Therapeutic drug monitoring (TDM) of the immunosuppressive drug tacrolimus is essential to avoid side effects and rejection of the allograft after transplantation. In the blood circulation, tacrolimus is largely located inside erythrocytes or bound to plasma proteins and less than 0.1% is protein-unbound (free). One basic principle of clinical pharmacology is that only free drug is pharmacologically active and monitoring this portion has the potential to better reflect the drug effect than conventional measurements of total tacrolimus in whole blood. To address this, a highly sensitive and straightforward on-line liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed, validated and applied to patient plasma samples. The sample preparation included ultracentrifugation and addition of the stable isotope labeled drug analogue D2,13C-tacrolimus, followed by on-line sample extraction and measurement using a Sciex QTRAP^® 6500 in the multiple reaction monitoring mode. Due to very low concentrations of protein-unbound tacrolimus, it was important to develop a highly sensitive, precise and accurate assay. Here, we first report the efficient formation of tacrolimus lithium adduct ions, which greatly increased assay sensitivity. A lower limit of quantification (LLOQ) of 1 pg/mL (10 fg on column) was achieved and the assay was linear between 1 and 200 pg/mL. There was no carry-over detected. The inaccuracy ranged from −9.8 to 7.4% and the greatest imprecision was 7.5%. The matrix factor was found to be smaller than 1.1%. In summary, this method represents a suitable tool to investigate the potential clinical value of free tacrolimus monitoring in organ transplant recipients.

免疫抑制药物他克莫司的治疗药物监测（TDM）对于避免副作用和移植后异体移植的排斥是必不可少的。在血液循环中，他克莫司主要位于红细胞内部或与血浆蛋白结合，而少于0.1％的蛋白未结合（游离）。临床药理学的一个基本原理是，只有游离药物才具有药理活性，与传统的全血他克莫司测量方法相比，监测此部分有可能更好地反映药物作用。为了解决这个问题，开发了一种高度灵敏，简单的在线液相色谱-串联质谱（LC-MS / MS）方法，并将其验证并应用于患者血浆样品。^® 6500在多反应监测模式。由于未结合他克莫司的蛋白质浓度非常低，因此开发高度灵敏，精确和准确的测定非常重要。在这里，我们首先报告他克莫司锂加合物离子的有效形成，这大大提高了测定的灵敏度。定量下限（LLOQ）为1 pg / mL（柱上为10 fg），该测定法在1到200 pg / mL之间呈线性关系。没有检测到残留。误差范围为-9.8至7.4％，最大不准确性为7.5％。发现基质因子小于1.1％。总而言之，该方法代表了一种合适的工具，可用于研究器官移植受者中免费他克莫司监测的潜在临床价值。