Kaisar Mohammad A, Kallem Raja Reddy, Sajja Ravi K, Sifat Ali Ehsan, Cucullo Luca
Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, 1300 S. Coulter Street, Amarillo, TX, 79106, USA.
Office of Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX, 79106, USA.
BMC Neurosci. 2017 Oct 11;18(1):71. doi: 10.1186/s12868-017-0389-5.
A sensitive, rapid and selective UHPLC-MS/MS method has been developed and validated for the quantification of Nicotine (NT) and Cotinine (CN) using Continine-d as internal standard (IS) as per FDA guidelines. Sample preparation involved simple protein precipitation of 20 µL mouse plasma or brain homogenate using acetonitrile at 1:8 ratio. Mass Spectrometer was operated in positive polarity under the multiple reaction-monitoring mode using electro spray ionization technique and the transitions of m/z 163.2 → 132.1, 177.2 → 98.0 and 180.2 → 101.2 were used to measure the NT, CN and IS, respectively. The elution of NT, CN and IS are at 1.89, 1.77 and 1.76 min, respectively. This was achieved with a gradient mobile phase consisting of 5 mM ammonium bicarbonate, acetonitrile and methanol (3:1, v/v) at a flow rate of 0.3 mL/min on a Kinetex EVO C18 column. The method was validated with a lower limit of quantitation 3.0 ng/mL in mouse plasma and brain for both the analytes.
A linear response function was established for the range of concentrations 3-200 (r > 0.995) for NT and 3-600 ng/mL (r > 0.995) for CN. The intra- and inter-day precision values met the acceptance criteria. NT and CN are stable in the battery of stability studies viz., stock solution, bench-top and auto-sampler.
This method was successfully utilized to validate a newly developed preclinical smoking model in mice.
根据美国食品药品监督管理局（FDA）的指导方针，已开发并验证了一种灵敏、快速且具有选择性的超高效液相色谱-串联质谱（UHPLC-MS/MS）方法，用于以可替宁-d作为内标（IS）对尼古丁（NT）和可替宁（CN）进行定量分析。样品制备过程包括使用乙腈按1:8的比例对20微升小鼠血浆或脑匀浆进行简单的蛋白质沉淀。质谱仪采用电喷雾电离技术在正离子模式下的多反应监测模式下运行，分别使用m/z 163.2→132.1、177.2→98.0和180.2→101.2的跃迁来测量NT、CN和IS。NT、CN和IS的洗脱时间分别为1.89、1.77和1.76分钟。这是通过在Kinetex EVO C18柱上以0.3毫升/分钟的流速使用由5毫摩尔/升碳酸氢铵、乙腈和甲醇（3:1，v/v）组成的梯度流动相实现的。该方法在小鼠血浆和脑中对两种分析物的定量下限均为3.0纳克/毫升时得到验证。
建立了NT在3 - 200纳克/毫升（r > 0.995）和CN在3 - 600纳克/毫升（r > 0.995）浓度范围内的线性响应函数。日内和日间精密度值符合验收标准。NT和CN在一系列稳定性研究中，即储备溶液、台式和自动进样器中均稳定。
该方法成功用于验证一种新开发的小鼠临床前吸烟模型。
