Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul 06351, Korea.
Health Promotion Center, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul 06351, Korea.
J Anal Toxicol. 2022 Feb 14;46(1):25-36. doi: 10.1093/jat/bkaa177.
Measuring nicotine metabolites is the most objective method for identifying smoke exposure. Liquid chromatography--tandem mass spectrometry (LC-MS-MS) can measure multiple metabolites and is sensitive enough to detect low concentrations of metabolites. Therefore, we developed a simple and high-throughput method for measuring nicotine, cotinine, trans-3'-hydroxycotinine (3-OH cotinine), nornicotine and anabasine for population-based studies using LC-MS-MS. Each 30 µL of urine sample was diluted with 90 µL of acetonitrile containing five deuterated internal standards. Chromatographic separation used a C18 column, and LC-MS-MS analysis was performed with a multiple reaction monitoring mode. The chromatographic run time for each sample was 6.5 min. The method was validated by evaluating selectivity, interference, limit of detection, lower limit of quantification, precision, accuracy, linearity, extraction recovery, matrix effect and carryover according to guidelines. Our methods required a short preparation time (∼20 min) while simultaneously measuring five markers for smoking status. No endogenous or exogenous interference was found. Our method showed excellent precision and accuracy: within-run coefficient of variation (CV) 2.9-9.4%, between-run CV 4.8-8.7% and bias -10.1 to 5.3%. Linear dynamic ranges were 1-10,000 ng/mL for nicotine, nornicotine and anabasine; 2-5,000 ng/mL for cotinine and 5-15,000 ng/mL for 3-OH cotinine. Extraction recovery was consistent (87-109%) across concentrations. No significant matrix effect or carryover was observed. The validated method was applied to 849 urine samples. In samples from the 125 current smokers, nicotine, cotinine, 3-OH cotinine, nornicotine and anabasine were detected in 97.6, 99.2, 98.4, 96.8 and 87.2%, respectively. No markers were detected in 93.9% of 609 nonsmokers. The overlapping detection of multiple markers made it possible to identify the smoking status even in current smokers with a low concentration of cotinine. Our LC-MS-MS method using a simple sample preparation technique is sensitive and effective for screening of smoking status in the general population.
测量尼古丁代谢物是识别吸烟暴露最客观的方法。液相色谱-串联质谱法（LC-MS-MS）可以测量多种代谢物，并且足够灵敏，可以检测到低浓度的代谢物。因此，我们开发了一种简单高效的基于 LC-MS-MS 的人群研究用尼古丁、可替宁、反式-3'-羟基可替宁（3-OH 可替宁）、去甲烟碱和新烟碱的测量方法。每个 30μL 的尿样用 90μL 含 5 种氘代内标物的乙腈稀释。色谱分离采用 C18 柱，LC-MS-MS 分析采用多反应监测模式。每个样品的色谱运行时间为 6.5 分钟。该方法通过根据指南评估选择性、干扰、检测限、定量下限、精密度、准确度、线性、提取回收率、基质效应和交叉污染来验证。我们的方法准备时间短（约 20 分钟），同时可测量吸烟状态的 5 种标志物。未发现内源性或外源性干扰。我们的方法显示出良好的精密度和准确度：批内变异系数（CV）为 2.9-9.4%，批间 CV 为 4.8-8.7%，偏差为-10.1%至 5.3%。尼古丁、去甲烟碱和新烟碱的线性动态范围为 1-10,000ng/mL；可替宁为 2-5,000ng/mL；3-OH 可替宁为 5-15,000ng/mL。提取回收率在浓度范围内一致（87-109%）。未观察到明显的基质效应或交叉污染。验证后的方法应用于 849 个尿样。在 125 名当前吸烟者的样本中，尼古丁、可替宁、3-OH 可替宁、去甲烟碱和新烟碱的检出率分别为 97.6%、99.2%、98.4%、96.8%和 87.2%。609 名非吸烟者中 93.9%的样本未检出标志物。多种标志物的重叠检测使得即使在可替宁浓度低的当前吸烟者中也能识别吸烟状态成为可能。我们使用简单的样品制备技术的 LC-MS-MS 方法对于一般人群的吸烟状态筛查具有灵敏性和有效性。
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