基于蜂蜜中氨基酸的毛细管电泳指纹图谱构建
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摘要
通过以细菌纤维素(Bacterial cellulose,BC)为流动相添加剂,提高毛细管电泳的分离能力,构建基于蜂蜜中氨基酸的指纹图谱。系统考察了电泳缓冲液种类、离子强度、pH值、分离电压及BC添加量对氨基酸分离效果的影响。结果表明:以30 mmol/L,p H 9.8硼砂-磷酸盐缓冲液为电泳缓冲液,BC添加量为0.5%,25 kV作为分离电压时,17种常见氨基酸的异硫氰酸荧光素的衍生物能在25 min内实现基线分离。最优电泳条件下,17种氨基酸的异硫氰酸荧光素的衍生物在相应浓度范围内线性关系良好,相关系数大于0.993,检测限为2.5×10~(-4)μmol/L~30.0×10~(-4)μmol/L。精密度试验表明:17种氨基酸衍生物峰面积相对标准偏差(Relative standard deviations,RSDs)(n=11)分别为1.84%~5.50%(日间,n=5)和0.94%~3.10%(日内,n=11)。洋槐蜜中17种氨基酸添加回收率在81.10%~96.15%,RSDs(n=3)小于8.22%。
Fingerprint of amino acids in honey was constructed by capillary electrophoresis. Herein,bacterial cellulose(BC)was used as additive in running buffer to improve the separation efficiency. Experimental parameters such as the p H and concentration of the running buffer,separation voltage as well as content of BC in running buffer were systematically investigated. The results showed that 17 amino acids derivatized with fluorescein isothiocyanate(FITC)could be baseline separated within 25 min using Na_2B_4O_7-NaH_2PO_4 buffer(30 mmol/L,pH 9.8)containing 0.5 % BC for background electrolyte,25 kV for the separation voltage. Under the optimized conditions,17 amino acid derivatives showed good linearities with correlation coefficients greater than 0.993.The limit detections of this developed method for 17 amino acid derivatives were ranging from 2.5×10~(-4) to 30.0×10~(-4)μmol/L. The relative standard deviations(RSDs)for peak areas were 1.84 %-5.50 %(inter-day)and 0.94 %-3.10 %(intra-day),respectively. The developed method was further applied to determine the amino acids in Acacia honey with the satisfactory recovery in the range of 81.10 %-96.15 % with RSDs(n=3)below 8.22 %.
Fingerprint of amino acids in honey was constructed by capillary electrophoresis. Herein,bacterial cellulose(BC)was used as additive in running buffer to improve the separation efficiency. Experimental parameters such as the p H and concentration of the running buffer,separation voltage as well as content of BC in running buffer were systematically investigated. The results showed that 17 amino acids derivatized with fluorescein isothiocyanate(FITC)could be baseline separated within 25 min using Na_2B_4O_7-NaH_2PO_4 buffer(30 mmol/L,pH 9.8)containing 0.5 % BC for background electrolyte,25 kV for the separation voltage. Under the optimized conditions,17 amino acid derivatives showed good linearities with correlation coefficients greater than 0.993.The limit detections of this developed method for 17 amino acid derivatives were ranging from 2.5×10~(-4) to 30.0×10~(-4)μmol/L. The relative standard deviations(RSDs)for peak areas were 1.84 %-5.50 %(inter-day)and 0.94 %-3.10 %(intra-day),respectively. The developed method was further applied to determine the amino acids in Acacia honey with the satisfactory recovery in the range of 81.10 %-96.15 % with RSDs(n=3)below 8.22 %.
引文
[1]杜宗绪.蜂蜜掺假鉴别检测方法研究进展[J].保鲜与加工,2015,15(5):67-71
[2]胡玥珊,孙国祥,刘迎春.系统指纹定量法评价牛黄解毒片毛细管电泳指纹图谱[J].中南药学,2015,13(9):897-990
[3]王荧荧,孙国祥.天王补心丸毛细管电泳指纹图谱[J].中国实验方剂学杂志,2012,18(16):97-101
[4]孙国祥,蔡新凤.补中益气丸的毛细管电泳指纹图谱研究[J].中南药学,2012,10(4):307-311
[5]周贤婧,师彦平.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
[6]CHEN S,SHEN W,YU F,HU W,et al.Preparation of amidoximated bacterial cellulose and its adsorption mechanism for Cu2+and Pb2+[J].Journal of Applied Polymer Science,2010(117):8-15
[7]WANG J,LU X,NG P F,et al.Polyethylenimine coated bacterial cellulose nanofiber membrane and application as adsorbent and catalyst[J].journal of colloid and interface science,2015(440):32-38
[8]LIN Q,ZHENG Y,WANG G,et al.Protein adsorption behaviors of carboxymethylated bacterial cellulose membranes[J].International Journal of Biological Macromolecules,2015(73):264-269
[9]尹东光,吴明红,谢春娟,等.新型毛细管电泳化学发光系统应用于氨基酸分离测定[J].分析化学,2009,37(1):152-156
[10]陈彦,殷学军,王东芝,等.氨基酸衍生物的非水毛细管电泳分离[J].曲阜师范大学学报,2008,34(1):80-83
[11]李延灏,牛千雪,范金石.纳晶纤维素的制备及其应用展望[J].中华纸业,2012,33(22):20-23
[12]黄蕴芝,祁龙凯,林励,等.蜂蜜中氨基酸类成分高效液相指纹图谱研究[J].广州中医药大学学报,2015,32(4):745-750
[2]胡玥珊,孙国祥,刘迎春.系统指纹定量法评价牛黄解毒片毛细管电泳指纹图谱[J].中南药学,2015,13(9):897-990
[3]王荧荧,孙国祥.天王补心丸毛细管电泳指纹图谱[J].中国实验方剂学杂志,2012,18(16):97-101
[4]孙国祥,蔡新凤.补中益气丸的毛细管电泳指纹图谱研究[J].中南药学,2012,10(4):307-311
[5]周贤婧,师彦平.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
[6]CHEN S,SHEN W,YU F,HU W,et al.Preparation of amidoximated bacterial cellulose and its adsorption mechanism for Cu2+and Pb2+[J].Journal of Applied Polymer Science,2010(117):8-15
[7]WANG J,LU X,NG P F,et al.Polyethylenimine coated bacterial cellulose nanofiber membrane and application as adsorbent and catalyst[J].journal of colloid and interface science,2015(440):32-38
[8]LIN Q,ZHENG Y,WANG G,et al.Protein adsorption behaviors of carboxymethylated bacterial cellulose membranes[J].International Journal of Biological Macromolecules,2015(73):264-269
[9]尹东光,吴明红,谢春娟,等.新型毛细管电泳化学发光系统应用于氨基酸分离测定[J].分析化学,2009,37(1):152-156
[10]陈彦,殷学军,王东芝,等.氨基酸衍生物的非水毛细管电泳分离[J].曲阜师范大学学报,2008,34(1):80-83
[11]李延灏,牛千雪,范金石.纳晶纤维素的制备及其应用展望[J].中华纸业,2012,33(22):20-23
[12]黄蕴芝,祁龙凯,林励,等.蜂蜜中氨基酸类成分高效液相指纹图谱研究[J].广州中医药大学学报,2015,32(4):745-750