Department of Laboratory Medicine, University-Hospital, Padova, Italy.
Clin Chem Lab Med. 2011 Jul;49(7):1151-8. doi: 10.1515/CCLM.2011.192. Epub 2011 May 17.
Immunosuppressant therapeutic drug monitoring (TDM) is an important requirement in post-transplant patient care. In recent years, high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has become a valid alternative to antibody-based immunoassays in TDM due to its high specificity and sensitivity. Furthermore, this technology allows for the simultaneous measurement of several immunosuppressive drugs. The aim of the present study was to establish a straightforward, robust, and high-throughput LC-MS/MS method for the simultaneous determination of sirolimus and everolimus in whole blood in order to replace immunoassays in our routine practice.
Five-level blood calibrators were employed for assay development, while three materials at different concentrations were used for internal quality control. The proposed method uses protein precipitation for sample preparation. Analyses were performed using a triple quadrupole LC-MS/MS, with a C18 held at 60°C. Using an appropriate gradient elution profile and SPE on-line, elution times for all compounds analysed were 2.6 min with a total run-time of 3.5 min.
Calibration curves were linear throughout the selected ranges. The intra- and inter-assay CVs (<7%), the limit of quantification (0.2 μg/L) and accuracy were highly satisfactory. On testing the results using the international proficiency testing scheme (UK-NEQAS), the performance of the proposed method was found to be highly reliable.
The findings made by us indicate that the proposed method is of value, since it is speedy, straightforward, accurate, and applicable to different LC-MS/MS instruments for the routine TDM of organ transplant recipients.
免疫抑制剂治疗药物监测（TDM）是移植后患者护理的重要要求。近年来，由于其高特异性和灵敏度，高效液相色谱-串联质谱（LC-MS/MS）已成为 TDM 中基于抗体的免疫分析的有效替代方法。此外，该技术允许同时测量几种免疫抑制剂。本研究旨在建立一种简单、稳健、高通量的 LC-MS/MS 方法，用于同时测定全血中的西罗莫司和依维莫司，以替代我们常规实践中的免疫分析。
采用五级血校准品进行方法开发，同时采用三种不同浓度的材料进行内部质量控制。所提出的方法使用蛋白质沉淀进行样品制备。分析采用三重四极杆 LC-MS/MS 进行，C18 在 60°C 下保持。采用适当的梯度洗脱程序和在线 SPE，所有分析物的洗脱时间为 2.6 分钟，总运行时间为 3.5 分钟。
校准曲线在所选范围内呈线性。日内和日间 CV（<7%）、定量限（0.2 μg/L）和准确度均非常令人满意。使用国际能力验证计划（英国-NEQAS）测试结果，发现所提出方法的性能非常可靠。
我们的研究结果表明，所提出的方法具有价值，因为它快速、简单、准确，适用于不同的 LC-MS/MS 仪器，用于器官移植受者的常规 TDM。
