超高压液相色谱-串联质谱法快速测定牛肉、牛肾中3种甾类同化激素药物残留量
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摘要
建立了一种以叔丁基甲醚为提取溶剂,无需固相萃取净化,即可采用超高压液相色谱-串联质谱法快速测定牛肉和牛肾中3种甾类同化激素(群勃龙、睾酮、黄体酮)的方法。结果表明:3种甾类同化激素在0.0～50.0μg/L浓度范围内的线性关系良好,相关系数大于0.996 7。在1.0、2.0、10.0μg/kg3个浓度水平下对牛肉和牛肾基质做添加回收实验,以外标法进行定量,平均回收率为73.6%～82.8%,相对标准偏差小于10%。使用该方法检测实际样品,具有操作简便、回收率高,阳性样品检出准确等优点,适用于大批量样品的快速检测。
Trenbolone,testosterone and progesterone are steroid-assimilating hormone drugs,which are widely used in animal husbandry,especially in ruminants.Most hormones have potential carcinogenic effects.Long-term intake of assimilative hormones can lead to metabolic disorders,dysplasia or tumors.The residues of assimilating hormone drugs in liver,kidney and adipose tissue are higher,and the residues in muscle and plasma are lower.Therefore,the development of detection method for steroid assimilation hormone prototype drug in animal-derived food has significance.In this study,a high pressure liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)method was established for determination of the amount of hormones residues in beef and kidney products.The methyl tert-butyl ester solution was used for the extraction of steroid residues in beef and kidney products without solid phase extraction column.External standard method was used to quantify,so reducing pretreatment steps as much as possible,using high-efficiency extraction solvent,and then reducing sample loss,is the key factor to improve the detection limit of the method.The results showed that higher recovery could be obtained even without further purification and separation with tert-butyl methyl ether as extraction solvent,and the matrix interference had little effect.However,when the sample matrix was more complex(such as bovine kidney samples),especially in the case of more fat,direct detection without purification and separation will inevitably cause pollution to the instrument.Therefore,the method of centrifugal degreasing by freezing the sample at low temperature after rotary evaporation was used.This procedure not only plays an important role in purification,but also shortens the pretreatment time.The linearity was satisfactory with the correlation coefficient more than 0.996 7 in the range of 0.0-50.0μg/L.Average recoveries for the steroid residues were in the range of 74%-83% with RSD below 10% in beef and kidney.The method is convenient,rapid and sensitive,which is suit for simultaneously determine three steroid residues in beef and kidney.
Trenbolone,testosterone and progesterone are steroid-assimilating hormone drugs,which are widely used in animal husbandry,especially in ruminants.Most hormones have potential carcinogenic effects.Long-term intake of assimilative hormones can lead to metabolic disorders,dysplasia or tumors.The residues of assimilating hormone drugs in liver,kidney and adipose tissue are higher,and the residues in muscle and plasma are lower.Therefore,the development of detection method for steroid assimilation hormone prototype drug in animal-derived food has significance.In this study,a high pressure liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)method was established for determination of the amount of hormones residues in beef and kidney products.The methyl tert-butyl ester solution was used for the extraction of steroid residues in beef and kidney products without solid phase extraction column.External standard method was used to quantify,so reducing pretreatment steps as much as possible,using high-efficiency extraction solvent,and then reducing sample loss,is the key factor to improve the detection limit of the method.The results showed that higher recovery could be obtained even without further purification and separation with tert-butyl methyl ether as extraction solvent,and the matrix interference had little effect.However,when the sample matrix was more complex(such as bovine kidney samples),especially in the case of more fat,direct detection without purification and separation will inevitably cause pollution to the instrument.Therefore,the method of centrifugal degreasing by freezing the sample at low temperature after rotary evaporation was used.This procedure not only plays an important role in purification,but also shortens the pretreatment time.The linearity was satisfactory with the correlation coefficient more than 0.996 7 in the range of 0.0-50.0μg/L.Average recoveries for the steroid residues were in the range of 74%-83% with RSD below 10% in beef and kidney.The method is convenient,rapid and sensitive,which is suit for simultaneously determine three steroid residues in beef and kidney.
