分散式固相萃取结合高效液相色谱法快速测定油脂制品中苯并a)芘
杨悠悠 王浩 刘佟 杨永坛
1引言
苯并(a)芘(BaP)具有强致癌和致突变作用\[1\]。油脂制品中的苯并(a)芘主要源自不合理的加工方式:如温度过高的油料焙炒、高温压榨等工艺过程均可导致油脂受热分解,环化聚合生成苯并(a)芘\[2\]。目前,关于食品中苯并(a)芘较为普遍的检测方法主要有薄层色谱法\[3\]、高效液相色谱法 (PLC)\[\]、气相色谱质谱法\[5\]等。提取净化方法主要有凝胶渗透色谱法\[6\]、液液萃取法\[7\]、固相萃取法\[8\]、层析柱法等\[9\]。本研究采用高效液相色谱荧光检测法,应用分散式固相萃取技术对油脂制品进行提取净化,极大简化了处理步骤,降低了实验成本和有机试剂排放,实现了油脂制品中苯并(a)芘的快速精确定量。
2实验部分
2.1仪器与材料UltiMate 3000 PLC(配荧光检测器,美国hermo公司);S 16R型高速离心机(美国hermo公司);QL902型涡旋混合器(其林贝尔仪器制造有限公司);DCⅡ 型氮吹仪(北京同泰联科技发展有限公司)。正己烷、乙腈、四氢呋喃(色谱纯,美国isher公司);苯并(a)芘标准品(纯度99.6%±0.5%,美国Sigma公司);中性氧化铝(分析纯,100~300目,00 ℃ 灼烧8 h,天津光复科技发展有限公司);植物油、动物油、辣椒酱样品均购于本地超市。
2.2样品处理方法
取0.2 g油脂样品,于50 mL离心管中加入吸附剂中性氧化铝9 g,加入50 μL去离子水,20 mL正己烷,涡旋振荡1 min。取提取液10 mL于5 ℃ 水浴中氮吹至干,用乙腈四氢呋喃(9∶1, V/V)混合溶剂0.5 mL复溶。再经0.5 μm滤膜过滤后,进样20 μL,用荧光检测器(LD)进行色谱分析。
2.3液相色谱条件Eclipse XDBC18反相色谱柱(150 mm×.6 mm×5 μm,美国Agilent公司);XDBC18预柱(12.5 mm×.6 mm×5 μm,美国Agilent公司);流速为1.0 mL/min;柱温25 ℃ ;荧光检测器激发波长38 nm、发射波长06 nm;流动相:乙腈水(88∶12, V/V)。
3结果与讨论
3.1中性氧化铝、水分及萃取剂用量的选择影响回收率和净化效果的3个主要因素为:吸附剂中性氧化铝的用量、影响中性氧化铝活度的加水量、萃取剂正己烷使用体积。首先对三因素的使用水平进行了初步筛选,对个水平的中性氧化铝的用量(7、8、9和10 g)、个水平的加水量(5%, 6%, 7%和8%)、个水平的萃取剂用量(16, 18, 20和22 mL)进行正交设计实验,得到回收率最佳的优选条件:中性氧化铝9 g,去离子水50 μL,正己烷20 mL。
3.2标准曲线的绘制和检出限标准品用甲苯溶解后,用乙腈四氢呋喃(9∶1, V/V)配成10 mg/L储备液,分取储备液再稀释成0.2,1.0,2.0,5.0和10.0 μg/L,各进样20 μL测定其峰面积,以峰面积为纵坐标(Y),质量浓度为横坐标(X),绘制标准曲线,采用外标法定量。苯并(a)芘在0.2~10.0 μg/L范围内符合线性关系,回归方程为Y=121770X-699,相关系数R2=0.9997。进空白样品,以信噪比S/N=3计,测得方法的最低检出浓度为1.0 μg/kg。
3.3方法的加标回收率和精密度按照2.2节的方法对阴性油脂样品的三水平加标样 (5.0, 25.0和50.0 μg/kg) 各进行6次分析,加标回收率为81.5%~97.5%,精密度为2.6%~.9%(n=6)。在9 min内可分离植物油加标样品(加标量25 μg/kg)。
3.实际样品测定采用本方法对购于市场的2个油脂样品进行测定。样品种类包括:菜籽油、葵花籽油、玉米油、花生油、大豆油、芝麻油、橄榄油、动物油、辣椒酱油等,部分阳性结果见表1。
与液液萃取、固相萃取、凝胶渗透色谱等传统的前处理方法相比,本方法更为简便快速,高效环保,成本低廉。“一步式”的样品前处理过程集成了传统的吸附剂活化、样品提取和样品净化等样品处理步骤,整个样品处理过程仅需10~15 min, 尤其适合批量油脂样品中苯并(a)芘的检测。
References
1imko P. J. Chromatogr. B, 2002, 770(1): 3-18
2LU ongMei, ZANG aieng. Culinary Science Journal of Yangzhou University, 2008, 25(2): 0-2
陆红梅, 章海风. 扬州大学烹饪学报, 2008, 25(2): 0-2
3Determination of Benzo(a) pyrene in oods. National Standards of the People′s Republic of China. GB/5009.272003
