高效液相色谱-大气压化学电离串联质谱法研究4-(甲基亚硝胺基)-1-(3-吡啶基)-1-丁酮体外模拟代谢作用

烟草特异亚硝胺4-(甲基亚硝胺基)-1-(3-吡啶基)-1-丁酮(NNK)是一种存在于烟草烟气及烟草制品中的强致癌物，其致癌机理是通过细胞色素P450酶的代谢活化生成活泼亲电试剂，导致DNA损伤，因此，建立NNK体外模拟代谢产物的定量分析方法对于研究烟草致癌物具有重要意义。本实验利用高效液相色谱-大气压化学电离串联质谱法（HPLC-APCI-MS/MS）对NNK代谢产物4-羟基-1-(3-吡啶基)-1-丁酮(HPB)进行定量分析。使用选择反应扫描（SRM）模式监测了HPB和内标[3,3,4,4-D₄]HPB，分析方法在0.2~400 nmol/L范围内线性关系良好，线性相关系数R²=0.999 9，检出限(LOD)为0.025 nmol/L (S/N=3)，定量限(LOQ)为0.05 nmol/L(S/N=10)。方法的日内和日间准确度为96.6%~101.8%，回收率为98.1%~102.6%。所建立的NNK体外模拟代谢模型可用于烟草特异亚硝胺代谢活化分子机制的研究，为定量分析吸烟致癌相关生物标志物奠定基础。

Study on In vitro Simulation of the Metabolism of 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone Using HPLC-APCI-MS/MS

Tobacoo-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent carcinogen present in tobacco products and tobacco smoke. The carcinogenic mechanism of NNK is involved in the DNA damage induced by the active electrophiles generated from the metabolic activation of NNK catalyzed by cytochrome P450. It is significant to establish an in vitro metabolism model of NNK and the quantitation method for the investigation of tabacco carcinogens motabolites. In this work, the NNK metabolite of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) was quantitatively analyzed by high performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry (HPLC-APCI-MS/MS). Selective reaction monitoring (SRM) was employed for determining HPB and the internal standard [3,3,4,4-D₄]HPB simultaneously. The method shows good linearity within 0.2-400 nmol/L with the correlation coefficient (R₂) 0.999 9. The limit of detection (LOD) and the limit of quantitation (LOQ) are 0.025 nmol/L (S/N=3) and 0.05 nmol/L (S/N=10), respectively. The inter-day and intra day accuracy range from 96.6% to 101.8%, and the recovery is 98.1%-102.6%. The in vitro metabolism model of NNK can be used to elucidate the molecular mechanism of the metabolic activation of tobacco specific nitrosamine. This work layed a foundation for the quantification of biomarkers for tobacco carcinogenesis.