HPLC-MS/MS法同时测定血浆中贝达喹啉、普托马尼和利奈唑胺的方法学建立及应用  

Development and application of an HPLC-MS/MS method for simultaneous determination of bedaquiline,pretomanid,and linezolid in plasma

作　　者：程文 朱慧[2] 付雷[1] 张炜焱 张立群[3] 陆宇[1] Cheng Wen;Zhu Hui;Fu Lei;Zhang Weiyan;Zhang Liqun;Lu Yu(Beijing Key Laboratory of Drug Resistance Tuberculosis Research,Department of Pharmacology,Beijing Tuberculosis and Thoracic Tumor Research Institute,Beijing Chest Hospital,Capital Medical University,Beijing 101149,China;Office for the Scientific and Technological Achievements,Beijing Chest Hospital,Capital Medical University/Beijing Tuberculosis and Thoracic Tumor Research Institute,Beijing 101149,China;Department of Tuberculosis,Beijing Chest Hospital,Capital Medical University/Beijing Tuberculosis and Thoracic Tumor Research Institute,Beijing 101149,China)

机构地区：[1]首都医科大学附属北京胸科医院耐药结核病研究北京市重点实验室/北京市结核病胸部肿瘤研究所药物研究室,北京101149 [2]首都医科大学附属北京胸科医院转化办公室,北京101149 [3]首都医科大学附属北京胸科医院结核科,北京101149

出　　处：《中国防痨杂志》2025年第5期613-622,共10页Chinese Journal of Antituberculosis

基　　金：北京市医院管理中心“登峰”人才培养计划(DFL20221402)。

摘　　要：目的:建立同时测定血浆中贝达喹啉(Bdq)、普托马尼(Pa)、利奈唑胺(Lzd)药物浓度的液相色谱串联质谱法(HPLC-MS/MS),为临床治疗药物监测提供测定方法。方法:血浆样品以普萘洛尔(propranolol,PR)为内标,使用乙腈沉淀血浆蛋白并离心取上清液。设置流动相为0.1%甲酸(A)和5 mM甲酸铵的纯水与乙腈(B),采用梯度洗脱方式进行上清液中Bdq、Pa和Lzd浓度的HPLC-MS/MS分析。根据峰型和分离度确定色谱分离条件为柱温35℃、流速0.4 ml/min、分析时间4 min。质谱检测采用电喷雾离子源(ESI),在多反应监测(MRM)模式下进行正离子扫描。通过专属性、建立标准曲线与定量限测定、精密度与准确度、回收率与基质效应、稳定性考察对所建立的HPLC-MS/MS进行方法学考察,最后使用实验小鼠进行方法学建立与验证。结果:MRM模式扫描血浆样本结果显示:Bdq、Pa、Lzd和PR的去簇电压分别为80、91、80、100 V,碎裂电压分别为80、32、28、30 V,母离子/子离子分别为555.2/58.2、360.2/175.2、338.1/296.3和260.2/116.2。方法学考察结果显示,Bdq、Pa、Lzd的保留时间分别为1.93、1.79、1.53 min,峰形良好,出峰位置均无干扰峰信号;线性相关系数(R 2)分别为0.993、0.999和0.999,且分别在0.05~12.5、0.1~25、0.2~50μg/ml浓度范围内线性关系良好;3种药物的日内和日间准确度均在90%以上,精密度波动均小于15%;不同浓度的3种药物及内标PR的提取回收率达到78.45%~108.78%,受血浆处理方法的影响小,基质效应均高于90%,且符合±15%的要求;样品在不同储存条件下的稳定性误差均在±15%以内;应用该方法成功检测36份小鼠血浆样品中Bdq、Pa、Lzd的血药浓度范围分别为0.03~1.82、0.11~14.21、0.02~16.54μg/ml。结论:本研究所建立的HPLC-MS/MS方法可稳定快速地同时检测血浆样本中Bdq、Pa和Lzd的药物浓度,方法学考察证实该方法稳定、准确、灵敏,可供临床参考应用。Objective:To establish a high-performance liquid chromatography tandem mass spectrometry(HPLC-MS/MS)method for simultaneous determination of the plasma concentrations of bedaquiline(Bdq),pretomanid(Pa),and linezolid(Lzd),providing a method for monitoring clinical therapeutic drugs.Methods:Plasma samples were precipitated using acetonitrile,meanwhile,propranolol(PR)was added as an internal standard,followed by centrifugation to obtain the supernatant.Using a gradient elution method for HPLC-MS/MS analysis of Bdq,Pa,and Lzd concentrations in the supernatant with mobile phases A(0.1%formic acid and 5 mM ammonium formate in water)and B(acetonitrile).Chromatographic separation conditions were determined based on peak shape and resolution with a column temperature of 35℃,flow rate of 0.4 ml/min,and analysis time of 4 min.Mass spectrometry detection was performed using an electrospray ionization source(ESI)in positive ion mode with multiple reaction monitoring(MRM).The established HPLC-MS/MS method was validated through specificity,standard curve establishment and limit of quantification determination,precision and accuracy,recovery and matrix effect,and stability studies.Finally,the method was applied and validated using experimental mice.Results:MRM mode scanning of plasma samples showed that the declustering voltages for Bdq,Pa,Lzd,and PR were 80,91,80,and 100 V,respectively,and the collision energies were 80,32,28,and 30 V,respectively,with parent ion/product ion pairs of 555.2/58.2,360.2/175.2,338.1/296.3,and 260.2/116.2,respectively.Methodological validation results showed that the retention times for Bdq,Pa,and Lzd were 1.93,1.79,and 1.53 min,respectively,with good peak shapes and no interfering peaks signals at the elution positions;the linear correlation coefficients R 2 were 0.993,0.999,and 0.999,respectively,with good linear relationships in the concentration ranges of 0.05-12.5,0.1-25,and 0.2-50μg/ml,respectively;the intra-day and inter-day accuracies for the three drugs were above 90%and the precision fl

关 键 词：结核 色谱法 液相 质谱分析法 剂量效应关系 药物 贝达喹啉 普托马尼 利奈唑胺 

分 类 号：R521[医药卫生—内科学] R927.2[医药卫生—临床医学]