Urate anion exchanger 1 (URAT1) expressed in the proximal renal tubules is responsible for about 90% of the reabsorption of uric acid. URAT1 is identified as an important target of uricosuric drugs. Here we present an LC-MS/MS-based approach, combined with URAT1-transgenic MDCK cells, for the assessment of uric acid. Cell lysis was executed with 50 mM NaOH to release uric acid. 1,3-¹⁵N₂ uric acid was employed as the internal standard. The harvested uric acid, along with the stable isotope-labeled uric acid, was analyzed by LC-MS/MS in multiple reactions monitoring and negative modes. Validation, i.e. determination of selectivity, precision, accuracy, extraction recovery, and matrix effect, and feasibility was evaluated by use of the approach developed. The linearity was observed in the range of 1.0–250 μM (r = 0.9960) with limit of detection of 50 nM and limit of quantitation of 200 nM. The precision and accuracy were found to be RSD ≤ 20% and 80–120% of the nominal value, respectively. Uric acid uptake showed concentration and time dependency in URAT1-transgenic cells. The observed inhibitory effects of three URAT1-targeted uricosuric drugs were consistent with those reported in literature. The stable isotope dilution-based approach was proven to be selective, sensitive, and convenient, which is a good in vitro model for URAT1-targeted drug candidate screening.

在近端肾小管中表达的尿酸根阴离子交换剂1（URAT1）负责大约90％的尿酸重吸收。URAT1被确定为尿酸排泄药物的重要靶标。在这里，我们提出了一种基于LC-MS / MS的方法，结合了URAT1转基因MDCK细胞，用于评估尿酸。用50 mM NaOH进行细胞裂解以释放尿酸。1,3- ¹⁵ N ₂尿酸用作内标。通过LC-MS / MS在多个反应监测和阴性模式下分析了收获的尿酸以及稳定的同位素标记的尿酸。验证，即确定选择性，精密度，准确度，提取回收率和基质效应以及可行性，通过使用开发的方法进行了评估。在1.0–250μM（r = 0.9960）范围内观察到线性，检测限为50 nM，定量限为200 nM。发现精度和准确度分别为标称值的RSD≤20％和80–120％。尿酸摄取在URAT1转基因细胞中显示浓度和时间依赖性。观察到的三种靶向URAT1的尿酸排泄药物的抑制作用与文献报道的一致。URAT1靶向药物候选物筛选的体外模型。