反向胶束电动毛细管色谱-直接紫外法测定天花粉中的氨基酸
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摘要
目的建立检测天花粉中氨基酸的高效反向胶束电动毛细管色谱-直接紫外法,并进行验证。方法样品分离采用未涂层毛细管(75μm×60 cm,有效长度为49.5 cm),通过运行缓冲液,在一定电压下进样,检测波长为200 nm,样品浓度为50μg/m L,操作温度为25℃。对分离条件中运行缓冲溶液p H(10、11和12)、缓冲体系浓度(40 mmol/L KH_2PO_4-110 mmol/L Na OH、60 mmol/L KH2PO4-130 mmol/L Na OH、70 mmol/L KH_2PO_4-140 mmol/L Na OH)、缓冲液阳离子表面活性剂浓度[0.2、0.6、1、1.2 mmol/L十六烷基三甲基氯化铵(cetyltrimethylammonium chloride,CTAC)]、缓冲溶液有机添加剂浓度(5%、10%、15%、20%乙腈)、进样方式(电动进样:-10 k V进样5 s,压力进样:34.5 k Pa进样5 s)、分离电压(15、20、25 k V)进行优化。同时验证方法的线性、精密性及准确性。采用最适条件检测供试品天花粉中3种氨基酸分离情况。结果最适检测条件为:运行缓冲液为5%(v/v)乙腈、1 mmol/L CTAC、60 mmol/L KH2PO4-130 mmol/L Na OH(p H 11);压力进样:34.5 k Pa×5 s,分离电压:20 k V。瓜氨酸、天冬酰胺和γ-氨基丁酸3种氨基酸的峰面积和浓度均呈良好的线性关系,R~2均>0.99;峰面积RSD分别为4.7%、4.5%和3.8%,迁移时间RSD分别为0.62%、0.93%和1.10%;加标回收率在82.48%~111.16%之间。该方法可有效分离天花粉中的3种氨基酸。结论本实验建立的方法快速有效,且具有良好的精密性及准确性,可用于天花粉中氨基酸的快速测定。
Objective To develop and verify a high performance reverse micellar electrokinetic capillary chromatographydirect ultraviolet(UV) spectrophotometry method for determination of amino acids in trichosanthes. Methods Uncoated capillary tube(75 μm × 60 cm,effective length 49. 5 cm) was used for sample separation with running buffer at a certain voltage. The detection wavelength,sample concentration and temperature were 200 nm,50 μg/m L and 25 ℃respectively. The p H values of running buffer(10,11 and 12),concentration of buffer system(40 mmol/L potassium dihydrogen phosphate-110 mmol/L sodium hydroxide,60 mmol/L potassium dihydrogen phosphate-130 mmol/L sodium hydroxide and 70 mmol/L potassium dihydrogen phosphate-140 mmol/L sodium hydroxide),concentrations of cationic surfactant in buffer [0. 2,0. 6,1. 0 and 1. 2 mmol/L cetyltrimethylammonium chloride(CTAC)],concentration of organic additives(5%,10%,15% and 20% acetonitrile),method for sample loading(electric injection:-10 k V for 5 s;pressure injection:34. 5 k Pa for 5 s) and voltage(15,20 and 25 k V) were optimized. The developed method was verified for linearity,precision and accuracy,and used for determination of amino acid in trichosanthin under the optimized condition. Results The optimal running buffer consisted of 5%(v/v)acetonitrile,1 mmol/L CTAC,and60 mmol/L potassium dihydrogen phosphate-130 mmol/L sodium hydroxide. The optimal method of sample loading was pressure injection at 34. 5 k Pa for 5 s,and the optimal voltage for separation was 20 k V. The peak areas of citrulline,asparagines and γ-aminobutyric acid showed good linear relationship to their concentrations,with R~2 values of more than0. 99. The RSDs of peak area were 4. 7%,4. 5% and 3. 8%,while those of migration time were 0. 62%,0. 93% and1. 10%,respectively. The spike recovery rates were 82. 48% ~ 111. 16%. Three amino acids in trichosanthin were effectively separated by the developed method. Conclusion The developed method was rapid,effective,precise and accurate,which might be used for the rapid determination of amino acids in trichosanthin.
