微波水解-液相色谱串联质谱法测定16种氨基酸的含量
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摘要
目的建立微波水解-液相色谱串联质谱法(liquid chromatography tandem mass spectrometry,LC-MS)测定保健食品中16种氨基酸的含量。方法取适量样品,加入6 mol/L盐酸溶液15 m L,加入适量苯酚,于150℃微波消解4 h,冷却稀释至80℃减压干燥1 h。样品经0.1%甲酸溶解后经Agilent-C_(18)柱分离,以乙腈-10 mmol/L乙酸铵溶液为流动相进行梯度洗脱,以串联质谱的多反应监测模式进行检测,外标法定量。结果方法的线性范围0.01~0.05 nmol/μL;平均回收率为95.6%~100.7%,相对标准偏差(relative standard deviation,RSD)小于3.0%。结论该方法分析速度快、灵敏度高、准确和重复性好,与现有技术对比,该方法通过微波技术辅助水解,缩短了样品的前处理时间,可适用于保健食品中16种氨基酸的快速检测。
Objective To establish a method for determination of 16 kinds of amino acids in health food by microwave hydrolysis-liquid chromatography tandem mass spectrometry. Methods Adding 15 m L 6 mol/L hydrochloric acid solution and appropriate amount of phenol to sample, it was hydrolyzed by microwave at 150 ℃for 4 h, cooled to 80 ℃ and dried for 1 h under reduced pressure. The sample was dissolved in 0.1% formic acid and separated by Agilent-C18 column. The gradient elution was performed with acetonitrile-10 mmol/L ammonium acetate as the mobile phase. The sample was detected by multi-reaction monitoring mode of tandem mass spectrometry and quantified by external standard method. Results The linear range of the method was 0.01-0.05 nmol/μL; the average recovery was 95.6%-100.7%, and the relative standard deviation(RSD) was less than 3.0%. Conclusion This method is rapid, sensitive, accurate and repeatable. Comparing with the current technology, it is assisted by microwave hydrolysis to shorten the sample pretreatment time, which can be used for the simultaneous determination of 16 amino acids in health food.
Objective To establish a method for determination of 16 kinds of amino acids in health food by microwave hydrolysis-liquid chromatography tandem mass spectrometry. Methods Adding 15 m L 6 mol/L hydrochloric acid solution and appropriate amount of phenol to sample, it was hydrolyzed by microwave at 150 ℃for 4 h, cooled to 80 ℃ and dried for 1 h under reduced pressure. The sample was dissolved in 0.1% formic acid and separated by Agilent-C18 column. The gradient elution was performed with acetonitrile-10 mmol/L ammonium acetate as the mobile phase. The sample was detected by multi-reaction monitoring mode of tandem mass spectrometry and quantified by external standard method. Results The linear range of the method was 0.01-0.05 nmol/μL; the average recovery was 95.6%-100.7%, and the relative standard deviation(RSD) was less than 3.0%. Conclusion This method is rapid, sensitive, accurate and repeatable. Comparing with the current technology, it is assisted by microwave hydrolysis to shorten the sample pretreatment time, which can be used for the simultaneous determination of 16 amino acids in health food.
引文
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[7]陈霞.微波消解法在测定新疆8种葡萄干中氨基酸的应用[J].食品工业,2015,36(4):266-270.Chen X.Application of microwave digestion method in determination of amino acids in 8 kinds of raisins in Xinjiang[J].Food Ind,2015,36(4):266-270.
[8]苗雨田.全自动氨基酸分析仪法测定不同年份黄酒中游离氨基酸的含量[J].食品安全质量检测学报,2015,6(4):1154-1161.Miao YT.Determination of amino acids content in yellow rice wine of different years with automatic amino acid analyzer[J].J Food Saf Qual,2015,6(4):1154-1161.
[9]杨远帆.茚三酮法测定蜂蜜及果葡糖浆中的氨基酸含量[J].中国食品学报,2013,13(2):171-176.Yang YF.Determination of amino acid in honey and high fructose corn syrup(HFCS)by the method of ninhydrin colorization[J].J Chin Inst Food Sci Technol,2013,13(2):171-176.
[10]周贤婧.毛细管电泳-间接紫外检测法测定蜂蜜中的氨基酸[J].色谱,2013,31(7):661-666.Zhou XJ.Determination of amino acids in honey by capillary electrophoresis with indirect ultraviolet detection[J].Chin J Chromatogr,2013,31(7):661-666.
[11]赫欣睿.高效液相色谱法测定氨基酸的研究进展[J].分析测试学报,2016,35(7):922-928.Hao XR.Research progress on detection of amino acids by high performance liquid chromatography[J].J Instrum Anal,2016,35(7):922-928.
[12]任晓宇.微波水解衍生高效液相色谱法测定饲料中的氨基酸[J].分析测试学报,2017,36(10):1255-1259.Ren XY.Determination of amino acids in feed by high performance liquid chromatography with microwave hydrolysis derivatization[J].J Instrum Anal,2017,36(10):1255-1259.
