超高效液相色谱-三重四极杆质谱法同时测定饲料中8种类固醇激素
|
摘要
建立了同时测定饲料中勃地酮、甲睾酮、诺龙、群勃龙、甲羟孕酮乙酸酯、美仑孕酮、乙酸甲地孕酮和17α-羟基孕酮8种类固醇激素的超高效液相色谱-三重四极杆质谱(UPLC-MS/MS)检测方法。考察了提取溶剂,除蛋白和除脂条件对8种类固醇激素回收率的影响,最终选择乙腈为提取溶剂,用三氯乙酸和氢氧化钠除蛋白,用氯化镁和正己烷除脂,样品经Athena C18-WP(2.1×150 mm,5μm)色谱柱分离,以5 mmol/L乙酸铵(1‰甲酸)和乙腈(1‰甲酸)进行梯度洗脱,正离子电喷雾电离多反应监测模式测定,内标法进行定量分析。结果显示,8种化合物在0～10μg/L范围内线性相关系数(r)≥0.9995;检出限为0.10～0.34μg/kg(S/N≥3),定量限为0.35～0.98μg/kg(S/N≥10)。方法的平均回收率范围为70.4%～109%,相对标准偏差为0.38%～10.3%(n=6)。该方法操作简单,灵敏度高,重复性好,适用于饲料中8种类固醇激素的快速检测。
A method was developed for the screening and quantification of 8 steroid hormones including(boldenone,17-alpha-Methyltestosterone,19-Nortestosterone,trenbolone,medroxyprogesterone,melengestrol,megestrol-17-acetate,17-alpha-Hydroxyprogesterone)in feed by ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS). The effects of extraction solvent,protein removal and degreasing conditions on the recovery of 8 steroid hormones were investigated. Finally,acetonitrile was selected as the extraction solvent,protein was removed with trichloroacetic acid and sodium hydroxide,and fat was removed with magnesium chloride and n-hexane. In the chromatographic analysis,the 8 target compounds were separated by Athena C18-WP(2.1×150 mm,5 μm)column and gradient elution with 5 mmol/L ammonium acetate(1‰ formic acid)and acetonitrile(1‰ formic acid),detected in positive multiple reaction monitoring(MRM)and quantified with internal standards. The results showed that the 8 steroid hormones exhibited good linear relationships in the range of 0～10 μg/L with correlation coefficients(r)of more than 0.9995;the limits of detection(LODs,S/N≥3)were 0.10～0.34 μg/kg and the limits of quantification(LOQs,S/N≥10)were 0.35～0.98 μg/kg. Average recoveries were 70.4%～109% with relative standard deviation of 0.38%～10.3%(n=6). The method was simple,rapid,sensitive and reproducible,and was suitable for rapid screening and detection of 8 steroid hormones in feed.
A method was developed for the screening and quantification of 8 steroid hormones including(boldenone,17-alpha-Methyltestosterone,19-Nortestosterone,trenbolone,medroxyprogesterone,melengestrol,megestrol-17-acetate,17-alpha-Hydroxyprogesterone)in feed by ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS). The effects of extraction solvent,protein removal and degreasing conditions on the recovery of 8 steroid hormones were investigated. Finally,acetonitrile was selected as the extraction solvent,protein was removed with trichloroacetic acid and sodium hydroxide,and fat was removed with magnesium chloride and n-hexane. In the chromatographic analysis,the 8 target compounds were separated by Athena C18-WP(2.1×150 mm,5 μm)column and gradient elution with 5 mmol/L ammonium acetate(1‰ formic acid)and acetonitrile(1‰ formic acid),detected in positive multiple reaction monitoring(MRM)and quantified with internal standards. The results showed that the 8 steroid hormones exhibited good linear relationships in the range of 0～10 μg/L with correlation coefficients(r)of more than 0.9995;the limits of detection(LODs,S/N≥3)were 0.10～0.34 μg/kg and the limits of quantification(LOQs,S/N≥10)were 0.35～0.98 μg/kg. Average recoveries were 70.4%～109% with relative standard deviation of 0.38%～10.3%(n=6). The method was simple,rapid,sensitive and reproducible,and was suitable for rapid screening and detection of 8 steroid hormones in feed.
