Clinical and Experimental Pharmacokinetics Unit, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy.
Department of Cardiac Surgery, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; Department of Clinical, Surgical, Diagnostic and Paediatric Sciences, University of Pavia, Italy.
J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Sep 1;1152:122215. doi: 10.1016/j.jchromb.2020.122215. Epub 2020 Jun 21.
Recent studies report strategies for analysing immunosuppressive drugs in brain, liver and renal tissue, mostly in animals: we developed and validated a two steps combined enzymatic digestion/mass spectrometry assay to quantify Tacrolimus (TAC) in heart biopsies. Our aims were to avoid sample loss and sample contamination during the laboratory preparation, and to limit matrix effects in the electrospray ionization source (ESI) of the mass spectrometer. Enzymatic tissue digestion followed by a liquid-liquid drug extraction in the same vial of reaction allowed us to reach both our aims. The assay was assessed for selectivity, matrix effect, linearity, Lower Limit of Quantification (LLOQ) and Detection (LOD), accuracy and precision, according to the "Guideline on Bioanalytical Method Validation (EMA). A stable isotopically labelled (SIL) analogue (CD-TAC) was used as internal standard. The chromatographic separation of the analyte took 6 min. The observed linear range of quantification was 0.0162-0.520 ng in terms of TAC added to the biopsies (by 50 μL of the corresponding working solutions). The limit of detection and the lower limit of quantification (LLOQ) were 0.008 and 0.0162 ng, respectively. Both the mobile phases contained ammonium acetate and formic acid that promote the formation of ammoniated precursor ions that can be easily fragmented ([M + NH], TAC m/z 821.3; CD-TAC m/z 824.3). The calibration curves were generated by plotting analyte-to-internal standard peak area ratios versus TAC amount (ng) added to the biopsies, and using a weighted (1/x) linear regression. Curves were not forced to pass through the origin. Swine hearts were employed as blank matrix for all the analytical method validation procedures but, after approval by the ethics committee (by "Fondazione IRCCS Policlinico San Matteo": Protocol 20190032933), TAC was also quantified in endomyocardial biopsies from informed and consenting heart transplant patients. The study was funded by Fondazione IRCCS Policlinico San Matteo (RC08017617), as a part of the clinical studies on the maintenance of immunosuppressive therapy in cardiac transplant patients. Tacrolimus concentrations in patients biopsies were expressed as ratio between the detected amount of TAC (ng) in the tissue and the weight of the tissue itself (mg).
最近的研究报告了在大脑、肝脏和肾脏组织中分析免疫抑制药物的策略，这些策略主要在动物中进行：我们开发并验证了一种两步酶解/质谱联用测定法，用于定量心脏活检中的他克莫司（TAC）。我们的目标是避免在实验室准备过程中样品损失和样品污染，并限制质谱电喷雾电离源（ESI）中的基质效应。酶组织消化后，在同一反应瓶中进行液-液药物提取，使我们达到了这两个目标。该测定法根据“生物分析方法验证指南（EMA）”评估了选择性、基质效应、线性、定量下限（LLOQ）和检测限（LOD）、准确度和精密度。使用稳定同位素标记（SIL）类似物（CD-TAC）作为内标。分析物的色谱分离耗时 6 分钟。通过向活检中添加（50 μL 相应工作溶液）TAC 的方式，观察到的定量线性范围为 0.0162-0.520ng。检测限和定量下限（LLOQ）分别为 0.008 和 0.0162ng。两种流动相均含有乙酸铵和甲酸，可促进氨化前体离子的形成，这些离子很容易碎裂([M+NH]，TAC m/z 821.3；CD-TAC m/z 824.3)。校准曲线是通过绘制分析物与内标峰面积比与向活检中添加的 TAC 量（ng）之间的关系，使用加权（1/x）线性回归生成的。曲线没有强制通过原点。猪心被用作所有分析方法验证程序的空白基质，但在伦理委员会批准后（Fondazione IRCCS Policlinico San Matteo：Protocol 20190032933），TAC 也在知情同意的心脏移植患者的心肌活检中进行了定量。该研究由 Fondazione IRCCS Policlinico San Matteo（RC08017617）资助，作为心脏移植患者免疫抑制治疗维持的临床研究的一部分。患者活检中的他克莫司浓度表示为组织中检测到的 TAC（ng）量与组织本身重量（mg）之间的比值。
