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目的建立一种快速、准确地检测人血清中阿米卡星浓度的LC-MS/MS法,并将该法应用于临床样本检测。方法以卡那霉素为内标,100μL血清样品经含0.1%甲酸的甲醇-乙腈(2∶1)沉淀蛋白后离心,取上清液稀释后进样。色谱柱为AtlantisHilic Silica柱(50 mm×2.1 mm,3.0μm)。采用电喷雾离子源、正离子选择反应监测进行分析扫描。检测离子通道为m/z 586.3→163.0(阿米卡星)和m/z 485.2→163.0(内标)。经方法学验证后用于临床患者血清标本中阿米卡星浓度的检测。结果阿米卡星在0.50~100.00 mg·L~(-1)内线性良好(R~2=0.993 8);批内、批间精密度良好(变异系数均<15.0%);准确度为101.0%~110.1%;内标归一化阿米卡星基质效应因子(MF)为91.2%~101.2%,变异系数均<15.0%;阿米卡星提取回收率为92.6%~105.9%,内标提取回收率为78.1%;样品在4℃、-70℃下分别放置56、86 d稳定,室温、35℃下放置21 d稳定。本研究共收集、检测19例临床患者谷浓度血清标本34份,16份样品浓度低于定量下限,其余样品浓度范围为0.52~19.12 mg·L~(-1)。结论本分析方法快速、简单,适用于人血清样品阿米卡星浓度分析。测定患者谷浓度样本结果差异较大,提示在临床应用阿米卡星时应进行治疗药物浓度监测,调整给药方案。
Objective To establish a rapid and accurate LC-MS / MS method for the determination of amikacin in human serum and apply the method to clinical samples. Methods With kanamycin as internal standard, 100μL serum samples were centrifuged after precipitation of protein by methanol-acetonitrile (2:1) containing 0.1% formic acid. The supernatant was diluted and injected. The column was an Atlantis Hilic Silica column (50 mm × 2.1 mm, 3.0 μm). An electrospray ionization source and positive ion selective reaction monitoring were used for scanning analysis. The ion channels tested were m / z 586.3 → 163.0 (amikacin) and m / z 485.2 → 163.0 (internal standard). After verification by methodological clinical serum levels of amikacin in patients with the detection. Results Amikacin had a good linearity (R ~ 2 = 0.993 8) within the range of 0.50-100.00 mg · L -1. The intra-assay and inter-assay precision were good (the coefficients of variation were all <15.0%); the accuracy was 101.0 % ~ 110.1%; the internal standard normalized amikacin matrix effect factor (MF) was 91.2% ~ 101.2%, the coefficient of variation was <15.0%; the recoveries of amikacin were 92.6% ~ 105.9% The extraction recovery was 78.1%. The samples were stored at 4 ℃ and -70 ℃ for 56 and 86 d, respectively. The samples were stable at 35 ℃ for 21 days at room temperature. In this study, 34 serum samples from 19 clinical patients were collected and tested. The concentration of 16 samples was lower than the lower limit of quantitation, and the other samples ranged from 0.52 to 19.12 mg · L -1. Conclusion This method is rapid and simple and is suitable for the analysis of amikacin concentrations in human serum samples. The results of the determination of patients with trough concentrations of large differences in the results suggest that the clinical application of amikacin drug concentration monitoring should be adjusted to adjust the drug program.