Li Qin, Cao Di, Huang Yue, Xu Hong, Yu Chen, Li Zhiping
Department of Pharmacy, Children's Hospital of Fudan University, Shanghai, China.
Biomed Chromatogr. 2013 Mar;27(3):327-34. doi: 10.1002/bmc.2795. Epub 2012 Aug 15.
A bioanalytical method for the quantification of tacrolimus (TAC) on dried blood spots (DBS) using liquid chromatography, electrospray ionization coupled with tandem mass spectrometry (LC-ESI-MS/MS) was developed and validated. It involves solvent extraction of a punch disk of DBS followed by liquid-liquid extraction. The analyte and the internal standard (IS, ascomycin) were separated on a phenyl column using an isocratic mobile phase elution at a flow rate of 0.3 mL/min. The assay was linear from 1 to 80 ng/mL. The mean recovery of TAC was 76.6%. Intra-assay, inter-assay imprecision and biases were all less than 15%. TAC on DBS was stable for at least 10 days at room temperature, and at least 24 h at 50°C. A chromatographic effect of the filter paper (Whatman 903) was not detected. The volume of blood (15-50 μL) and hematocrit of blood (ranging from 23.2 to 48.6%) did not show a significant influence on detection of TAC concentration by DBS-LC-MS/MS. Fifty samples from patients were detected by both DBS-LC-MS/MS and microparticle enzyme-linked immunoassay (MEIA). TAC concentrations measured by DBS-LC-MS/MS method tended to be lower than those by MEIA.
建立并验证了一种采用液相色谱 - 电喷雾电离串联质谱法（LC - ESI - MS/MS）对干血斑（DBS）中他克莫司（TAC）进行定量分析的生物分析方法。该方法包括对DBS冲孔圆盘进行溶剂萃取，然后进行液 - 液萃取。分析物和内标（IS，子囊霉素）在苯基柱上使用等度流动相洗脱，流速为0.3 mL/min进行分离。该测定在1至80 ng/mL范围内呈线性。TAC的平均回收率为76.6%。批内、批间不精密度和偏差均小于15%。DBS上的TAC在室温下至少稳定10天，在50°C下至少稳定24小时。未检测到滤纸（Whatman 903）的色谱效应。血液体积（15 - 50 μL）和血液血细胞比容（范围为23.2%至48.6%）对DBS - LC - MS/MS检测TAC浓度未显示出显著影响。采用DBS - LC - MS/MS和微粒酶联免疫分析（MEIA）对50例患者样本进行检测。DBS - LC - MS/MS法测得的TAC浓度往往低于MEIA法测得的浓度。
