of forensic toxicology.