食品中苯并(a)芘的测定. 中华人民共和国国家标准. GB/ 5009.272003
Kishikawa N, Wada M, Kuroda N, Akiyama S, Nakashima K. J. Chromatogr. B, 2003, 789(2): 257-26
5Veyrand B, Brosseaud A, Sarcher L, Varlet V, Monteau , Marchand P, Andre , Bizec B L. J. Chromatogr. A, 2007, 119(2): 333-3
6WANG Jianua, GUO Cui, PANG Guoang, ZANG YiBing. Journal of Instrumental Analysis, 2009, 28(3): 267-271
王建华, 郭 翠, 庞国芳, 张艺兵. 分析测试学报, 2009, 28(3): 267-271
7Determination of Benzo\[a\]pyrene in Meat Products igh Performance Liquid Chromatography Method. National Standards of the People′s Republic of China. NY/ 16662008
肉制品中苯并\[a\]芘的测定高效液相色谱法, 中华人民共和国国家标准. NY/ 16662008
8ANG ua, CEN DaZhou, WU Xue, XU Ruieng, ZONG ShiLin, SAO MingWu, WANG an. Journal of Chinese Mass Spectrometry Society, 2010, 31(): 208-213
汤 桦, 陈大舟, 吴 雪, 徐锐锋, 钟石林, 邵明武, 王 覃. 质谱学报, 2010, 31(): 208-213
9Animal andVegetable ats and OilsDetermination of Benzo(a)pyreneReverse igh Performance Liquid Chromatography Method. National Standards of the People′s Republic of China. GB/ 225092008
动植物油脂苯并(a)芘的测定反相高效液相色谱法, 中华人民共和国国家标准. GB/ 225092008
AbstractA dispersive solid phase extraction method combined with reversed phase liquid chromatography was investigated for the determination of benzo(a)pyrene (BaP) residues in edible oil and oil products. he developed “onestep” sample preparation method integrated with the adsorbent activation, sample extraction and sample cleaning. Using AlumiaN as the adsorbent, good cleaning effect and high recovery ranging from 81.5%-97.5% was achieved. In addition, coupled with fluorescence detector (LD), high sensitivity was obtained with LOD of 1.0 μg/kg (S/N=3). urthermore, SPSS was used to design orthogonal optimization experiments and establish the optimal condition. Under the optimized condition, the standard calibration curve was linear over the concentration range of 0.5-10.0 μg/L, with the regression efficiency (R2) of 0.9997. he relative standard deviation (RSD) of peak area was between 2.6%-.9%, showing good repeatability and the reliability.
