LC-HRMS法检测C57BL/6小鼠血液中NNK及其7种代谢物
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摘要
为准确测定小鼠血液中4-(N-甲基-N-亚硝氨基)-1-(3-吡啶基)-1-丁酮(NNK)及其代谢物,了解小鼠体内NNK的代谢途径,利用Q Exactive轨道阱高分辨质谱仪,建立了一种同时检测C57BL/6小鼠血液中NNK及其7种代谢物[4-(N-甲基-N-亚硝氨基)-1-(3-吡啶基)-1-丁醇(NNAL)、NNK-N-氧化物(NNK-N-oxide)、NNAL-N-氧化物(NNAL-N-oxide)、4-羟基-1-(3-吡啶基)-1-丁酮(HPB)、1-(3-吡啶基)-1,4-二醇(PBD)、1-(3-吡啶基)-1-丁酮-4-羧酸(吡啶基丁酮酸)和1-(3-吡啶基)-1-丁醇-4-羧酸(吡啶基羟基酸)]的液相色谱-高分辨质谱(LC-HRMS)方法。以NNK-d4、NNAL-d3和HPB-d4为内标,小鼠血清样品经乙腈沉淀蛋白后,直接进行亲水相互作用色谱(HILIC)柱分离和质谱分析,质谱扫描模式采用一级全扫描和数据依赖二级扫描(Full MS/dd MS2)模式。结果表明:各目标物的定量限在0.042~0.263 ng/m之间,线性相关系数为0.999 1~1.000 0,加标回收率在89.2%~116.5%之间,日内精密度在1.54%~8.06%之间。该方法选择性好,分析效率高,目标物线性范围大,适合于小鼠血液中NNK及其代谢物检测与分析。
In order to accurately determine levels of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone(NNK) and its metabolites in mouse blood and the metabolic pathways of NNK in mice, a liquid chromatography-high resolution mass spectrometry(LC-HRMS) method for simultaneous determination of NNK and its seven metabolites [including 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanol(NNAL),NNK-N-oxide, NNAL-N-oxide, 4-hydroxy-1-(3-pyridyl)-1-butanone(HPB), 1-(3-pyridyl)-1,4-diol(PBD), 1-(3-pyridyl)-1-butanone-4-carboxylic acid and 1-(3-pyridyl)-1-butanol-4-carboxylic acid] in the blood ofC57BL/6 mice was established by Q Exactive Orbitrap High Resolution Mass Spectrometer. With NNK-d4,NNAL-d3, and HPB-d4 as internal standards, after protein precipitation with acetonitrile, the serum samples of C57BL/6 mice were separated by a HILIC column and determined by a mass spectrometer under full scan/data dependent MS2 mode. The results showed that the limits of quantification of the target analytes ranged from 0.042 to 0.263 ng/m L with correlation coefficients of 0.999 1 to 1.000 0, and the recoveries ranged from 89.2% to116.5% with the intra-day precision of 1.54% to 8.06%. The developed method features good selectivity, high efficiency and large linear range, and is suitable for the simultaneous determination of NNK and its seven metabolites in mouse blood.
In order to accurately determine levels of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone(NNK) and its metabolites in mouse blood and the metabolic pathways of NNK in mice, a liquid chromatography-high resolution mass spectrometry(LC-HRMS) method for simultaneous determination of NNK and its seven metabolites [including 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanol(NNAL),NNK-N-oxide, NNAL-N-oxide, 4-hydroxy-1-(3-pyridyl)-1-butanone(HPB), 1-(3-pyridyl)-1,4-diol(PBD), 1-(3-pyridyl)-1-butanone-4-carboxylic acid and 1-(3-pyridyl)-1-butanol-4-carboxylic acid] in the blood ofC57BL/6 mice was established by Q Exactive Orbitrap High Resolution Mass Spectrometer. With NNK-d4,NNAL-d3, and HPB-d4 as internal standards, after protein precipitation with acetonitrile, the serum samples of C57BL/6 mice were separated by a HILIC column and determined by a mass spectrometer under full scan/data dependent MS2 mode. The results showed that the limits of quantification of the target analytes ranged from 0.042 to 0.263 ng/m L with correlation coefficients of 0.999 1 to 1.000 0, and the recoveries ranged from 89.2% to116.5% with the intra-day precision of 1.54% to 8.06%. The developed method features good selectivity, high efficiency and large linear range, and is suitable for the simultaneous determination of NNK and its seven metabolites in mouse blood.
引文
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[12]姜树银,郭常川,石峰,等.Q-Orbitrap高分辨质谱用于降血脂类中成药和保健食品中非法添加物的快速筛查、鉴定和定量[J].药物分析杂志,2015,35(8):1447-1452.JIANG Shuyin,GUO Changchuan,SHI Feng,et al.Application of Q-Orbitrap high resolution mass spectrometry to the detection of illegal adulterated antihyperlipidemics in herbal medicines and dietary supplements[J].Chinese Journal of Pharmaceutical Analysis,2015,35(8):1447-1452.