引文
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[7]SHAO B,ZHAO R,MENG J.Simultaneous determination of residual hormonal chemicals in meat,kidney,liver tissues and milk by liquid chromatography-tandem mass spectrometry[J].Analytica Chimica Acta,2005,548(1/2):41-50.
[8]徐锦忠,张晓燕,丁涛,吴斌,沈崇钰,蒋原,刘飞.高效液相色谱-串联质谱法同时检测鸡肉和鸡蛋中合成类固醇类激素和糖皮质激素[J].分析化学,2009,37(3):341-346.XU Jinzhong,ZHANG Xiaoyan,DING Tao,WU Bin,SHEN Chongyu,JIANG Yuan,LIUFei.Determination of anabolic steroid and glucocorticoid hormones in chicken and egg using liquid chromatography tandem mass spectrometry[J].Chinese Journal of Analytical Chemistry,2009,37(3):341-346(in Chinese).
[9]班付国,陈蔷,宋志超,贾振民,吴宁鹏,彭丽.超高效液相色谱-串联质谱法测定水产品中群勃龙残留方法研究[J].中国兽药杂志,2010,44(10):46-47.BAN Fuguo,CHEN Qiang,SONG Zhichao,JIAZhenmin,WU Ningpeng,PENG Li.Determination of trenbolone residues in fishery products by ultra performance liquid chromatography-tandem mass spectrometry[J].Chinese Journal of Veterinary Drug,2010,44(10):46-47(in Chinese).
[10]牛晋阳,孙焕,李莹莹.高效液相色谱串联质谱法测定猪肉中10种类固醇类激素残留[J].食品科学,2010,31(4):230-232.NIU Jinyang,SUN Huan,LI Yingying.Highperformance liquid chromatography coupled with tandem mass spectrometric determination of steroid hormone multi-residues in pork[J].Food Science,2010,31(4):230-232(in Chinese).
[11]SEO J,KIM H Y,CHUNG B C.Simultaneous determination of anabolic steroids and synthetic hormones in meat by freezing-lipid filtration,solid-phase extraction and gas chromatography-mass spectrometry[J].J Chromatogr.A,2005,1 067(1/2):303-309.
[12]湛嘉,俞雪钧,李佐卿,谢东华,樊苑牧,陈树兵.黄鳝肌肉中的20种性激素同时测定的气相色谱串联质谱法[J].食品科学,2008,(6):298-303.ZHAN Jia,YU Xuejun,LI Zuoqing,XIE Donghua,FAN Yuanmu,CHEN Shubing.Simultaneous determination of residue of sex hormones in eel muscle by gas chromatography-mass spectrometry[J].Food Science,2008,(6):298-303(in Chinese).
[13]于慧娟,张珊珊,张晓玲,惠云华,顾润润,冯兵.同位素稀释-液相色谱-串联质谱法同时测定水产品中雌激素、孕激素和雄激素[J].质谱学报,2014,35(2):170-178.YU Huijuan,ZHANG Shanshan,ZHANG Xiaoling,HUI Yunhua,GU Runrun,FENG Bing.Simultaneous determination of the estrogen,progestogen and androgen in aquatic products by isotope dilution LC-MS/MS[J].Journal of Chinese Mass Spectrometry Society,2014,35(2):170-178(in Chinese).
[14]李宁,张玉龙,林涛,王继良,刘宏程.UPLC-MS法同时测定牛奶中磺胺类、喹诺酮类、甾体激素类及四环素类兽药残留[J].分析测试学报,2016,35(6):714-718.LI Ning,ZHANG Yulong,LIN Tao,WANGJiliang,LIU Hongcheng.Analysis of sulfonamide,quinolones,steroid hormones and tetracyclines residues in milk by ultra performance liquid chromatography-tandem mass spectrometry[J].Journal of Instrumental Analysis,2016,35(6):714-718(in Chinese).
[2]STOLKER A M,ZOONTJES P W,VAN G LA.The use of supercritical fluid extraction for the determination of steroids in animal tissues[J].Analyst,1998,123(12):2 671-2 676.
[3]DAESELEIRE E,VANDEPUTTE R,VAN PC.Validation of multi-residue methods for the detection of anabolic teroids by GC-MS in muscle tissues and urine samples from cattle[J].Analyst,1998,123(12):2 595-2 598.