KeywordsBenzo(a)pyrene; igh performance liquid chromatography; Dispersive solid phase extraction; Oil product
王建华, 郭 翠, 庞国芳, 张艺兵. 分析测试学报, 2009, 28(3): 267-271
7Determination of Benzo\[a\]pyrene in Meat Products igh Performance Liquid Chromatography Method. National Standards of the People′s Republic of China. NY/ 16662008
肉制品中苯并\[a\]芘的测定高效液相色谱法, 中华人民共和国国家标准. NY/ 16662008
8ANG ua, CEN DaZhou, WU Xue, XU Ruieng, ZONG ShiLin, SAO MingWu, WANG an. Journal of Chinese Mass Spectrometry Society, 2010, 31(): 208-213
汤 桦, 陈大舟, 吴 雪, 徐锐锋, 钟石林, 邵明武, 王 覃. 质谱学报, 2010, 31(): 208-213
9Animal andVegetable ats and OilsDetermination of Benzo(a)pyreneReverse igh Performance Liquid Chromatography Method. National Standards of the People′s Republic of China. GB/ 225092008
动植物油脂苯并(a)芘的测定反相高效液相色谱法, 中华人民共和国国家标准. GB/ 225092008
AbstractA dispersive solid phase extraction method combined with reversed phase liquid chromatography was investigated for the determination of benzo(a)pyrene (BaP) residues in edible oil and oil products. he developed “onestep” sample preparation method integrated with the adsorbent activation, sample extraction and sample cleaning. Using AlumiaN as the adsorbent, good cleaning effect and high recovery ranging from 81.5%-97.5% was achieved. In addition, coupled with fluorescence detector (LD), high sensitivity was obtained with LOD of 1.0 μg/kg (S/N=3). urthermore, SPSS was used to design orthogonal optimization experiments and establish the optimal condition. Under the optimized condition, the standard calibration curve was linear over the concentration range of 0.5-10.0 μg/L, with the regression efficiency (R2) of 0.9997. he relative standard deviation (RSD) of peak area was between 2.6%-.9%, showing good repeatability and the reliability.
KeywordsBenzo(a)pyrene; igh performance liquid chromatography; Dispersive solid phase extraction; Oil product
王建华, 郭 翠, 庞国芳, 张艺兵. 分析测试学报, 2009, 28(3): 267-271
7Determination of Benzo\[a\]pyrene in Meat Products igh Performance Liquid Chromatography Method. National Standards of the People′s Republic of China. NY/ 16662008
肉制品中苯并\[a\]芘的测定高效液相色谱法, 中华人民共和国国家标准. NY/ 16662008
8ANG ua, CEN DaZhou, WU Xue, XU Ruieng, ZONG ShiLin, SAO MingWu, WANG an. Journal of Chinese Mass Spectrometry Society, 2010, 31(): 208-213
汤 桦, 陈大舟, 吴 雪, 徐锐锋, 钟石林, 邵明武, 王 覃. 质谱学报, 2010, 31(): 208-213
9Animal andVegetable ats and OilsDetermination of Benzo(a)pyreneReverse igh Performance Liquid Chromatography Method. National Standards of the People′s Republic of China. GB/ 225092008
动植物油脂苯并(a)芘的测定反相高效液相色谱法, 中华人民共和国国家标准. GB/ 225092008
AbstractA dispersive solid phase extraction method combined with reversed phase liquid chromatography was investigated for the determination of benzo(a)pyrene (BaP) residues in edible oil and oil products. he developed “onestep” sample preparation method integrated with the adsorbent activation, sample extraction and sample cleaning. Using AlumiaN as the adsorbent, good cleaning effect and high recovery ranging from 81.5%-97.5% was achieved. In addition, coupled with fluorescence detector (LD), high sensitivity was obtained with LOD of 1.0 μg/kg (S/N=3). urthermore, SPSS was used to design orthogonal optimization experiments and establish the optimal condition. Under the optimized condition, the standard calibration curve was linear over the concentration range of 0.5-10.0 μg/L, with the regression efficiency (R2) of 0.9997. he relative standard deviation (RSD) of peak area was between 2.6%-.9%, showing good repeatability and the reliability.
