摘要目的 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.

abstractsObjective 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.