Methimazole (MMI, 1-methyl-2-mercaptoimidazole) is widely used for the treatment of hyperthyroidisms. There are methods available for the measurement of MMI concentration in human serum or plasma from the past, but none meet the current regulatory standards for bioanalytical method validations. In this paper, we developed and validated a total MMI measurement method using liquid chromatography-tandem mass spectrometry (LC-MS/MS), a technique that conforms to current bioanalytical method validation. To form a free sulfhydryl group on MMI, sodium bisulfite was added to 50 µl of plasma or serum samples containing MMI before the derivatization step. The internal standard (MMI-D3) was spiked into samples, then these samples were derivatized with 4-chloro-7-nitro-2,1,3-benzoxadiazole. After derivatization, these samples were extracted by supported liquid extraction. Then, the organic solvent was evaporated and the residue was dissolved in 50% methanol and injected into the LC-MS/MS system. A calibration curve was plotted over the concentration range 1-1000 ng/mL (r2 = 0.999). The intra-day and inter-day precisions were less than 10.2% and 9.8%, respectively. The intra-day and inter-day accuracies were between 89.5% and 101.1%, and 96.0% and 99.7%, respectively. The long-term stability of samples showed good precision and accuracy. The validated method was successfully applied to determine serum total MMI concentration in Graves' disease patients after oral administration of 5, 10 or 15 mg MMI. The range of circulating total MMI concentrations was found to be between 2.69 and 304.27 ng/mL in this study. It was shown that the measured serum total MMI concentrations changed in a dose-dependent manner.

中文翻译：

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建立高效液相色谱-串联质谱法测定人血中甲硝唑的方法。

甲基咪唑（MMI，1-甲基-2-巯基咪唑）被广泛用于甲状腺功能亢进症的治疗。过去有一些方法可用于测量人血清或血浆中MMI的浓度，但没有一种方法符合当前的生物分析方法验证法规标准。在本文中，我们开发并验证了使用液相色谱-串联质谱（LC-MS / MS）的总MMI测量方法，该技术符合当前的生物分析方法验证。为了在MMI上形成游离巯基，在衍生步骤之前，将亚硫酸氢钠添加到50μl含MMI的血浆或血清样品中。将内标（MMI-D3）加标到样品中，然后用4-氯-7-硝基-2,1,3-苯并恶二唑衍生化这些样品。衍生化之后 这些样品通过支持液萃取法萃取。然后，蒸发有机溶剂，并将残余物溶解在50％甲醇中，然后注入LC-MS / MS系统。在1-1000 ng / mL的浓度范围内绘制校准曲线（r2 = 0.999）。日内和日间精度分别低于10.2％和9.8％。日内和日间精度分别在89.5％和101.1％之间，以及96.0％和99.7％之间。样品的长期稳定性显示出良好的精度和准确性。经验证的方法已成功应用于口服5、10或15 mg MMI的Graves病患者的血清总MMI浓度测定。在这项研究中，发现循环中总MMI浓度范围在2.69至304.27 ng / mL之间。