KeywordsBenzo(a)pyrene; igh performance liquid chromatography; Dispersive solid phase extraction; Oil product
1引言
苯并(a)芘(BaP)具有强致癌和致突变作用\[1\]。油脂制品中的苯并(a)芘主要源自不合理的加工方式:如温度过高的油料焙炒、高温压榨等工艺过程均可导致油脂受热分解,环化聚合生成苯并(a)芘\[2\]。目前,关于食品中苯并(a)芘较为普遍的检测方法主要有薄层色谱法\[3\]、高效液相色谱法 (PLC)\[\]、气相色谱质谱法\[5\]等。提取净化方法主要有凝胶渗透色谱法\[6\]、液液萃取法\[7\]、固相萃取法\[8\]、层析柱法等\[9\]。本研究采用高效液相色谱荧光检测法,应用分散式固相萃取技术对油脂制品进行提取净化,极大简化了处理步骤,降低了实验成本和有机试剂排放,实现了油脂制品中苯并(a)芘的快速精确定量。
2实验部分
2.1仪器与材料UltiMate 3000 PLC(配荧光检测器,美国hermo公司);S 16R型高速离心机(美国hermo公司);QL902型涡旋混合器(其林贝尔仪器制造有限公司);DCⅡ 型氮吹仪(北京同泰联科技发展有限公司)。正己烷、乙腈、四氢呋喃(色谱纯,美国isher公司);苯并(a)芘标准品(纯度99.6%±0.5%,美国Sigma公司);中性氧化铝(分析纯,100~300目,00 ℃ 灼烧8 h,天津光复科技发展有限公司);植物油、动物油、辣椒酱样品均购于本地超市。
2.2样品处理方法
取0.2 g油脂样品,于50 mL离心管中加入吸附剂中性氧化铝9 g,加入50 μL去离子水,20 mL正己烷,涡旋振荡1 min。取提取液10 mL于5 ℃ 水浴中氮吹至干,用乙腈四氢呋喃(9∶1, V/V)混合溶剂0.5 mL复溶。再经0.5 μm滤膜过滤后,进样20 μL,用荧光检测器(LD)进行色谱分析。
2.3液相色谱条件Eclipse XDBC18反相色谱柱(150 mm×.6 mm×5 μm,美国Agilent公司);XDBC18预柱(12.5 mm×.6 mm×5 μm,美国Agilent公司);流速为1.0 mL/min;柱温25 ℃ ;荧光检测器激发波长38 nm、发射波长06 nm;流动相:乙腈水(88∶12, V/V)。
3结果与讨论
3.1中性氧化铝、水分及萃取剂用量的选择影响回收率和净化效果的3个主要因素为:吸附剂中性氧化铝的用量、影响中性氧化铝活度的加水量、萃取剂正己烷使用体积。首先对三因素的使用水平进行了初步筛选,对个水平的中性氧化铝的用量(7、8、9和10 g)、个水平的加水量(5%, 6%, 7%和8%)、个水平的萃取剂用量(16, 18, 20和22 mL)进行正交设计实验,得到回收率最佳的优选条件:中性氧化铝9 g,去离子水50 μL,正己烷20 mL。
3.2标准曲线的绘制和检出限标准品用甲苯溶解后,用乙腈四氢呋喃(9∶1, V/V)配成10 mg/L储备液,分取储备液再稀释成0.2,1.0,2.0,5.0和10.0 μg/L,各进样20 μL测定其峰面积,以峰面积为纵坐标(Y),质量浓度为横坐标(X),绘制标准曲线,采用外标法定量。苯并(a)芘在0.2~10.0 μg/L范围内符合线性关系,回归方程为Y=121770X-699,相关系数R2=0.9997。进空白样品,以信噪比S/N=3计,测得方法的最低检出浓度为1.0 μg/kg。
3.3方法的加标回收率和精密度按照2.2节的方法对阴性油脂样品的三水平加标样 (5.0, 25.0和50.0 μg/kg) 各进行6次分析,加标回收率为81.5%~97.5%,精密度为2.6%~.9%(n=6)。在9 min内可分离植物油加标样品(加标量25 μg/kg)。
3.实际样品测定采用本方法对购于市场的2个油脂样品进行测定。样品种类包括:菜籽油、葵花籽油、玉米油、花生油、大豆油、芝麻油、橄榄油、动物油、辣椒酱油等,部分阳性结果见表1。
与液液萃取、固相萃取、凝胶渗透色谱等传统的前处理方法相比,本方法更为简便快速,高效环保,成本低廉。“一步式”的样品前处理过程集成了传统的吸附剂活化、样品提取和样品净化等样品处理步骤,整个样品处理过程仅需10~15 min, 尤其适合批量油脂样品中苯并(a)芘的检测。
References
1imko P. J. Chromatogr. B, 2002, 770(1): 3-18
2LU ongMei, ZANG aieng. Culinary Science Journal of Yangzhou University, 2008, 25(2): 0-2
陆红梅, 章海风. 扬州大学烹饪学报, 2008, 25(2): 0-2
