色谱与质谱参数的优化对免疫抑制剂定量灵敏度的影响

Influence of optimization of chromatographic and mass spectrometric parameters on the quantitative sensitivity of immunosuppressive drugs

目的·考察色谱条件中的流动相及质谱离子源参数对环孢菌素A(cyclosporine A,CsA)、他克莫司(tacrolimus,TaC)、西罗莫司(sirolimus,SiR)及依维莫司(everolimus,EvE)4种常见免疫抑制剂药物的液相色谱-质谱分析定量结果的影响。方法·基于超高效液相色谱-串联质谱(ultra-high performance liquid chromatography-tandem mass spectrometry,UPLC-MS/MS)技术,通过改变流动相及离子源参数,评价不同参数设置对免疫抑制剂峰形及色谱峰响应的影响;通过考察标准曲线线性及最低定量限(limit of quantitation,LOQ)检验不同离子源温度下方法的灵敏度。结果·甲醇作为有机相能提供更窄的峰宽;水相中添加甲酸铵(ammonium formate,AF)能够明显提高色谱峰响应,高浓度的AF反而能一定程度地抑制色谱峰响应,因此选择5 mmol/L的AF作为流动相改性剂。离子源温度的优化能够明显改善色谱峰峰形和定量灵敏度,250℃下方法具良好的线性(r20.99)和灵敏度(LOQ=0.05 ng/mL)。结论·建立了基于UPLC-MS/MS技术的4种免疫抑制剂同步定量分析方法,并通过优化流动相及离子源参数便捷且显著地提高了定量灵敏度。该方法中AF的添加和离子源温度是影响定量分析的关键参数,这为基于液相色谱-质谱法的其他化合物的高灵敏度定量方法开发和优化提供新的思路。

Objective·To investigate the influence of mobile phase in chromatographic conditions and mass spectrometry ion source parameters on the quantitative results of four common immunosuppressant drugs including cyclosporine A(CsA),tacrolimus(TaC),sirolimus(SiR)and everolimus(EvE)by liquid chromatography-mass spectrometry.Methods·Based on ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)technology,the effect on peak shape and response under different parameter settings were evaluated by changing the mobile phase and ion source parameters;the sensitivity of the method under different ion source temperatures was detected by linearity of the standard curve and limit of quantitation(LOQ).Results·The narrower peak width was obtained when methanol was used as organic phase;the addition of ammonium formate(AF)to the aqueous phase observably improved the peak response but inhibited partly when the concentration of AF excessed;therefore,5 mmol/L AF was chosen as the mobile phase modifier.The optimization of the ion source temperature significantly improved the chromatographic peak shape and quantitative sensitivity.The quantitative method had nice linearity(r2>0.99)and sensitivity(LOQ=0.05 ng/mL)under 250℃ion source temperature.Conclusion·A simultaneous quantitative analytical method for four immunosuppressant drugs is established based on UPLC-MS/MS technology and the quantitative sensitivity has been conveniently and significantly improved by optimizing the mobile phase and ion source parameters.The addition of AF in the mobile phase and the ion source temperature are key parameters that affect the quantitative analysis and provide new ideas for the development and optimization of high-sensitivity quantitative methods for other compounds based on liquid chromatography-mass spectrometry.