应用超高效液相色谱-高分辨质谱法检测血样中秋水仙碱

Determination of Colchicine in Blood by UPLC-Q/Orbitrap HRMS

秋水仙碱是一种强效生物碱,因其具有特殊的结构及药效,受到人们广泛的关注。多项研究都表明秋水仙碱体内血药浓度较低,因此对其检测分析方法的灵敏度要求较高。本文建立了使用超高效液相色谱-高分辨质谱仪(UPLC-Q/Orbitrap HRMS)测定血样中秋水仙碱的检测方法。考察不同流动相和色谱柱对秋水仙碱分离效果的影响,结果表明在甲醇水体系作为流动相条件下,使用Accucore^(TM)Phenyl-Hexyl(2.1 mm×100 mm×2.6μm)色谱柱,仪器响应值高且分离效果较好。采用乙腈为蛋白沉淀剂,有机微孔过滤膜净化提取液,用UPLC-Q/Orbitrap HRMS对血样中秋水仙碱进行定性定量检验。分析采用Accucore^(TM)Phenyl-Hexyl(2.1 mm×100 mm×2.6μm)色谱柱,电喷雾离子源正离子(FullMS/ddMS^(2))模式,使用阶梯碰撞能量:35、60和85 eV,用5 mmol/L甲酸铵水溶液和甲醇作为流动相梯度洗脱。本方法血样中的秋水仙碱定量限为0.5 ng/mL;秋水仙碱在0.5~100 ng/mL范围内线性良好(R^(2)=0.9985),3个浓度平均回收率为92.8%~98.3%,日内精密度为1.6%~7.1%(n=6)。这一定性定量分析方法简易可靠,灵敏度高,适用于血样中秋水仙碱的检验鉴定。

As a potent alkaloid,colchicine has attracted much attention due to its unique structure and pharmacological properties.Colchicine has many medical applications,not only for the prevention and treatment of gouty arthritis,but also for the treatment of tumors,hepatitis and so on.Studies indicated that colchicine had low blood concentrations,so the method of determination of colchicine in blood should be more sensitive.Therefore,the objective of this article is to establish a sensitive method for analysis of colchicine in blood by liquid chromatogram-high resolution mass spectrum(UPLC-Q/Orbitrap HRMS).The effects of different mobile phases and chromatographic columns on the separation of colchicine were investigated.The results showed that the instrument had higher response value and better separation efficiency when Accucore^(TM)Phenyl hexyl analytical column(2.1 mm×100 mm×2.6μm)was used with methanol-water system as mobile phase.The blood sample(1.0 mL)was extracted with acetonitrile(1.0 mL)to precipitate protein.Then,its well-treated supernatant was separated on a Accucore^(TM)Phenyl hexyl analytical column(2.1 mm×100 mm×2.6μm)with gradient elution that was run through the mobile phases consisting of 5 mmol/L ammonium formate aqueous solution(mobile phase A)and methanol(mobile phase B).The analytes were analyzed with UPLC-Q/Orbitrap HRMS under the mode of positive-ion ESI in full MS/ddMS^(2).Step collision energies of 35 eV,60 eV and 85 eV were used.Colchicine was linear with its calibration curve among the range of 0.5 to 100 ng/mL(R^(2)=0.9985),which showed good recovery and inter-day precision less than 7.1%.The method established here is simple,reliable,sensitive and appropriate for determination of colchicine in blood.In addition,colchicine is unstable when exposed to sunlight in solution.Attention should be paid to avoiding sunlight during the experiment so as to make the analysis results more reliable.