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[14]LI Chenchen,WEN Dawei,ZHANG Jianxun,et al.Study of the metabolism on tobacco-specific N-nitrosamines in the rabbit by solid-phase extraction and liquid chromatography-tandem mass spectrometry[J].Analytical and Bioanalytical Chemistry,2006,386(7):1985-1993.
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[16]Jorquera R,Castonguay A,Schuller M H.Placental transfer of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone instilled intratracheally in Syrian golden hamsters[J].Cancer Research,1992,52(12):3273-3280.
[2]International Agency for Research on Cancer(IARC).Monographs on the evaluation of carcinogenic risk to humans,Volume 89:Smokeless tobacco and some tobacco-specific nitrosamines[M].Lyon:World Health Organization,2007.
[3]Hecht S S.Biochemistry,biology,and carcinogenicity of tobacco-specific N-nitrosamines[J].Chemical Research in Toxicology,1998,11(6):559-603.
[4]Lee H L,Wang C Y,Lin S S,et al.Liquid chromatography/tandem mass spectrometric method for the simultaneous determination of tobacco-specific nitrosamine NNK and its five metabolites[J].Talanta,2007,73(1):76-80.
[5]Zhou X,D’Agostino J,Xie F,et al.Role of CYP2A5 in the bioactivation of the lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in mice[J].Journal of Pharmacology and Experimental Therapeutics,2012,341(1):233-241.
[6]郎海磊,王昇,赵贝贝,等.HILIC-MS/MS测定家兔血液中的NNK及其7种代谢物[J].烟草科技,2013(3):31-36.LANG Hailei,WANG Sheng,ZHAO Beibei,et al.Determination of NNK and its 7 metabolites in rabbit blood with HILIC-MS/MS[J].Tobacco Science&Technology,2013(3):31-36.
[7]CAO Baojun,ZHANG Qidong,JI Houwei,et al.Simultaneous determination of NNK and its metabolites in mouse tissue for evaluating the effects of chronic alcohol consumption on the metabolism of NNK in mouse liver and lung[J].Analytical and Bioanalytical Chemistry,2014,406(18):4465-4471.
[8]安卓玲,史忱,赵瑞,等.基于超高效液相色谱-质谱的药物性肝损伤患者血清代谢组学研究[J].分析化学,2015,43(9):1408-1414.AN Zhuoling,SHI Chen,ZHAO Rui,et al.Ultra high performance liquid chromatography coupled with QExactive hybrid quadrupole-orbitrap mass spectrometry for serumal metabonomics study of drug-induced liver injury in patients[J].Chinese Journal of Analytical Chemistry,2015,43(9):1408-1414.
[9]叶芳挺,王勇为,Chang J S.利用Thermo Scientific全新高分辨台式、四极杆静电场轨道阱Q-Exactive分析19种β-受体激动剂[J].中国食品,2011(18):62-65.
[10]冯明飞,刘俊辉,卢斌斌,等.UPLC-Q Exactive轨道阱高分辨质谱法测定小鼠血液中的NNN及其7种代谢物[J].烟草科技,2015,48(6):40-44.FENG Mingfei,LIU Junhui,LU Binbin,et al.Determination of NNN and its seven metabolites in mouse blood with UPLC-Q Exactive orbit trap high-resolution mass spectrometry[J].Tobacco Science&Technology,2015,48(6):40-44.
[11]Ngonggang D A,Duy V S,SauvéS.Analysis of nine N-nitrosamines using liquid chromatography-accurate mass high resolution-mass spectrometry on a Q-Exactive instrument[J].Analytical Methods,2015,7(14):5748-5759.
[12]姜树银,郭常川,石峰,等.Q-Orbitrap高分辨质谱用于降血脂类中成药和保健食品中非法添加物的快速筛查、鉴定和定量[J].药物分析杂志,2015,35(8):1447-1452.JIANG Shuyin,GUO Changchuan,SHI Feng,et al.Application of Q-Orbitrap high resolution mass spectrometry to the detection of illegal adulterated antihyperlipidemics in herbal medicines and dietary supplements[J].Chinese Journal of Pharmaceutical Analysis,2015,35(8):1447-1452.
[13]Concheiro M,Anizan S,Ellefsen K,et al.Simultaneous quantification of 28 synthetic cathinones and metabolites in urine by liquid chromatography-high resolution mass spectrometry[J].Analytical and Bioanalytical Chemistry,2013,405(29):9437-9948.
[14]LI Chenchen,WEN Dawei,ZHANG Jianxun,et al.Study of the metabolism on tobacco-specific N-nitrosamines in the rabbit by solid-phase extraction and liquid chromatography-tandem mass spectrometry[J].Analytical and Bioanalytical Chemistry,2006,386(7):1985-1993.
[15]Staretz E M,Hecht S S.Effect of phenethyl isothiocyanate on the tissue distribution of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and metabolites in F344 rats[J].Cancer Research,1995,55:5580-5588.
[16]Jorquera R,Castonguay A,Schuller M H.Placental transfer of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone instilled intratracheally in Syrian golden hamsters[J].Cancer Research,1992,52(12):3273-3280.