二维液相色谱法测定两性霉素B的血药浓度方法研究

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目的使用二维液相色谱(two-dimensional liquid chromatography/UV detector,2D-LC/UV)建立一种快速准确的两性霉素B的检测方法,并将所建立的方法用于临床患者两性霉素B血药浓度监测。方法本研究基于新型二维液相色谱仪(2D-LC/UV),通过去蛋白剂处理血浆样品,使用一维色谱柱SX1-1A柱(3.5 mm×25 mm,5μm),对血清中两性霉素B进行在线固相萃取,实现初步的富集分离。随后转移到二维色谱柱Aston SCB柱(4.6 mm×125 mm,5μm)上对目标分析物进一步分离检测。一维流动相为CAA-1D酸性移动相;流速0.7 mL/min;二维流动相为BPI-3碱性移动相;流速为1.0 mL/min。紫外检测波长:380 nm;柱温:40℃;对方法的选择性、线性范围与定量下限、精密度与回收率、基质效应及稳定性进行验证后,将该方法应用于临床患者两性霉素B的血药浓度监测。结果血清中两性霉素B的标准曲线方程为y=7204.6+914550x(R^(2)=0.9999),在0.07~12.31μg/mL范围内与峰面积呈良好的线性关系,定量下限为0.07μg/mL;低、中、高三个浓度的相对回收率在97.4~105.3%,日内和日间精密度均低于6.1%,说明本方法检测性能良好。采集20例使用两性霉素B患者血清样本,患者血药浓度范围在0.25~0.98μg/mL。结论本方法简便快捷,准确高效,实现了自动化分析,适用于临床血药浓度高通量分析监测;使用两性霉素B的患者个体差异较大,个体化治疗时需要进行血药浓度监测。

Objective Establish a rapid and accurate detection method for amphotericin B using two-dimensional liquid chromatography(2D-LC/UV)and apply the established method to monitor the blood concentration of amphotericin B in clinical patients.Methods This study was based on a new two-dimensional liquid chromatograph(2D-LC/UV).Plasma samples were processed by deproteinizing agents.On line solid phase extraction of amphotericin B in plasma was performed using a one-dimensional chromatographic column SX1-1A(3.5mm×25 mm,5μm)to achieve preliminary enrichment and separation.Subsequently,it was transferred to a two-dimensional chromatographic column,Aston SCB column(4.6 mm×125 mm,5μm),for further separation and detection of the target analytes.The one-dimensional mobile phase was CAA-1D acidic mobile phase,with a flow rate of 0.7 mL/min;the two-dimensional mobile phase is BPI-3 alkaline mobile phase,with a flow rate of 1.0 mL/min.UV detection wavelength:380nm;Column temperature:40℃;after verifying the selectivity,linear range and lower limit of quantification,precision and recovery,matrix effect and stability of the method,the method was applied to the monitoring of amphotericin B blood concentration in clinical patients.Results The standard curve equation of amphotericin B in plasma was■,There was a good linear relationship between concentration and peak area within the range of 0.07-12.31μg/mL,and the lower limit of quantification was 0.07μg/mL;the relative recoveries of low,medium,and high concentrations were between 97.4%and 105.3%,and the intra-day and inter-day precision were all below 6.1%,indicating good detection performance of this method.Serum samples were collected from 20 patients using amphotericin B,and the patients'blood drug concentration ranged from 0.25 to 0.98μg/mL.Conclusion The method is simple,fast,accurate and efficient,it realizes automatic analysis,and is suitable for high-throughput analysis and monitoring of clinical blood drug concentration;individual differences in patients using amphotericin B are large,and blood drug concentration monitoring is required during individualized treatment.