Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, 32545 Bad Oeynhausen, Germany.
J Pharm Biomed Anal. 2012 Aug-Sep;67-68:137-43. doi: 10.1016/j.jpba.2012.04.036. Epub 2012 May 3.
We developed a stable isotope dilution ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry assay to measure nicotine and cotinine, the major oxidative and pharmacologically less active metabolite of nicotine, in human urine. A simple dilution step was used as sample preparation and the measurement of nicotine and cotinine was performed during a 1.5-min run-time using nicotine-D₄ and cotinine-D₄ as internal standards. Multiple calibration curves for the analysis of both nicotine and cotinine exhibited a consistent excellent linearity and reproducibility in the range of 5-35,000 μg/L (r>0.999). Limits of Detection were 0.7 μg/L for nicotine and 0.4 μg/L for cotinine, and Lower Limits of Quantification were 1.7 μg/L for nicotine and 1.1 μg/L for cotinine. The intraassay coefficients of variation (CVs) for nicotine and cotinine were <4% and <2%, respectively, the interassay CVs were <6% for nicotine and <4% for cotinine. The inaccuracy was <6% for both substances. The mean recovery was 103.2% (range 96.8-105.1%) for nicotine and 97.4% (range 94.3-99.2%) for cotinine. A method comparison showed that the values of nicotine metabolites in human urine samples (n=98) measured by a commercially available chemiluminescent immunoassay tested on analyzer IMMULITE 2000 were much higher than the cotinine concentration in the same urine samples measured by our UPLC-MS/MS assay. The Passing-Bablok regression line was: immunoassay=4.62 (UPLC-MS/MS)+3.64 [μg/L]; r=0.75. This robust, sensitive and interference-free UPLC-MS/MS assay permits rapid and accurate determination of nicotine and cotinine in human urine.
我们开发了一种稳定同位素稀释超高效液相色谱-电喷雾串联质谱法，用于测量人尿液中的尼古丁和可替宁，尼古丁的主要氧化和药理学活性较低的代谢物。采用简单的稀释步骤作为样品制备，使用尼古丁-D₄和可替宁-D₄作为内标，在 1.5 分钟的运行时间内测量尼古丁和可替宁。尼古丁和可替宁的分析多校准曲线在 5-35,000μg/L（r>0.999）范围内均表现出一致的优异线性和重现性。尼古丁和可替宁的检测限分别为 0.7μg/L 和 0.4μg/L，定量下限分别为 1.7μg/L 和 1.1μg/L。尼古丁和可替宁的日内变异系数（CV）均<4%，<2%，日间 CV 分别<6%和<4%。两种物质的不准确度均<6%。尼古丁的平均回收率为 103.2%（范围为 96.8-105.1%），可替宁的回收率为 97.4%（范围为 94.3-99.2%）。方法比较表明，在 IMMULITE 2000 分析仪上使用市售化学发光免疫分析法测定的 98 个人尿液样本中尼古丁代谢物的值远高于相同尿液样本中可替宁的浓度，由我们的 UPLC-MS/MS 测定。通过 Passing-Bablok 回归线为：免疫分析法=4.62（UPLC-MS/MS）+3.64[μg/L]；r=0.75。这种强大、灵敏且无干扰的 UPLC-MS/MS 测定法可快速准确地测定人尿液中的尼古丁和可替宁。