[4]兽药管理条例实施细则.中华人民共和国农业部令第28号.
[5]动物性食品中兽药最高残留限量.农业部公告第235号.
[6]Food and Agriculture Organization of the United Nations.Residues of some veterinary drugs in animals and foods(FAO Food and Nutrition Paper 41)[M].1st Edition.Rome:Bernan’s Annotated Reporter Press,1994,13.
[7]SHAO B,ZHAO R,MENG J.Simultaneous determination of residual hormonal chemicals in meat,kidney,liver tissues and milk by liquid chromatography-tandem mass spectrometry[J].Analytica Chimica Acta,2005,548(1/2):41-50.
[8]徐锦忠,张晓燕,丁涛,吴斌,沈崇钰,蒋原,刘飞.高效液相色谱-串联质谱法同时检测鸡肉和鸡蛋中合成类固醇类激素和糖皮质激素[J].分析化学,2009,37(3):341-346.XU Jinzhong,ZHANG Xiaoyan,DING Tao,WU Bin,SHEN Chongyu,JIANG Yuan,LIUFei.Determination of anabolic steroid and glucocorticoid hormones in chicken and egg using liquid chromatography tandem mass spectrometry[J].Chinese Journal of Analytical Chemistry,2009,37(3):341-346(in Chinese).
[9]班付国,陈蔷,宋志超,贾振民,吴宁鹏,彭丽.超高效液相色谱-串联质谱法测定水产品中群勃龙残留方法研究[J].中国兽药杂志,2010,44(10):46-47.BAN Fuguo,CHEN Qiang,SONG Zhichao,JIAZhenmin,WU Ningpeng,PENG Li.Determination of trenbolone residues in fishery products by ultra performance liquid chromatography-tandem mass spectrometry[J].Chinese Journal of Veterinary Drug,2010,44(10):46-47(in Chinese).
[10]牛晋阳,孙焕,李莹莹.高效液相色谱串联质谱法测定猪肉中10种类固醇类激素残留[J].食品科学,2010,31(4):230-232.NIU Jinyang,SUN Huan,LI Yingying.Highperformance liquid chromatography coupled with tandem mass spectrometric determination of steroid hormone multi-residues in pork[J].Food Science,2010,31(4):230-232(in Chinese).
[11]SEO J,KIM H Y,CHUNG B C.Simultaneous determination of anabolic steroids and synthetic hormones in meat by freezing-lipid filtration,solid-phase extraction and gas chromatography-mass spectrometry[J].J Chromatogr.A,2005,1 067(1/2):303-309.
[12]湛嘉,俞雪钧,李佐卿,谢东华,樊苑牧,陈树兵.黄鳝肌肉中的20种性激素同时测定的气相色谱串联质谱法[J].食品科学,2008,(6):298-303.ZHAN Jia,YU Xuejun,LI Zuoqing,XIE Donghua,FAN Yuanmu,CHEN Shubing.Simultaneous determination of residue of sex hormones in eel muscle by gas chromatography-mass spectrometry[J].Food Science,2008,(6):298-303(in Chinese).
[13]于慧娟,张珊珊,张晓玲,惠云华,顾润润,冯兵.同位素稀释-液相色谱-串联质谱法同时测定水产品中雌激素、孕激素和雄激素[J].质谱学报,2014,35(2):170-178.YU Huijuan,ZHANG Shanshan,ZHANG Xiaoling,HUI Yunhua,GU Runrun,FENG Bing.Simultaneous determination of the estrogen,progestogen and androgen in aquatic products by isotope dilution LC-MS/MS[J].Journal of Chinese Mass Spectrometry Society,2014,35(2):170-178(in Chinese).
[14]李宁,张玉龙,林涛,王继良,刘宏程.UPLC-MS法同时测定牛奶中磺胺类、喹诺酮类、甾体激素类及四环素类兽药残留[J].分析测试学报,2016,35(6):714-718.LI Ning,ZHANG Yulong,LIN Tao,WANGJiliang,LIU Hongcheng.Analysis of sulfonamide,quinolones,steroid hormones and tetracyclines residues in milk by ultra performance liquid chromatography-tandem mass spectrometry[J].Journal of Instrumental Analysis,2016,35(6):714-718(in Chinese).