Objective To develop and verify a high performance reverse micellar electrokinetic capillary chromatographydirect ultraviolet(UV) spectrophotometry method for determination of amino acids in trichosanthes. Methods Uncoated capillary tube(75 μm × 60 cm,effective length 49. 5 cm) was used for sample separation with running buffer at a certain voltage. The detection wavelength,sample concentration and temperature were 200 nm,50 μg/m L and 25 ℃respectively. The p H values of running buffer(10,11 and 12),concentration of buffer system(40 mmol/L potassium dihydrogen phosphate-110 mmol/L sodium hydroxide,60 mmol/L potassium dihydrogen phosphate-130 mmol/L sodium hydroxide and 70 mmol/L potassium dihydrogen phosphate-140 mmol/L sodium hydroxide),concentrations of cationic surfactant in buffer [0. 2,0. 6,1. 0 and 1. 2 mmol/L cetyltrimethylammonium chloride(CTAC)],concentration of organic additives(5%,10%,15% and 20% acetonitrile),method for sample loading(electric injection:-10 k V for 5 s;pressure injection:34. 5 k Pa for 5 s) and voltage(15,20 and 25 k V) were optimized. The developed method was verified for linearity,precision and accuracy,and used for determination of amino acid in trichosanthin under the optimized condition. Results The optimal running buffer consisted of 5%(v/v)acetonitrile,1 mmol/L CTAC,and60 mmol/L potassium dihydrogen phosphate-130 mmol/L sodium hydroxide. The optimal method of sample loading was pressure injection at 34. 5 k Pa for 5 s,and the optimal voltage for separation was 20 k V. The peak areas of citrulline,asparagines and γ-aminobutyric acid showed good linear relationship to their concentrations,with R~2 values of more than0. 99. The RSDs of peak area were 4. 7%,4. 5% and 3. 8%,while those of migration time were 0. 62%,0. 93% and1. 10%,respectively. The spike recovery rates were 82. 48% ~ 111. 16%. Three amino acids in trichosanthin were effectively separated by the developed method. Conclusion The developed method was rapid,effective,precise and accurate,which might be used for the rapid determination of amino acids in trichosanthin.
引文
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[7]LIU Y,JIANG Y X.Comparison of amino acid analysis methods and test instruments[J].Modern Silk Sci&Technol,2012,27(1):10-12.(in Chinese)刘雨,蒋耀兴.氨基酸分析方法及测试仪器的比较[J].现代丝绸科学与技术,2012,27(1):10-12.
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[9]RAMOS-PAY魣N M,OCA譙A-GONZALEZ J A,FERN魣NDEZTORRES R M,et al.Recent trends in capillary electrophoresis for complex samples analysis:a review[J].Electrophoresis,2018,39(1):111-125.
[10]DONG X,WANG L Y.The determination of amino acids of ultraviolet absorption spectrum[J].J Dezhou Univ,2015,31(2):44-46.(in Chinese)董欣,王丽燕.氨基酸紫外光谱的再测定[J].德州学院学报,2015,31(2):44-46.
[2]LIU W,CHEN W Q,PAN J,et al.Content determination of citrulline andγ-aminobutyric acid in trichosanthin[J].Chin Pharm,2012,21(4):26-28.(in Chinese)刘文,陈文秋,潘建,等.天花粉中瓜氨酸与γ-氨基丁酸的含量测定[J].中国药业,2012,21(4):26-28.
[3]PAN J,LIU W,CHEN W Q,et al.Content determination of aspartic acid and glutamic acid in Trichosanthis radix by precolumn derivatization RP-HPLC[J].Chin Pharm,2012,23(35):3326-3328.(in Chinese)潘建,刘文,陈文秋,等.柱前衍生RP-HPLC法同时的天花粉中天门冬氨酸与古氨酸的含量[J].中国药房,2012,23(35):3326-3328.
[4]Chinese Pharmacopoeia Comission.Pharmacopoeia of People′s Republic of China[S].Beijing:China Medical Science Press,2015:56.(in Chinese)国家药典委员会.中华人民共和国药典[S].北京:中国医药科技出版社,2015:56.
[5]PAN J,CHEN W Q,YU S X,et al.Content determination ofγ-aminobutyric acid in Trichosanthis radix by pre-column derivatization RP-HPLC[J].Chin Pharm,2008,17(2):37-38.(in Chinese)潘建,陈文秋,余施筱,等.柱前衍生反相高效液相色谱法测定天花粉中γ-氨基丁酸含量[J].中国药业,2008,17(2):37-38.
[6]ZHANG B,LIU H Y,LI J,et al.Content comparision of amino acid in female and male plants of Trichosanthes kirilowii[J].China Pharmacist,2014,17(9):1517-1519.(in Chinese)张波,刘红燕,李佳,等.基于性别差异的栝楼药用部位氨基酸含量比较研究[J].中国药师,2014,17(9):1517-1519.
[7]LIU Y,JIANG Y X.Comparison of amino acid analysis methods and test instruments[J].Modern Silk Sci&Technol,2012,27(1):10-12.(in Chinese)刘雨,蒋耀兴.氨基酸分析方法及测试仪器的比较[J].现代丝绸科学与技术,2012,27(1):10-12.
[8]DEEB S E,魧W魧TZIG H,EL-HADY D A,et al.Recent advances in capillary electrophoretic migration techniques for pharmaceutical analysis[J].Electrophoresis,2014,35(1):170-189.
[9]RAMOS-PAY魣N M,OCA譙A-GONZALEZ J A,FERN魣NDEZTORRES R M,et al.Recent trends in capillary electrophoresis for complex samples analysis:a review[J].Electrophoresis,2018,39(1):111-125.
[10]DONG X,WANG L Y.The determination of amino acids of ultraviolet absorption spectrum[J].J Dezhou Univ,2015,31(2):44-46.(in Chinese)董欣,王丽燕.氨基酸紫外光谱的再测定[J].德州学院学报,2015,31(2):44-46.