液液萃取—高效液相色谱串联质谱法测定血清中可替宁及3-羟基可替宁含量  

Determination of cotinine and 3-hydroxycotinine in serum using liquid-liquid extraction-high performance liquid chromatography-tandem mass spectrometry

作　　者：张志荣[1] 张来颖[1] 李韵冰 刘裕婷[1] 王玉江[1] 陈曦[2] ZHANG Zhi-rong;ZHANG Lai-ying;LI Yun-bing;LIU Yu-ting;WANG Yu-jiang;CHEN Xi(Fengtai District Center for Disease Control and Prevention,Beijing 100071,China;National Institute of Environmental Health,Chinese Center for Disease Control and Prevention)

机构地区：[1]北京市丰台区疾病预防控制中心,北京100071 [2]中国疾病预防控制中心环境与健康相关产品安全所

出　　处：《环境卫生学杂志》2024年第2期167-172,共6页JOURNAL OF ENVIRONMENTAL HYGIENE

摘　　要：目的建立全自动固体介质液液萃取—高效液相色谱串联质谱法(HPLC-MS/MS)同时测定血清中可替宁及3-羟基可替宁含量的检测方法。方法采用液液萃取进行前处理,样品中加入50μL氢氧化钾溶液,50μL同位素内标溶液,混匀加入到固体介质液液萃取96孔板中,先后加入2次750μL萃取溶剂(5%异丙醇二氯甲烷混合液)后,收集萃取液,氮气吹干,100μL纯水复溶后上机测定。以HSS T3液相色谱柱(2.1 mm×100 mm,1.8μm)、0.05%氨水溶液和乙腈为流动相进行梯度洗脱分离目标物。采用正离子电喷雾模式电离,多反应离子监测方式(MRM)检测,保留时间和离子对定性,内标法定量。结果2种目标物在0.1~50、5~300μg/L范围内线性关系好,相关系数(r)均优于0.9990;可替宁与3-羟基可替宁方法检出限分别为0.016与0.023μg/L;2种化合物在添加水平为2.0、15.0、40.0μg/L时,平均回收率为86.4%~117.2%,相对标准偏差(RSD)为2.03%~8.03%。结论该方法样品前处理有机试剂消耗少,可实现自动化操作,准确性、精密度及灵敏度均较好,适用于吸烟及非吸烟人群血清中可替宁及3-羟基可替宁含量的检测。Objective To establish a method for the simultaneous determination of the contents of cotinine and 3-hydroxycotinine in serum using automatic liquid-liquid extraction-high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)in a solid medium.Methods The sample was pretreated with liquid-liquid extraction by adding 50μL of potassium hydroxide solution and 50μL of isotope internal standard.The solution was mixed well and added into a 96-well plate used for liquid-liquid extraction in a solid medium.After 750μL of extraction solvent(5%isopropanol dichloromethane mixture)was added twice at an interval of time,the extraction solution was collected and blow-dried with nitrogen gas.The sample was reconstituted with 100μL of pure water and then loaded onto the machine for determination.The compounds of interest were separated on an HSS T3 liquid-phase column(2.1 mm×100 mm,1.8μm),with 0.05%ammonia solution and acetonitrile used as a mobile phase for gradient elution separation.The sample underwent positive electrospray ionization and multiple reaction monitoring.The retention time and ion pairs were measured using quali-tative method,and quantitative measurements were performed using the internal standard method.Results The two compounds of in-terest had a good linear relationship within the ranges of 0.1-50 and 5-300μg/L,with a correlation coefficient(r)of greater than 0.9990.The limits of detection of cotinine and 3-hydroxycotinine were 0.016 and 0.023μg/L,respectively.The average recoveries of the two compounds were between 86.4%and 117.2%,with relative standard deviations falling between 2.03%and 8.03%,when the spiking levels were 2.0,15.0,and 40.0μg/L.Conclusion The pretreatment of samples using this method consumes less organic reagents,most steps can be automated,and it is accurate,precise,and sensitive,making it suitable for the determination of the con-tent of cotinine and 3-hydroxycotinine in the serum of smokers and non-smokers.

关 键 词：高效液相色谱—串联质谱 血清 可替宁 3-羟基可替宁 

分 类 号：R115[医药卫生—公共卫生与预防医学]