3Determination of Benzo(a) pyrene in oods. National Standards of the People′s Republic of China. GB/5009.272003
食品中苯并(a)芘的测定. 中华人民共和国国家标准. GB/ 5009.272003
Kishikawa N, Wada M, Kuroda N, Akiyama S, Nakashima K. J. Chromatogr. B, 2003, 789(2): 257-26
5Veyrand B, Brosseaud A, Sarcher L, Varlet V, Monteau , Marchand P, Andre , Bizec B L. J. Chromatogr. A, 2007, 119(2): 333-3
6WANG Jianua, GUO Cui, PANG Guoang, ZANG YiBing. Journal of Instrumental Analysis, 2009, 28(3): 267-271
王建华, 郭 翠, 庞国芳, 张艺兵. 分析测试学报, 2009, 28(3): 267-271
7Determination of Benzo\[a\]pyrene in Meat Products igh Performance Liquid Chromatography Method. National Standards of the People′s Republic of China. NY/ 16662008
肉制品中苯并\[a\]芘的测定高效液相色谱法, 中华人民共和国国家标准. NY/ 16662008
8ANG ua, CEN DaZhou, WU Xue, XU Ruieng, ZONG ShiLin, SAO MingWu, WANG an. Journal of Chinese Mass Spectrometry Society, 2010, 31(): 208-213
汤 桦, 陈大舟, 吴 雪, 徐锐锋, 钟石林, 邵明武, 王 覃. 质谱学报, 2010, 31(): 208-213
9Animal andVegetable ats and OilsDetermination of Benzo(a)pyreneReverse igh Performance Liquid Chromatography Method. National Standards of the People′s Republic of China. GB/ 225092008
动植物油脂苯并(a)芘的测定反相高效液相色谱法, 中华人民共和国国家标准. GB/ 225092008
AbstractA dispersive solid phase extraction method combined with reversed phase liquid chromatography was investigated for the determination of benzo(a)pyrene (BaP) residues in edible oil and oil products. he developed “onestep” sample preparation method integrated with the adsorbent activation, sample extraction and sample cleaning. Using AlumiaN as the adsorbent, good cleaning effect and high recovery ranging from 81.5%-97.5% was achieved. In addition, coupled with fluorescence detector (LD), high sensitivity was obtained with LOD of 1.0 μg/kg (S/N=3). urthermore, SPSS was used to design orthogonal optimization experiments and establish the optimal condition. Under the optimized condition, the standard calibration curve was linear over the concentration range of 0.5-10.0 μg/L, with the regression efficiency (R2) of 0.9997. he relative standard deviation (RSD) of peak area was between 2.6%-.9%, showing good repeatability and the reliability.
