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 (¹³CD₂-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 + NH₄]⁺, TAC m/z 821.3; ¹³CD₂-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).

最近的研究报告了分析大脑，肝脏和肾脏组织（主要是动物）中的免疫抑制药物的策略：我们开发并验证了两步组合酶消化/质谱联用测定心脏活检中他克莫司（TAC）的方法。我们的目标是在实验室准备过程中避免样品损失和样品污染，并限制质谱仪电喷雾电离源（ESI）中的基质效应。酶促组织消化，然后在同一反应瓶中进行液-液药物提取，使我们能够达到我们的两个目标。

根据“生物分析方法验证指南”（EMA）评估分析的选择性，基质效应，线性，定量下限（LLOQ）和检测（LOD），准确性和精密度。稳定的同位素标记（SIL）类似物（¹³ CD ₂ -TAC）被用作内标。

分析物的色谱分离用了6分钟。观察到的定量线性范围为0.0162-0.520 ng（根据添加到活组织检查中的TAC值）（50μL相应的工作溶液）。检测限和定量下限（LLOQ）分别为0.008和0.0162 ng。流动相均包含乙酸铵和甲酸，促进形成易碎的氨化前体离子（[M + NH ₄ ] ⁺，TAC m / z 821.3；¹³ CD ₂ -TAC m / z824.3）。通过绘制分析物与内部标准品的峰面积之比与添加到活组织检查中的TAC量（ng）并使用加权（1 / x）线性回归来绘制校准曲线。曲线不被迫通过原点。

猪心脏被用作所有分析方法验证程序的空白矩阵，但是，在伦理委员会批准（“ Fondazione IRCCS Policlinico San Matteo”：协议20190032933）之后，TAC还被量化了来自知情同意的心脏移植患者的心内膜活检。

该研究由Fondazione IRCCS Policlinico San Matteo（RC08017617）资助，是维持心脏移植患者免疫抑制疗法临床研究的一部分。

患者活检中他克莫司的浓度表示为组织中TAC的检测量（ng）与组织本身重量（mg）之比。