中华预防医学杂志
中華預防醫學雜誌
중화예방의학잡지
CHINESE JOURNAL OF
2012年
12期
1117-1120
,共4页
黄晓%陈佳文%贺莉萍%康学军
黃曉%陳佳文%賀莉萍%康學軍
황효%진가문%하리평%강학군
色谱法,液相%神经递质%丹酰氯,柱前衍生化
色譜法,液相%神經遞質%丹酰氯,柱前衍生化
색보법,액상%신경체질%단선록,주전연생화
Chromatography,liquid%Neurotransmitter%Dansyl chloride,precolumn derivatization
目的 建立丹酰氯柱前衍生化-高效液相色谱法测定单胺类神经递质及其代谢产物的实验方法.方法 采用C18色谱柱(150 mm×4.6mm×5 μm)并以丹酰氯(10 mg/ml)为衍生试剂,在水浴50℃,缓冲液pH11时进行柱前衍生反应,20μl醋酸丙酮溶液(1.0mol/L.)被加入以终止反应,冷却至室温,进样,采用0.1 mol/L的醋酸锌乙腈四氢呋喃溶液,以体积比为62∶ 35∶3为流动相,在0～ 10 min时流速为1.0 ml/min,10 ～ 35 min时流速为2 0ml/min进行洗脱,紫外检测波长286 nm.采用上述方法分别测试了单胺类神经递质及其代谢物,并对方法的检测限、精准度和准确度进行评价.结果 在1～20 μg/ml范围内,各组分线性关系良好(r=0.999).去甲肾上腺素、多巴胺、5-羟色胺及其代谢产物3-甲氧-4-羟苯乙二醇、高香草酸、5-羟吲哚乙酸最低检出限分别为0.60、0.80、0.41、0.21、0.19、0.1 ug/ml,各待测物质的平均回收率为78.5％～95.9％,RSD分别为6.62％、7.64％、2.98％、3.60％、5.09％、3.09％.在色谱条件的选择和优化过程中,观察到金属离子对分离的重要作用.并对衍生反应的温度、缓冲液pH及丹酰氯用量进行了探讨.在上述条件下衍生反应完全,各待测物质基线分离.结论 建立了丹酰氯柱前衍生化-高教液相色谱法测定单胺类神经递质及其代谢产物的实验方法.
目的 建立丹酰氯柱前衍生化-高效液相色譜法測定單胺類神經遞質及其代謝產物的實驗方法.方法 採用C18色譜柱(150 mm×4.6mm×5 μm)併以丹酰氯(10 mg/ml)為衍生試劑,在水浴50℃,緩遲液pH11時進行柱前衍生反應,20μl醋痠丙酮溶液(1.0mol/L.)被加入以終止反應,冷卻至室溫,進樣,採用0.1 mol/L的醋痠鋅乙腈四氫呋喃溶液,以體積比為62∶ 35∶3為流動相,在0～ 10 min時流速為1.0 ml/min,10 ～ 35 min時流速為2 0ml/min進行洗脫,紫外檢測波長286 nm.採用上述方法分彆測試瞭單胺類神經遞質及其代謝物,併對方法的檢測限、精準度和準確度進行評價.結果 在1～20 μg/ml範圍內,各組分線性關繫良好(r=0.999).去甲腎上腺素、多巴胺、5-羥色胺及其代謝產物3-甲氧-4-羥苯乙二醇、高香草痠、5-羥吲哚乙痠最低檢齣限分彆為0.60、0.80、0.41、0.21、0.19、0.1 ug/ml,各待測物質的平均迴收率為78.5％～95.9％,RSD分彆為6.62％、7.64％、2.98％、3.60％、5.09％、3.09％.在色譜條件的選擇和優化過程中,觀察到金屬離子對分離的重要作用.併對衍生反應的溫度、緩遲液pH及丹酰氯用量進行瞭探討.在上述條件下衍生反應完全,各待測物質基線分離.結論 建立瞭丹酰氯柱前衍生化-高教液相色譜法測定單胺類神經遞質及其代謝產物的實驗方法.
목적 건립단선록주전연생화-고효액상색보법측정단알류신경체질급기대사산물적실험방법.방법 채용C18색보주(150 mm×4.6mm×5 μm)병이단선록(10 mg/ml)위연생시제,재수욕50℃,완충액pH11시진행주전연생반응,20μl작산병동용액(1.0mol/L.)피가입이종지반응,냉각지실온,진양,채용0.1 mol/L적작산자을정사경부남용액,이체적비위62∶ 35∶3위류동상,재0～ 10 min시류속위1.0 ml/min,10 ～ 35 min시류속위2 0ml/min진행세탈,자외검측파장286 nm.채용상술방법분별측시료단알류신경체질급기대사물,병대방법적검측한、정준도화준학도진행평개.결과 재1～20 μg/ml범위내,각조분선성관계량호(r=0.999).거갑신상선소、다파알、5-간색알급기대사산물3-갑양-4-간분을이순、고향초산、5-간신타을산최저검출한분별위0.60、0.80、0.41、0.21、0.19、0.1 ug/ml,각대측물질적평균회수솔위78.5％～95.9％,RSD분별위6.62％、7.64％、2.98％、3.60％、5.09％、3.09％.재색보조건적선택화우화과정중,관찰도금속리자대분리적중요작용.병대연생반응적온도、완충액pH급단선록용량진행료탐토.재상술조건하연생반응완전,각대측물질기선분리.결론 건립료단선록주전연생화-고교액상색보법측정단알류신경체질급기대사산물적실험방법.
Objective To develop a high performance liquid chromatography (HPLC) for detection of monoamine neurotransmitters and its metabolites after pre-column derivatization witb dansyl chloride.Methods The C18 chromatograph column (150 mm × 4.6 mm × 5 μm) was selected for detection,and derived by dansyl chloride (10 mg/ml) under the condition of 50 ℃ water bath by pH11 buffer solution.20 μl acetic acid acetone solution (1.0 mol/L) was then mixed in for termination of the reaction.Then the solution was cooling to room temperature,0.1 mol/L acetic acid zinc-acetonitrile-tetrahydrofuran solution was adopted for mobile phrase,with tbe volume ratio at 62∶ 35∶ 3.The flow rate was 1.0 ml/min between 0-10 min,2.0 ml/min between 10-35 min.The ultraviolet detection wavelength was 286 nm.The above method separately detected monoamine neurotransmitters and its metabolites and evaluated the limit of detection,accurate degree and accuracy degree.Results The linear relations between each component was good in the range of 1-20 μg/ml (r =0.999).The lowest detection limit of norepinephrine,dopamine,5-hydroxytryptamine and the metabolites 3-methoxy-4-benzoglycols,homovanillic acid and 5-heteroauxin were separately 0.60,0.80,0.41,0.21,0.19 and 0.1 μg/ml; while the average recovery rates were between 78.5％-95.9％,and the relative standard deviation (RSD) was 6.62％,7.64％,2.98％,3.60％,5.09％ and 3.09％,respectively.In the process of selection and optimization of the chromatographic conditions,we observed the importance of metal ions to discretion,and discussed the temperature,pH of the buffer solution and dosage of dansyl chloride in derivation.Under the above conditions,the reaction was perfect,and the baseline of the detected materials thoroughly separated.Conclusion The method to detect monoamine neurotransmitters and its metabolites by HPLC and pre-column derivatization with dansyl chloride was established ; and this method could provide reference for the detection of polyamine by HPLC.
