Hotha Kishore Kumar, Ravindranath L K, Veera K N Jaya, Kumar K Kishore, Reddy Y Ramachandra, Jagadeesh B, Rao D V Narayana, Bharathi D Vijaya, Mullangi Ramesh
Bioanalytical Department, Integrated Product Development, Dr Reddy's Laboratories Ltd, Bachupalli, Hyderabad-500 072, India.
Biomed Chromatogr. 2010 Oct;24(10):1113-9. doi: 10.1002/bmc.1413.
A highly sensitive and specific LC-MS/MS method has been developed for simultaneous estimation of nortriptyline (NTP) and 10-hydroxynortriptyline (OH-NTP) in human plasma (250 µL) using carbamazepine as an internal standard (IS). LC-MS/MS was operated under the multiple reaction-monitoring mode using the electrospray ionization technique. A simple liquid-liquid extraction process was used to extract NTP, OH-NTP and IS from human plasma. The total run time was 2.5 min and the elution of NTP, OH-NTP and IS occurred at 1.44, 1.28 and 1.39 min, respectively; this was achieved with a mobile phase consisting of 20 mm ammonium acetate : acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a HyPURITY C(18) column. The developed method was validated in human plasma with a lower limit of quantitation of 1.09 ng/mL for both NTP and OH-NTP. A linear response function was established for the range of concentrations 1.09-30.0 ng/mL (r > 0.998) for both NTP and OH-NTP. The intra- and inter-day precision values for NTP and OH-NTP met the acceptance as per FDA guidelines. NTP and OH-NTP were stable in a battery of stability studies, i.e. bench-top, auto-sampler and freeze-thaw cycles. The developed assay was applied to a pharmacokinetic study in humans.
已开发出一种高灵敏度和特异性的液相色谱-串联质谱（LC-MS/MS）方法，以卡马西平作为内标（IS），同时测定人血浆（250 μL）中的去甲替林（NTP）和10-羟基去甲替林（OH-NTP）。LC-MS/MS采用电喷雾电离技术，在多反应监测模式下运行。采用简单的液-液萃取法从人血浆中提取NTP、OH-NTP和内标。总运行时间为2.5分钟，NTP、OH-NTP和内标的洗脱时间分别为1.44分钟、1.28分钟和1.39分钟；这是在HyPURITY C(18)柱上，以流速0.50 mL/min的由20 mM醋酸铵：乙腈（20:80，v/v）组成的流动相实现的。所开发的方法在人血浆中进行了验证，NTP和OH-NTP的定量下限均为1.09 ng/mL。为NTP和OH-NTP在1.09-30.0 ng/mL的浓度范围内建立了线性响应函数（r>0.998）。NTP和OH-NTP的日内和日间精密度值符合FDA指南的接受标准。在一系列稳定性研究中，即台式、自动进样器和冻融循环中，NTP和OH-NTP均稳定。所开发的测定方法应用于人体药代动力学研究。
