Drug Metabolism and Pharmacokinetics, Takeda Pharmaceutical Company Limited, Cambridge, Massachusetts.
Drug Metabolism and Pharmacokinetics, Takeda Pharmaceutical Company Limited, Cambridge, Massachusetts
Drug Metab Dispos. 2022 May;50(5):716-724. doi: 10.1124/dmd.122.000832. Epub 2022 Mar 3.
Glucuronidation is the most common phase II metabolic pathway to eliminate small molecule drugs from the body. However, determination of glucuronide structure is quite challenging by mass spectrometry due to its inability to generate structure-informative fragments about the site of glucuronidation. In this article, we describe a simple method to differentiate acyl-, , and glucuronides using chemical derivatization. The idea is that derivatization of acyl-, -, or glucuronides of a molecule results in predictable and different numbers of derivatized functional groups, which can be determined by the mass shift using mass spectrometry. The following two reactions were applied to specifically derivatize carboxyl and hydroxyl groups that are present on the aglycone and its glucuronide metabolite: Carboxyl groups were activated by thionyl chloride followed by esterification with ethanol. Hydroxyl groups were derivatized via silylation by 1-(trimethylsilyl)imidazole. The mass shift per derivatized carboxyl and hydroxyl group was +28.031 Da and +72.040 Da, respectively. This approach was successfully validated using commercial glucuronide standards, including benazepril acyl-glucuronides, raloxifene glucuronide, and silodosin glucuronide. In addition, this approach was applied to determine the type of glucuronide metabolites that were isolated from liver microsomal incubation, where alvimopan and diclofenac acyl-glucuronides; darunavir, haloperidol, and propranolol glucuronides; and darunavir glucuronide were identified. Lastly, this approach was successfully used to elucidate the definitive structure of a clinically observed metabolite, soticlestat glucuronide. In conclusion, a novel, efficient, and cost-effective approach was developed to determine acyl-, , and glucuronides using chemical derivatization coupled with liquid chromatography-high resolution mass spectrometry. SIGNIFICANCE STATEMENT: The method described in this study can differentiate acyl-, , and glucuronides and allow for elucidation of glucuronide structures when multiple glucuronidation possibilities exist. The type of glucuronidation information is particularly useful for a drug candidate containing carboxyl groups, which can form reactive acyl-glucuronides. Additionally, the method can potentially be used for definitive structure elucidation for a glucuronide with its aglycone containing a single carboxyl, hydroxyl, or amino group even when multiple types of functional groups are present for glucuronidation.
糖基化是消除体内小分子药物的最常见的 II 相代谢途径。然而,由于质谱法无法生成关于糖基化部位的结构信息片段,因此确定糖基化物的结构具有相当大的挑战性。在本文中,我们描述了一种使用化学衍生化来区分酰基-、-和葡萄糖醛酸苷的简单方法。其思路是,分子的酰基-、-或葡萄糖醛酸苷的衍生化会导致可预测的和不同数量的衍生化官能团,这可以通过质谱法的质量位移来确定。以下两个反应被应用于专门衍生化糖苷配基及其葡萄糖醛酸代谢物上存在的羧基和羟基:通过氯化亚砜激活羧基,然后用乙醇进行酯化。羟基通过 1-(三甲基甲硅烷基)咪唑进行硅烷化衍生化。每个衍生化的羧基和羟基的质量位移分别为+28.031 Da 和+72.040 Da。该方法使用商业葡萄糖醛酸苷标准品成功验证,包括贝那普利酰基-葡萄糖醛酸苷、雷洛昔芬葡萄糖醛酸苷和西洛多辛葡萄糖醛酸苷。此外,该方法还应用于确定从肝微粒体孵育中分离出的葡萄糖醛酸代谢物的类型,其中鉴定出阿立哌唑酰基-葡萄糖醛酸苷、双氯芬酸酰基-葡萄糖醛酸苷、达鲁那韦、氟哌啶醇和普萘洛尔葡萄糖醛酸苷以及达鲁那韦葡萄糖醛酸苷。最后,该方法成功用于阐明临床上观察到的代谢物,索替司他汀葡萄糖醛酸苷的明确结构。总之,开发了一种新颖、高效且具有成本效益的方法,通过化学衍生化结合液相色谱-高分辨质谱法来确定酰基-、-和葡萄糖醛酸苷。意义声明:本研究中描述的方法可以区分酰基-、-和葡萄糖醛酸苷,并在存在多种糖基化可能性的情况下允许阐明葡萄糖醛酸苷的结构。糖基化类型的信息对于含有羧基的候选药物特别有用,因为它可以形成反应性酰基-葡萄糖醛酸苷。此外,即使存在多种用于糖基化的官能团,该方法也可以潜在地用于具有单一羧基、羟基或氨基的糖苷配基的葡萄糖醛酸苷的明确结构阐明。