Ethoxyresorufin (ER)-O-deethylation (EROD) activity has been widely used to assess cytochrome P450 1A (CYP1A) activity. The kinetics of CYP1A activity have been well characterized in the liver microsomes. However, studies in kidney microsomes are limited due to the much lower EROD activity in this organ. Here, we developed and validated a sensitive UPLC-MS/MS assay for the characterization of the EROD activity in the rat kidney microsomes. In a 50 µL reaction mixture, rat kidney microsomes (0.25 mg/mL) were incubated with ER (0.1–5 µM) and NADPH (1 mM) for 10 min. Acidic solvents, such as trichloroacetic acid or formic acid, used for quenching of the metabolic reactions and precipitation of the proteins, unexpectedly caused a spontaneous formation of resorufin (RES) from ER. Therefore, the metabolic reactions were terminated by adding acetonitrile, containing a deuterated internal standard (IS). Chromatographic separation was achieved on a C₁₈ UPLC column, and the MS/MS ion transitions were 213.9/185.9 for RES and 220.0/192.0 for IS. The assay was validated in the linear range of 0.5 nM to 75 nM of RES and had a lower limit of quantitation of 0.5 nM. The overall recoveries of RES (90%–99%) and IS (85%–103%) were relatively high, with minimal matrix effect. The assay was successfully applied to the estimation of the Michaelis-Menten (MM) kinetics of EROD activity in the rat kidney microsomes (n = 3), which showed a maximum velocity of 2.68 ± 0.17 pmol/min/mg and a MM constant of 1.72 ± 0.24 µM (mean ± SD). It is concluded that our sensitive and specific analytical method, coupled with the optimized microsomal incubation conditions, provides a robust platform for further investigations of the effects of xenobiotics, environmental factors, or pathophysiologic conditions on the kinetics of EROD activity in the kidney microsomes.

乙氧基间苯二酚（ER）-O-去乙基化（EROD）活性已被广泛用于评估细胞色素P450 1A（CYP1A）的活性。CYP1A活性的动力学已在肝微粒体中得到很好的表征。但是，由于该器官中的EROD活性低得多，因此对肾脏微粒体的研究受到限制。在这里，我们开发并验证了用于表征大鼠肾脏微粒体中EROD活性的灵敏UPLC-MS / MS分析方法。在50 µL反应混合物中，将大鼠肾脏微粒体（0.25 mg / mL）与ER（0.1–5 µM）和NADPH（1 mM）孵育10分钟。用于猝灭代谢反应和蛋白质沉淀的酸性溶剂，例如三氯乙酸或甲酸，出乎意料地引起了ER自发形成试卤灵（RES）。因此，通过添加乙腈终止代谢反应，包含氘代内标（IS）。色谱分离在C柱上完成₁₈ UPLC柱，RES的MS / MS离子跃迁为213.9 / 185.9，IS的MS / MS离子跃迁为220.0 / 192.0。该方法在RES的0.5 nM至75 nM线性范围内得到验证，定量下限为0.5 nM。RES（90％–99％）和IS（85％–103％）的总体回收率相对较高，基质效应最小。该测定法已成功地用于估计大鼠肾脏微粒体内EROD活性的Michaelis-Menten（MM）动力学（n = 3），其最大速度为2.68±0.17 pmol / min / mg，MM常数为1.72±0.24 µM（均值±SD）。结论是，我们灵敏而特异的分析方法，加上优化的微粒体温育条件，为进一步研究异种生物，环境因素或病理生理条件对肾脏微粒体内EROD活性动力学的影响提供了一个可靠的平台。