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High-throughput protein precipitation method with 96-well plate for determination of doxepin and nordoxepin in human plasma using LC-MS/MS.
Biomedical Chromatography ( IF 1.8 ) Pub Date : 2020-04-06 , DOI: 10.1002/bmc.4844
Tigran Margaryan 1 , Mariam Sargsyan 1 , Arpine Gevorgyan 1 , Hasmik Zakaryan 1 , Armine Aleksanyan 1 , Armen Harutyunyan 1 , Yeghig Armoudjian 1 , Astghik Mikayelyan 1
Affiliation  

The aim of this study was to establish a high‐throughput and sensitive LC–MS/MS method for the determination of doxepin and its major active metabolite nordoxepin in human plasma. It has been designed for bioequivalence study for formulations containing 25 mg of doxepin. Doxepin and nordoxepin were extracted from human plasma samples by protein precipitation with acetonitrile by using protein precipitation 96‐well plates. The analyte was separated using a Phenomenex Kinetex Biphenyl column (100 × 2.1 mm, 2.6 μm) using isocratic elution with a mobile phase of 20 mM ammonium formate (30%) and acetonitrile:methanol 3:7 v:v (70%) at a flow rate of 0.5 mL/min and an injection volume of 10 μL. The detection was performed using a triple quadrupole mass spectrometer by multiple reaction monitoring mode to monitor the precursor‐to‐product ion transitions of m /z 280.4 → 107.0 and 283.4 → 235.0 for doxepin and doxepin‐D3, respectively, and 266.3 → 106.9 and 269.3 → 235.0 for nordoxepin and nordoxepin‐D3, respectively, in positive electrospray ionization mode. The total run time was 3.5 min. The method was validated over a concentration range of 50–10,000 pg/mL using a Triple Quad 4500 MS System (Sciex) for both analytes. The developed and validated method can be successfully used to study the bioequivalence/pharmacokinetics of doxepin and nordoxepin.

中文翻译:

使用LC-MS / MS的96孔板高通量蛋白质沉淀法测定人血浆中的多塞平和降氧平。

这项研究的目的是建立一种高通量且灵敏的LC-MS / MS方法,用于测定人血浆中的多塞平及其主要活性代谢物诺多塞平。它被设计用于生物等效性研究,用于含有25 mg多塞平的制剂。通过使用蛋白质沉淀96孔板用乙腈进行蛋白质沉淀,从人血浆样品中提取多塞平和诺多塞平。使用Phenomenex Kinetex联苯色谱柱(100×2.1 mm,2.6μm),使用20 mM甲酸铵(30%)和乙腈:甲醇3:7 v:v(70%)的流动相进行等度洗脱,分离分析物流速为0.5 mL / min,进样量为10μL。在正电喷雾电离模式下,多西平和doxepin-D3的m / z分别为280.4→107.0和283.4→235.0,诺多昔平和nordoxepin-D3的分别为266.3→106.9和269.3→235.0。总运行时间为3.5分钟。使用Triple Quad 4500 MS系统(Sciex)对两种分析物在50–10,000 pg / mL的浓度范围内对该方法进行了验证。所开发和验证的方法可以成功地用于研究多塞平和诺多塞平的生物等效性/药代动力学。
更新日期:2020-04-06
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