超高液相色谱串联质谱法测定贝类组织中环境雄激素睾酮的前处理方法优化

Optimization of Sample Pretreatment for Determination of Testosterone in Shellfish by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry

环境雄激素属于典型的内分泌干扰物,不仅干扰生物内分泌系统导致其生长发育受阻,还会对人体健康和生态安全造成威胁.为快速测定贝类组织中典型雄激素睾酮的质量浓度,分别在4种提取剂乙腈、乙酸乙酯、叔丁基甲醚、正己烷-二氯甲烷(1∶1)及3种净化方法超低温冷冻离心、超低温冷冻离心-净化剂、超低温冷冻离心-HLB固相萃取柱条件下提取河蚬组织中的睾酮,通过比较其回收率,优化超高液相色谱-串联质谱法测定睾酮的前处理方法.结果表明:4种提取剂中乙酸乙酯对睾酮提取效果最好,回收率高达101.6%;提取液经超低温(-80℃)冷冻离心法净化后回收率最高,且操作简便.在此优化条件下,睾酮在质量浓度为0.05~1.00μg/mL范围内与对应的峰面积呈现良好的线性正相关关系(R 2=0.9989),检出限为6.25 ng/g,定量限为20 ng/g.不同添加水平的加标回收率为66.4%~101.1%,相对标准偏差为2.3%~11.6%.经本研究优化后的前处理方法,操作简便、重现性好、稳定性高,具有良好的准确度和精密度,适用于贝类样品中睾酮的提取分析.

In the present study,the recoveries of testosterone from Corbicula fluminea extracted with acetonitrile,ethyl acetate,tert-butyl methyl ether and hexane-methylene chloride(1∶1)mixture were compared.The effects of three purification methods of ultra-low temperature refrigerated centrifugation,ultra-low temperature refrigerated centrifugation with purifying agent,and ultra-low temperature refrigerated centrifugation with HLB solid phase extraction column on the recovery of testosterone from Corbicula fluminea were researched to optimize the pretreatment method for determination of testosterone in shellfish by ultra-performance liquid chromatography-tandem mass spectrometry.The results showed that ethyl acetate had the best extracting effect among the four extracting agents,with a recovery of 101.6%.Among three purification methods,ultra-low temperature(-80℃)refrigerated centrifugation had the highest recovery rate.There was a good linear relationship between testosterone concentration and the peak area in the range of 0.05~1.00μg/mL,with the correlation coefficients all above 0.9989.The limit of detection was 6.25 ng/g,and the limit of quantitation was 20 ng/g.The average recoveries for testosterone at three concentration levels were 66.4%~101.1%,with the relative standard deviation between 2.3%~11.6%.The pretreatment method was easy to operate,also accurate and precision.It was suitable for the extraction of testosterone from shellfish.