某些芳香族氨基酸作探针荧光猝灭法测定安乃近及其代谢产物

在pH3.2的缓冲介质中,安乃近(ANG)及其代谢产物4-甲氨基安替比林(MAA)、4-乙酰氨基安替比林(AAA)与色氨酸(Trp)、酪氨酸(Tyr)和苯丙氨酸(Phe)等芳香族氨基酸反应并形成结合产物,引起上述氨基酸的荧光发生猝灭,最大猝灭波长分别位于352nm(ANG-Trp体系)、304nm(ANG-Tyr,MAA-Tyr和AAA-Tyr体系)和284nm(ANG-Phe体系).其荧光猝灭值(ΔF)在一定范围内与ANG,MAA和AAA成正比.荧光猝灭反应具有较高灵敏度,对于ANG,MAA和AAA的检出限为13.3ng/mL(ANG-Trp体系)、15.8ng/mL(ANG-Tyr体系)、64.5ng/mL(ANG-Phe体系)、150.0ng/mL(MAA-Tyr体系)和230.8ng/mL(AAA-Tyr体系).实验研究了荧光猝灭反应的适宜条件和影响因素,考察了共存物质的影响,表明方法具有良好的选择性,可用于ANG片剂及其代谢物尿药浓度的快速测定.从吸收光谱的变化、温度的影响以及Stern-Volmer作图,判断该反应为静态猝灭反应,氨基酸和安乃近通过静电引力和芳基堆积作用而形成1:1的复合物.

Fluorescence Quenching Method for the Determination of Analgin and Metabolin with Some Aromatic Amino Acids as Probes

In pH 3.2 Britton-Robinson(BR) buffer medium,analgin(ANG) and metabolin of 4-methylaminoantipyrine(MAA) and 4-acetylaminoantipyrine(AAA) can react with some aromatic amino acids such as tryptophane(Trp),tyrosine(Tyr) and phenylalanine(Phe) to form complexes,which lead to fluorescence quenching of above amino acids,and the maximum quenching wavelength is at 352 nm(ANG-Trp system),304 nm(ANG-Tyr,MAA-Tyr and AAA-Tyr system),284 nm(ANG-Phe system),re-spectively.Fluorescence quenching value(ΔF) is proportional to the concentration of ANG,MAA and AAA in a certain range.Fluorescence quenching methods for the determination of ANG,MAA and AAA have high sensitivity with the detection limits of 13.3 ng/mL(Trp-ANG system),15.8 ng/mL(Tyr-ANG system),64.5 ng/mL(Phe-ANG system),150.0 ng/mL(MAA-Tyr system) and 230.8 ng/mL(AAA-Tyr system),re-spectively.The optimum conditions of the reaction,influencing factors and the effect of coexisting sub-stances are investigated,and the results show this method has a good selectivity.This method has been suc-cessfully applied to the quick determination of ANG in tablets and its metabolin in urine samples.Judging from the change of the absorption spectra,the effect of temperature and the Stern-Volmer plots,the quench-ing of fluorescence of amino acids by ANG is a static quenching process.Experimental results show that a 1∶1 compound of ANG and amino acid is formed by electrostatic attraction and aromatic stacking interaction.