Zhang Jin, Geng Peiwu, Luo Xinhua, Zhou Genzhi, Lin Yingying, Zhang Lijing, Wang Shuanghu, Wen Congcong, Ma Jianshe, Ding Ting
Department of Clinical Lab Medicine, Taizhou Municipal Hospital affiliated with Taizhou University Taizhou 318000, Zhejiang, China.
The Laboratory of Clinical Pharmacy, The People's Hospital of Lishui Lishui 323000, Zhejiang, China.
Int J Clin Exp Med. 2015 Oct 15;8(10):18420-6. eCollection 2015.
It was reported that macitentan was metabolized predominantly by cytochrome P450 3A4, and ACT-132577, its pharmacologically active metabolite, is fivefold less potent at blocking ET receptors than macitentan. In this work, a sensitive and selective ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for determination of ACT-132577 in rat plasma was developed and validated. After addition of diazepam as an internal standard (IS), protein precipitation by acetonitrile was used to prepare samples. Chromatographic separation was achieved on a UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm) with 0.2% formic acid and methanol as the mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; multiple reactions monitoring (MRM) mode was used for quantification using target fragment ions m/z 546.9→200.6 for ACT-132577, and m/z 285.1→193.1 for IS. Calibration plots were linear throughout the range 10-4000 ng/mL for ACT-132577 in rat plasma. Mean recovery of ACT-132577 in rat plasma ranged from 82.6% to 90.6%, matrix effect of ACT-132577 in rat plasma ranged from 101.4% to 115.2%. RSD of intra-day and inter-day precision were both less than 11%. The accuracy of the method ranged from 96.1% to 103.5%. The method was successfully applied to pharmacokinetic study of ACT-132577 after oral and intravenous administration of macitentan.
据报道，马昔腾坦主要通过细胞色素P450 3A4代谢，其药理活性代谢物ACT-132577在阻断内皮素受体方面的效力比马昔腾坦低五倍。在本研究中，建立并验证了一种灵敏且选择性的超高效液相色谱-串联质谱（UPLC-MS/MS）法，用于测定大鼠血浆中的ACT-132577。加入地西泮作为内标（IS）后，采用乙腈沉淀蛋白法制备样品。在UPLC BEH C18柱（2.1 mm×100 mm，1.7μm）上进行色谱分离，以0.2%甲酸和甲醇为流动相进行梯度洗脱。采用电喷雾电离源，以正离子模式运行；采用多反应监测（MRM）模式进行定量分析，ACT-132577的目标碎片离子为m/z 546.9→200.6，内标的目标碎片离子为m/z 285.1→193.1。大鼠血浆中ACT-132577在10 - 4000 ng/mL范围内校准曲线呈线性。大鼠血浆中ACT-132577的平均回收率为82.6%至90.6%，基质效应为101.4%至115.2%。日内和日间精密度的相对标准偏差均小于11%。该方法的准确度为96.1%至103.5%。该方法成功应用于马昔腾坦口服和静脉给药后ACT-132577的药代动力学研究。
