用HPLC-MS/MS法同时测定人血清中异烟肼和乙酰异烟肼的浓度

Simultaneous determination of isoniazid and acetylisoniazid in human serum by HPLC-MS/MS

目的建立同时测定人血清中异烟肼和乙酰异烟肼药物浓度的高效液相色谱串联质谱(HPLC-MS/MS)法,并检测结核患者血药浓度。方法色谱柱为Agilent RRHDSB-Aq(50.0mm×3.0mm,1.8μm),柱温为40℃。流动相为纯化水和甲醇,流速为0.25mL·min^(-1),梯度洗脱。异烟肼和乙酰异烟肼的质谱检测方式均为电喷雾正离子模式,多反应监测。考察该方法的专属性、定量下限、标准曲线、精密度与回收率、基质效应、稳定性和残留效应。结果异烟肼和的乙酰异烟肼的保留时间分别为1.37和1.24min。异烟肼在0.2~20.0μg·mL^(-1)内呈良好线性关系,异烟肼的回归方程为y=0.29x+3.05×10^(-3)(r^(2)=0.9996),定量下限为200ng·mL^(-1);乙酰异烟肼在0.2~20μg·mL^(-1)内呈良好线性关系,乙酰异烟肼的回归方程为y=0.29x-3.21×10^(-3)(r^(2)=0.9997),定量下限为200ng·mL^(-1)。批内、批间精密度的相对标准偏差(RSD)均≤4.00%;提取回收率在100.34%~106.22%;内标归一化基质效应因子(MF)≤3.79%;样品具有良好的冻融循环稳定性及长期稳定性。该法成功用于本院72例结核患者血清样本分析。结论该方法简便、快速、准确,适用于同时测定结核患者体内异烟肼和乙酰异烟肼的血药浓度。

Objective To establish a high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)method for the simultaneous determination of isoniazid and acetylisoniazid in human serum,and to detect the concentration of isoniazid in the blood of tuberculosis patients.Methods The serum samples were achieved on an Agilent RRHD SB-Aq(50.0 mm×3.0 mm,1.8μm),whose column temperature was 40℃.The mobile phase consisted of purified water and methanol with a gradient program at a flow rate of 0.25 mL·min^(-1).Both isoniazid and acetyl-isoniazid mass spectrometry detection methods were electrospray positive ion mode,multiple reaction monitoring.The specificity,lower limit of quantitation,standard curve,precision and recovery,matrix effect,stability and residual effect of the method were investigated.Results Theretentiontimesof isoniazid1 and acetylisoniazid were 1.37 and 1.24 min,respectively.Isoniazid showed a good linear relationship in the range of 0.2-20.0μg·mL^(-1).The regression equation of isoniazid was y=0.29x+3.05×10^(-3)(r^(2)=0.9996),and the lower limit of quantitation was 200 ng:mL^(-1).The regression equation of acetyl isoniazid was y=0.29x-3.21×10^(-3)(r^(2)=0.9997),and the lower limit of quantitation was 200 ng·mL^(-1).The relative standard deviation(RSD)of intra-lot and inter-lot precision was less than 4.00%.The recovery rates were 100.34%-106.22%.Internal standard normalized matrix effect factor(MF)≤3.79%.The samples have good freeze-thaw cycle stability and long-term stability.The method has been successfully used to analyze serum samples of 72 patients with tuberculosis in our hospital.Conclusion This method is simple,rapid and accurate,and is suitable for simultaneous determination of isoniazid and acetylisoniazid in the blood of tuberculosis patients.