Hotha Kishore Kumar, Kumar S Sirish, Bharathi D Vijaya, Venkateswarulu V
Bioanalytical Department, Integrated Product Development, Dr Reddy's Laboratories Ltd, Bachupalli, Hyderabad 500072, India.
Biomed Chromatogr. 2012 Apr;26(4):491-6. doi: 10.1002/bmc.1692. Epub 2011 Sep 8.
A highly sensitive, specific and fully validated LC-MS/MS method as per general practices of industry has been developed for estimation of lamotrigine (LAM) with 100 μL of human plasma using flucanozole as an internal standard (IS). The API-4000 LC-MS/MS was operated under the multiple reaction-monitoring mode using electrospray ionization. A simple liquid-liquid extraction process was used to extract LAM and IS from human plasma. The total run time was 2.0 min and the elution of LAM and IS occurred at 1.25 and 1.45 min; this was achieved with a mobile phase consisting of 0.1% formic acid-methanol (20:40:40, v/v) at a flow rate of 0.50 mL/min on a Discovery CN (50 × 4.6 mm, 5 µm) column. The developed method was validated in human plasma with a lower limit of quantitation of 0.1 ng/mL for LAM. A linear response function was established for the range of concentrations 0.1-1500 ng/mL (r > 0.998) for LAM. The intra- and inter-day precision values for LAM met the acceptance as per Food and Drug Administration guidelines. LAM was stable in the set of stability studies, viz. bench-top, autosampler and freeze-thaw cycles. The developed assay method was applied to an oral bioequivalence study in humans.
按照行业惯例，已开发出一种高度灵敏、特异且经过充分验证的液相色谱-串联质谱（LC-MS/MS）方法，用于使用氟康唑作为内标（IS），以100 μL人血浆来测定拉莫三嗪（LAM）。API-4000 LC-MS/MS在多反应监测模式下运行，采用电喷雾电离。使用简单的液-液萃取过程从人血浆中萃取LAM和IS。总运行时间为2.0分钟，LAM和IS的洗脱分别在1.25分钟和1.45分钟出现；这是在Discovery CN（50×4.6 mm，5 µm）柱上，以0.1%甲酸-甲醇（20:40:40，v/v）为流动相，流速为0.50 mL/min的条件下实现的。所开发的方法在人血浆中进行了验证，LAM的定量下限为0.1 ng/mL。建立了LAM在0.1 - 1500 ng/mL浓度范围内的线性响应函数（r > 0.998）。LAM的日内和日间精密度值符合美国食品药品监督管理局指南的接受标准。LAM在一系列稳定性研究中，即台式、自动进样器和冻融循环条件下均稳定。所开发的测定方法应用于人体口服生物等效性研究。
