采用液相色谱-串联质谱法对人外周血单个核细胞中麦考酚酸进行定量分析。
Mycophenolic acid quantification in human peripheral blood mononuclear cells using liquid chromatography-tandem mass spectrometry.
机构信息
Clinical Chemistry Department, Cliniques Universitaires St. Luc, Brussels, Belgium; Louvain Center for Toxicology and Applied Pharmacology, Université Catholique de Louvain, UCL, Brussels, Belgium.
出版信息
Clin Biochem. 2013 Dec;46(18):1909-11. doi: 10.1016/j.clinbiochem.2013.09.009. Epub 2013 Sep 21.
BACKGROUND AND OBJECTIVES
Mycophenolic acid (MPA) is an immunosuppressant widely used to prevent organ rejection after organ transplantation. MPA therapeutic drug monitoring (TDM) is currently recommended by plasma C0 - or even better - by AUC determinations, but little is known about the potential interest of measuring the intra-lymphocyte MPA concentrations, representing the target site of action. The aim of this study is to develop and validate a selective and sensitive analytical method for quantification of MPA levels in human peripheral blood mononuclear cells (PBMCs).
METHODS
PBMCs were extracted from heparin blood samples by Leucosep®. Methanol and MPA-d3 were used as extraction solvent and internal standard, respectively. Chromatographic separation was obtained on a XBridge BEH C18 column (2.1mm×75mm, 2.5μm) maintained at 50°C on a Waters Alliance 2795 coupled to a QuattroMicro tandem mass-spectrometer. The multiple reaction monitoring transitions used for quantification were m/z 320.97→207.0 for MPA, and 324.2→210.1 for MPA-d3, in positive ESI mode.
RESULTS
The total HPLC run time was 6min. The retention times for MPA-d3 and MPA were 2.55 for both compounds. The method was linear from 0.1 to 50ng/mL MPA. The coefficient r(2) ranged from 0.996 to 0.998. Intra-assay and inter-assay imprecisions were <10% in the whole range of concentrations, and <20% at the LLOQ, and accuracy level was >90%. The matrix and ion suppression effects were <6%. The MPA limit of quantification was 0.1ng/mL. No interference was identified in the assay. From the preliminary results, intra-cellular MPA concentrations in kidney transplant patients ranged from 0.69 to 3.39ng/10(7) cells.
CONCLUSION
We described a robust, rapid and simple method suitable for the determination of MPA concentrations in PBMCs. This method is currently used in pharmacokinetics-pharmacodynamics (PK-PD) clinical trials, in comparison to standard plasma TDM.
背景与目的
霉酚酸（MPA）是一种广泛用于预防器官移植后器官排斥反应的免疫抑制剂。目前建议对 MPA 进行治疗药物监测（TDM），通过检测血浆 C0 水平，或更好的 AUC 水平，但对于测量淋巴细胞内 MPA 浓度（代表作用部位）的潜在意义知之甚少。本研究旨在开发和验证一种用于定量检测人外周血单个核细胞（PBMCs）中 MPA 水平的选择性和灵敏的分析方法。
方法
使用 Leucosep®从肝素血样中提取 PBMCs。甲醇和 MPA-d3 分别用作提取溶剂和内标。色谱分离在 Waters Alliance 2795 上的 XBridge BEH C18 柱（2.1mm×75mm，2.5μm）上进行，柱温为 50°C。定量采用正电喷雾模式下的多重反应监测转化，MPA 的质荷比为 320.97→207.0，MPA-d3 的质荷比为 324.2→210.1。
结果
整个 HPLC 运行时间为 6 分钟。MPA-d3 和 MPA 的保留时间均为 2.55。MPA 的线性范围为 0.1 至 50ng/mL。相关系数 r(2) 范围为 0.996 至 0.998。在整个浓度范围内，日内和日间精密度<10%，在定量下限（LLOQ）时<20%，准确度水平>90%。基质和离子抑制效应<6%。MPA 的定量下限为 0.1ng/mL。该检测方法无干扰。初步结果显示，肾移植患者的细胞内 MPA 浓度范围为 0.69 至 3.39ng/10(7)细胞。
结论
我们描述了一种稳健、快速和简单的方法，适用于 PBMCs 中 MPA 浓度的测定。该方法目前正在用于与标准血浆 TDM 进行比较的药代动力学-药效学（PK-PD）临床试验。
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