Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, UT 84112, United States.
J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Nov 15;879(30):3465-74. doi: 10.1016/j.jchromb.2011.09.026. Epub 2011 Sep 18.
A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of nicotine (NIC), cotinine (COT), nornicotine (NNIC), norcotinine (NCOT), nicotine-N-β-D-glucuronide (NIC GLUC), cotinine-N-β-D-glucuronide (COT GLUC), nicotine-1'-oxide (NNO), cotinine-N-oxide (CNO), trans-3'-hydroxycotinine (3-HC), anabasine (AB) and anatabine (AT) was modified and validated for quantification of these selected analytes in rat brain tissue. This analytical method provides support for preclinical NIC pharmacokinetic and toxicological studies after controlled dosing protocols. After brain homogenization and solid-phase extraction, target analytes and corresponding deuterated internal standards were chromatographically separated on a Discovery(®) HS F5 HPLC column with gradient elution and analyzed by LC-MS/MS in positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM) data acquisition. Method linearity was assessed and calibration curves were determined over the following ranges: 0.1-7.5 ng/mg for NIC, COT GLUC and AB; and 0.025-7.5 ng/mg for COT, NNIC, NCOT, NIC GLUC, NNO, CNO, 3-HC and AT (R(2)≥0.99 for all analytes). Extraction recoveries ranged from 64% to 115%, LC-MS/MS matrix effects were ≤21%, and overall process efficiency ranged from 57% to 93% at low and high quality control concentrations. Intra- and inter-assay imprecisions and accuracy for all analytes were ≤12.9% and ≥86%, respectively. The method was successfully applied to quantification of NIC and metabolites in the brain of post-natal day 90 rats that were sacrificed 2-h after a single 0.8 mg/kg s.c. administration of (-)NIC. In these tissues, striatal concentrations were 204.8±49.4, 138.2±14.2 and 36.1±6.1 pg/mg of NIC, COT and NNIC, respectively. Concentrations of NIC, COT and NNIC in the remaining whole brain (RWhB) were 183.3±68.0, 130.0±14.1 and 46.7±10.3 pg/mg, respectively. Quantification of these same analytes in plasma was also performed by a previously validated method. NIC, COT, NNIC, NCOT, NNO and CNO were detected in plasma with concentrations comparable to those reported in previous studies. However, and in contrast to brain tissues, COT concentrations in plasma were significantly higher than were those of NIC (194.6±18.6 ng/mL versus 52.7±12.9 ng/mL). Taken together, these results demonstrate that a sensitive and selective method has been developed for the determination of NIC biomarkers in rat brain.
一种液相色谱-串联质谱（LC-MS/MS）方法被修改并验证，用于同时定量大鼠脑组织中的尼古丁（NIC）、可替宁（COT）、去甲烟碱（NNIC）、去甲可替宁（NCOT）、尼古丁-N-β-D-葡萄糖醛酸（NIC GLUC）、可替宁-N-β-D-葡萄糖醛酸（COT GLUC）、尼古丁-1'-氧化物（NNO）、可替宁-N-氧化物（CNO）、反式-3'-羟基可替宁（3-HC）、假木贼碱（AB）和新烟草碱（AT）。这种分析方法为控制给药方案后进行 NIC 药代动力学和毒理学的临床前研究提供了支持。在脑匀浆和固相萃取后，目标分析物和相应的氘代内标在 Discovery(®) HS F5 HPLC 柱上进行色谱分离，采用正电喷雾电离（ESI）模式和多重反应监测（MRM）数据采集进行 LC-MS/MS 分析。方法线性通过评估和确定以下范围内的校准曲线来评估：NIC、COT GLUC 和 AB 的 0.1-7.5ng/mg；COT、NNIC、NCOT、NIC GLUC、NNO、CNO、3-HC 和 AT 的 0.025-7.5ng/mg（所有分析物的 R(2)均≥0.99）。提取回收率在 64%-115%之间，LC-MS/MS 基质效应在 21%以下，低、高质量控制浓度下的整体过程效率在 57%-93%之间。所有分析物的日内和日间精密度和准确度分别为≤12.9%和≥86%。该方法成功应用于单次皮下给予（-）NIC 0.8mg/kg 后 2 小时处死的 90 日龄大鼠脑内 NIC 和代谢物的定量。在这些组织中，纹状体中 NIC、COT 和 NNIC 的浓度分别为 204.8±49.4、138.2±14.2 和 36.1±6.1pg/mg。剩余全脑（RWhB）中 NIC、COT 和 NNIC 的浓度分别为 183.3±68.0、130.0±14.1 和 46.7±10.3pg/mg。还通过之前验证的方法对这些相同分析物在血浆中的定量进行了分析。血浆中检测到 NIC、COT、NNIC、NCOT、NNO 和 CNO，其浓度与之前的研究报告相似。然而，与脑组织不同的是，血浆中 COT 的浓度明显高于 NIC（194.6±18.6ng/mL 对 52.7±12.9ng/mL）。综上所述，这些结果表明，已经开发出一种用于测定大鼠脑组织中 NIC 生物标志物的灵敏和选择性方法。