[13]李秀英.微波水解/超高效液相色谱-串联质谱法快速测定乳粉中的胆碱和左旋肉碱[J].食品安全检测学报,2016,7(5):1877-1881.Li XY.Rapid determination of choline and L-carnitine in milk powder by ultra-high performance liquid chromatography tandem mass spectrometry using microwave digestion for sample preparation[J].J Food Saf Qual,2016,7(5):1877-1881.
[14]马戎.液相色谱串联质谱法测定烟草中的游离氨基酸[J].中国测试,2013,39(2):34-37.Ma R.Determination of free amino acids in tobaceo by liquid chromatography tandem mass spectrometry[J].Chin Measur Test,2013,39(2):34-37.
[15]GB 5009.124-2016食品中氨基酸的测定[S].GB 5009.124-2016 Determination of amino acids in foods[S].
[2]李梅.酸水解—氨基酸分析前处理方法的应用实践[J].安徽农业科学,2014,42(10):2846-2847,2862.Li M.Practice on pretreatment of acid hydrolysis of protein for amino acids analysis[J].J Anhui Agric Sci,2014,42(10):2846-2847,2862.
[3]周虹.烟叶样品中18种氨基酸的微波水解分析方法研究[J].西南农业学报,2014,27(6):2629-2634.Zhou H.Study on analysis method for 18 amino acids of tobacco samples[J].Southwest Chin J Agric Sci,2014,27(6):2629-2634.
[4]Damm M.Microwave-assisted high throughput acid hydrolysis in silicon carbide microtiter platforms-Arapid and low volume sample preparation technique for total amino acid analysis in proteins and peptides[J].J Chromatogr A,2010,1217(50):7826-7832.
[5]吴英婷.微波消解技术在氨基酸分析中的应用研究[J].食品科技,2016,41(8):260-264.Wu YT.Application of microwave digestion method in analysis of amino acid[J].Food Sci Technol,2016,41(8):260-264.
[6]陶玉.微波水解法测定人工虎骨粉中总氨基酸含量[J].中医药导报,2017,23(16):76-78.Tao Y.Determination of total amino acid content in artificial tiger bone powder by microwave hydrolysis[J].Chin Med Herald,2017,23(16):76-78.
[7]陈霞.微波消解法在测定新疆8种葡萄干中氨基酸的应用[J].食品工业,2015,36(4):266-270.Chen X.Application of microwave digestion method in determination of amino acids in 8 kinds of raisins in Xinjiang[J].Food Ind,2015,36(4):266-270.
[8]苗雨田.全自动氨基酸分析仪法测定不同年份黄酒中游离氨基酸的含量[J].食品安全质量检测学报,2015,6(4):1154-1161.Miao YT.Determination of amino acids content in yellow rice wine of different years with automatic amino acid analyzer[J].J Food Saf Qual,2015,6(4):1154-1161.
[9]杨远帆.茚三酮法测定蜂蜜及果葡糖浆中的氨基酸含量[J].中国食品学报,2013,13(2):171-176.Yang YF.Determination of amino acid in honey and high fructose corn syrup(HFCS)by the method of ninhydrin colorization[J].J Chin Inst Food Sci Technol,2013,13(2):171-176.
[10]周贤婧.毛细管电泳-间接紫外检测法测定蜂蜜中的氨基酸[J].色谱,2013,31(7):661-666.Zhou XJ.Determination of amino acids in honey by capillary electrophoresis with indirect ultraviolet detection[J].Chin J Chromatogr,2013,31(7):661-666.
[11]赫欣睿.高效液相色谱法测定氨基酸的研究进展[J].分析测试学报,2016,35(7):922-928.Hao XR.Research progress on detection of amino acids by high performance liquid chromatography[J].J Instrum Anal,2016,35(7):922-928.
[12]任晓宇.微波水解衍生高效液相色谱法测定饲料中的氨基酸[J].分析测试学报,2017,36(10):1255-1259.Ren XY.Determination of amino acids in feed by high performance liquid chromatography with microwave hydrolysis derivatization[J].J Instrum Anal,2017,36(10):1255-1259.
[13]李秀英.微波水解/超高效液相色谱-串联质谱法快速测定乳粉中的胆碱和左旋肉碱[J].食品安全检测学报,2016,7(5):1877-1881.Li XY.Rapid determination of choline and L-carnitine in milk powder by ultra-high performance liquid chromatography tandem mass spectrometry using microwave digestion for sample preparation[J].J Food Saf Qual,2016,7(5):1877-1881.
[14]马戎.液相色谱串联质谱法测定烟草中的游离氨基酸[J].中国测试,2013,39(2):34-37.Ma R.Determination of free amino acids in tobaceo by liquid chromatography tandem mass spectrometry[J].Chin Measur Test,2013,39(2):34-37.
[15]GB 5009.124-2016食品中氨基酸的测定[S].GB 5009.124-2016 Determination of amino acids in foods[S].