KeywordsBenzo(a)pyrene; igh performance liquid chromatography; Dispersive solid phase extraction; Oil product
王建华, 郭 翠, 庞国芳, 张艺兵. 分析测试学报, 2009, 28(3): 267-271
7Determination of Benzo\[a\]pyrene in Meat Products igh Performance Liquid Chromatography Method. National Standards of the People′s Republic of China. NY/ 16662008
肉制品中苯并\[a\]芘的测定高效液相色谱法, 中华人民共和国国家标准. NY/ 16662008
8ANG ua, CEN DaZhou, WU Xue, XU Ruieng, ZONG ShiLin, SAO MingWu, WANG an. Journal of Chinese Mass Spectrometry Society, 2010, 31(): 208-213
汤 桦, 陈大舟, 吴 雪, 徐锐锋, 钟石林, 邵明武, 王 覃. 质谱学报, 2010, 31(): 208-213
9Animal andVegetable ats and OilsDetermination of Benzo(a)pyreneReverse igh Performance Liquid Chromatography Method. National Standards of the People′s Republic of China. GB/ 225092008
动植物油脂苯并(a)芘的测定反相高效液相色谱法, 中华人民共和国国家标准. GB/ 225092008
AbstractA dispersive solid phase extraction method combined with reversed phase liquid chromatography was investigated for the determination of benzo(a)pyrene (BaP) residues in edible oil and oil products. he developed “onestep” sample preparation method integrated with the adsorbent activation, sample extraction and sample cleaning. Using AlumiaN as the adsorbent, good cleaning effect and high recovery ranging from 81.5%-97.5% was achieved. In addition, coupled with fluorescence detector (LD), high sensitivity was obtained with LOD of 1.0 μg/kg (S/N=3). urthermore, SPSS was used to design orthogonal optimization experiments and establish the optimal condition. Under the optimized condition, the standard calibration curve was linear over the concentration range of 0.5-10.0 μg/L, with the regression efficiency (R2) of 0.9997. he relative standard deviation (RSD) of peak area was between 2.6%-.9%, showing good repeatability and the reliability.
KeywordsBenzo(a)pyrene; igh performance liquid chromatography; Dispersive solid phase extraction; Oil product
王建华, 郭 翠, 庞国芳, 张艺兵. 分析测试学报, 2009, 28(3): 267-271
7Determination of Benzo\[a\]pyrene in Meat Products igh Performance Liquid Chromatography Method. National Standards of the People′s Republic of China. NY/ 16662008
肉制品中苯并\[a\]芘的测定高效液相色谱法, 中华人民共和国国家标准. NY/ 16662008
8ANG ua, CEN DaZhou, WU Xue, XU Ruieng, ZONG ShiLin, SAO MingWu, WANG an. Journal of Chinese Mass Spectrometry Society, 2010, 31(): 208-213
汤 桦, 陈大舟, 吴 雪, 徐锐锋, 钟石林, 邵明武, 王 覃. 质谱学报, 2010, 31(): 208-213
9Animal andVegetable ats and OilsDetermination of Benzo(a)pyreneReverse igh Performance Liquid Chromatography Method. National Standards of the People′s Republic of China. GB/ 225092008
动植物油脂苯并(a)芘的测定反相高效液相色谱法, 中华人民共和国国家标准. GB/ 225092008
AbstractA dispersive solid phase extraction method combined with reversed phase liquid chromatography was investigated for the determination of benzo(a)pyrene (BaP) residues in edible oil and oil products. he developed “onestep” sample preparation method integrated with the adsorbent activation, sample extraction and sample cleaning. Using AlumiaN as the adsorbent, good cleaning effect and high recovery ranging from 81.5%-97.5% was achieved. In addition, coupled with fluorescence detector (LD), high sensitivity was obtained with LOD of 1.0 μg/kg (S/N=3). urthermore, SPSS was used to design orthogonal optimization experiments and establish the optimal condition. Under the optimized condition, the standard calibration curve was linear over the concentration range of 0.5-10.0 μg/L, with the regression efficiency (R2) of 0.9997. he relative standard deviation (RSD) of peak area was between 2.6%-.9%, showing good repeatability and the reliability.
KeywordsBenzo(a)pyrene; igh performance liquid chromatography; Dispersive solid phase extraction; Oil product