引文
[1]Fornes R,Maliqueo M,Hu M,et al.The effect of androgen excess on maternal metabolism,placental function and fetal growth in obese dams[J].Scientific Reports,2017,7(1):8066.
[2]宫立红,李亚英,刘少华.合成类固醇药物对人体雄性激素的影响[J].现代中西医结合杂志,2002,11(17):1682-1683.
[3]Peng C F,Chen Y W,Chen H Q,et al.A rapid and sensitive enzyme-linked immunosorbent assay(ELISA)method and validation for progestogen multi-residues in feed[J].Journal of Animal & Feed Sciences,2008,17(3):434-441.
[4]Hashemi S R,Davoodi H.Phytogenics as new class of feed additive in poultry industry[J].Journal of Animal & Veterinary Advances,2010,9(17):2295-2304.
[5]Wang L,Wang X,Yuan X,et al.Simultaneous analysis of diosgenin and sarsasapogenin in Asparagus officinalis byproduct by thin-layer chromatography[J].Phytochem Anal,2011,22(1):14-17.
[6]中华人民共和国农业部,中华人民共和国卫生部,国家药品监督管理局..中华人民共和国农业部公告第193号《食品动物禁用的兽药及其它化合物清单》[S].北京:中国农业出版社,2002.
[7]Commission Decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methds and the interprettion of results[J].Official Journal,L 221,17/08/2002:8-36.
[8]NagayamaT.Positive list system for agricultural chemical residues in foods[J].Journal of Pesticide Science,2005,30(4):418-425.
[9]中华人民共和国业部,中华人民共和国卫生部,国家药品监督管理局.中华人民共和国农业部公告第176号《禁止在饲料和动物饮用水种使用的药物品种目录》[S].北京:中国农业出版社,2002.
[10]黄丽英,邹圆,陆强,等.分散固相萃取-超高效液相色谱-串联质谱法测定禽蛋中4种内外源性孕激素[J].食品科学,2018,39(16):295-300.
[11]连英杰,林升航,曾琪,等.QuEChERS/超高效液相色谱-电喷雾串联质谱法检测鸡肉中11种激素类药物残留[J].食品安全质量检测学报,2014(2):384-392.
[12]Muňiz-Valencia R,Ceballos-Magaňa S G,Gonzalo-Lumbreras R,et al.GC-MS method development and validation for anabolic steroids in feed samples[J].Journal of Separation Science,2015,31(4):727-734.
[13]刘红菊,闫冲,刘宏果.反相高效液相色谱法同时测定饲料中的10种性激素[J].中国饲料,2010(18):34-36.
[14]张峰,许成保,蓝芳,等.超高效液相色谱-四极杆飞行时间质谱法测定饲料中9种雄性激素类药物[J].分析化学,2012,40(1):101-106.
[15]张毅,蓝芳,张峰,等.液相色谱-串联质谱法测定谷物类饲料中的41种激素[J].色谱,2011,29(6):523-534.
[16]李敏青,徐娟,王岚,等.高效液相色谱-电喷雾电离串联质谱法测定植物性食品中氟虫腈及其代谢物的残留[J].食品工业科技,2019(2):245-250,256.
[17]Zhu Y,Xie S,Dongmei C,et al.Targeted analysis and determination of β-agonists,hormones,glucocorticoid,and psychiatric drugs in feed by liquid chromatography with electrospray ionization tandem mass spectrometry[J].Journal of Separation Science,2016,39(13):2584-2594.
[18]孙珊珊,朱丽君,胡延喜,等.超高效液相色谱-串联质谱测定饲料中镇静剂类和β-受体激素类药物残留[J].色谱,2018,36(2):150-158.
[19]姜光丽,何小琴,郗存显,等.QuEchERS/超高液相色谱-串联质谱法同时测定饲料中6种β-阻断剂类药物[J].中国饲料,2015(12):33-36.
[20]肖全伟,吴文林,杨万林,等.固相萃取-超高效液相色谱-串联质谱法同时测定饲料中的3种雌激素[J].色谱,2014,32(11):1209-1213.
