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目的:建立人外周血单核细胞(h PBMCs)内齐多夫定三磷酸化物(ZDV-TP)的固相萃取-液相色谱-串联质谱(SPE-LCMS/MS)测定方法,并将其应用于临床试验中ZDV-TP在h PBMCs内浓度的测定。方法:h PBMCs样本采用强阴离子交换固相萃取小柱实现ZDV-TP与齐多夫定(ZDV)、齐多夫定一磷酸化物(ZDV-MP)和齐多夫定二磷酸化物(ZDV-DP)的分离后,加入酸性磷酸酶将其去磷酸化为等物质的量的齐多夫定原药。去磷酸化后的样本经HLB固相萃取小柱脱盐处理后,采用Agilent XDB-C18色谱柱,以0.2%甲酸水溶液-甲醇为流动相进行等度洗脱。采用ESI正离子模式检测,离子监测方式为MRM,用于定量分析的离子反应分别为m/z 267.9→m/z 126.8(ZDV)和m/z 247.9→m/z120.8(替硝唑,内标)。结果:ZDV-TP在0.112 5~82.05 ng·m L-1(每106个细胞含ZDV-TP 0.06~43 pmol),范围内线性良好(r=0.997 1),定量下限为0.112 5 ng·m L-1。日内及日间精密度(RSD)均小于9.0%,回收率和基质效应均符合生物样本的测定要求。结论:本文所建立的方法灵敏度高,专属性强,且成功应用于临床试验中ZDV在h PBMCs内的ZDV-TP的测定。
OBJECTIVE: To establish a method for the determination of zidovudine triphosphate (ZDV-TP) in human peripheral blood mononuclear cells (h PBMCs) by SPE-LCMS / MS, Determination of the concentration of ZDV-TP in h PBMCs in clinical trials. METHODS: h PBMCs were characterized using ZHD-ZDV, ZDV-MP and ZDV- DP), the acid phosphatase is added to dephosphorylate it to a zidovudine equivalent. Dephosphorylated samples were desalted on an HLB SPE cartridge and eluted isocratically with an Agilent XDB-C18 column using 0.2% formic acid in methanol as the mobile phase. The ESI positive ion mode was used to detect the ion and the ion monitoring method was MRM. The ion reactions for quantitative analysis were m / z 267.9 → m / z 126.8 (ZDV) and m / z 247.9 → m / z120.8 Internal standard). RESULTS: ZDV-TP had a good linearity (r = 0.997 1) within the range of 0.112 5 ~ 82.05 ng · m L -1 (ZDV-TP 0.06 ~ 43 pmol per 106 cells) with a lower limit of quantitation of 0.112 5 ng · m L-1. Intra-day and inter-day precision (RSD) were less than 9.0%, and the recovery and matrix effects were in line with the biological sample determination requirements. Conclusion: The established method is highly sensitive and specific and has been successfully applied to the determination of ZDV-TP in h PBMCs in clinical trials.