[21]林小莉,李宁,霍峰,等.气相色谱-质谱法同时测定饲料中6种雌激素类药物[J].分析测试学报,2016,35(3):322-326.
[2]宫立红,李亚英,刘少华.合成类固醇药物对人体雄性激素的影响[J].现代中西医结合杂志,2002,11(17):1682-1683.
[3]Peng C F,Chen Y W,Chen H Q,et al.A rapid and sensitive enzyme-linked immunosorbent assay(ELISA)method and validation for progestogen multi-residues in feed[J].Journal of Animal & Feed Sciences,2008,17(3):434-441.
[4]Hashemi S R,Davoodi H.Phytogenics as new class of feed additive in poultry industry[J].Journal of Animal & Veterinary Advances,2010,9(17):2295-2304.
[5]Wang L,Wang X,Yuan X,et al.Simultaneous analysis of diosgenin and sarsasapogenin in Asparagus officinalis byproduct by thin-layer chromatography[J].Phytochem Anal,2011,22(1):14-17.
[6]中华人民共和国农业部,中华人民共和国卫生部,国家药品监督管理局..中华人民共和国农业部公告第193号《食品动物禁用的兽药及其它化合物清单》[S].北京:中国农业出版社,2002.
[7]Commission Decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methds and the interprettion of results[J].Official Journal,L 221,17/08/2002:8-36.
[8]NagayamaT.Positive list system for agricultural chemical residues in foods[J].Journal of Pesticide Science,2005,30(4):418-425.
[9]中华人民共和国业部,中华人民共和国卫生部,国家药品监督管理局.中华人民共和国农业部公告第176号《禁止在饲料和动物饮用水种使用的药物品种目录》[S].北京:中国农业出版社,2002.
[10]黄丽英,邹圆,陆强,等.分散固相萃取-超高效液相色谱-串联质谱法测定禽蛋中4种内外源性孕激素[J].食品科学,2018,39(16):295-300.
[11]连英杰,林升航,曾琪,等.QuEChERS/超高效液相色谱-电喷雾串联质谱法检测鸡肉中11种激素类药物残留[J].食品安全质量检测学报,2014(2):384-392.
[12]Muňiz-Valencia R,Ceballos-Magaňa S G,Gonzalo-Lumbreras R,et al.GC-MS method development and validation for anabolic steroids in feed samples[J].Journal of Separation Science,2015,31(4):727-734.
[13]刘红菊,闫冲,刘宏果.反相高效液相色谱法同时测定饲料中的10种性激素[J].中国饲料,2010(18):34-36.
[14]张峰,许成保,蓝芳,等.超高效液相色谱-四极杆飞行时间质谱法测定饲料中9种雄性激素类药物[J].分析化学,2012,40(1):101-106.
[15]张毅,蓝芳,张峰,等.液相色谱-串联质谱法测定谷物类饲料中的41种激素[J].色谱,2011,29(6):523-534.
[16]李敏青,徐娟,王岚,等.高效液相色谱-电喷雾电离串联质谱法测定植物性食品中氟虫腈及其代谢物的残留[J].食品工业科技,2019(2):245-250,256.
[17]Zhu Y,Xie S,Dongmei C,et al.Targeted analysis and determination of β-agonists,hormones,glucocorticoid,and psychiatric drugs in feed by liquid chromatography with electrospray ionization tandem mass spectrometry[J].Journal of Separation Science,2016,39(13):2584-2594.
[18]孙珊珊,朱丽君,胡延喜,等.超高效液相色谱-串联质谱测定饲料中镇静剂类和β-受体激素类药物残留[J].色谱,2018,36(2):150-158.
[19]姜光丽,何小琴,郗存显,等.QuEchERS/超高液相色谱-串联质谱法同时测定饲料中6种β-阻断剂类药物[J].中国饲料,2015(12):33-36.
[20]肖全伟,吴文林,杨万林,等.固相萃取-超高效液相色谱-串联质谱法同时测定饲料中的3种雌激素[J].色谱,2014,32(11):1209-1213.
[21]林小莉,李宁,霍峰,等.气相色谱-质谱法同时测定饲料中6种雌激素类药物[J].分析测试学报,2016,35(3):322-326.