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目的:建立共振瑞利散射法测定大黄素的含量。方法:在p H 7.3的B-R缓冲溶液中,铁(Ⅱ)与1,10-邻菲啰啉形成稳定的1∶3的配合物,再与大黄素(EMO)结合形成1∶2的离子缔合物。铁(Ⅱ)-1,10-邻菲啰啉溶液:称取0.278 0 g Fe SO4·7H2O溶于70 m L水中,加入邻菲啰啉(1,10-phen)0.595 0 g,加入适量的抗坏血酸,稀释至100 m L,得到1×10-2mol·L-1的储备液,用时稀释成2.0×10-3mol·L-1的工作液。室温下,于10 m L比色管中依次加入p H 7.3的B-R缓冲溶液2 m L,适量的EMO标准溶液,以及2.0×10-3mol·L-1Fe(phen)2+3溶液1.2 m L。每加一种试剂后混合均匀,用二次蒸馏水定容至刻度,摇匀,静置10 min后,在荧光分光光度计上以λex=λem方式进行同步扫描,记录共振瑞利散射(RRS)光谱,以λem=2λex和λem=1/2λex进行扫描,分别测量不同入射波长(λex)下的散射强度ISOS和IFDS,然后分别以ISOS和IFDS对应的波长作图,得到二级散射(SOS)光谱和倍频散射(FDS)光谱。分别在各自的最大散射波长处测量样品和试剂空白的散射强度IRRS,ISOS,IFDS及I0RRS,I0SOS,I0FDS,ΔI=I-I0。结果:体系的RRS、SOS和FDS显著增强并出现新的散射峰,相应的最大散射峰分别位于349、684和351 nm。EMO的质量浓度在0.8~10.4μg·m L-1时,与RRS、SOS和FDS的散射强度呈良好的线性关系,其检出限(3σ)依次分别为10.1、32.8、28.6 ng·m L-1。血样和尿样(各3批)中测定EMO的回收率分别在94.9%~102.4%和99.4%~102.7%之间,RSD分别在1.5%~3.1%和1.1%~3.0%之间。将本法与紫外可见分光光度法比较,结果满意。结论:经方法学验证,体系的共振瑞利散射方法可用于尿样和血清中大黄素的测定。
Objective: To establish a resonance Rayleigh scattering method for the determination of emodin. Methods: A stable 1: 3 complex of iron (Ⅱ) with 1,10-phenanthroline was formed in BR buffer solution of p H 7.3, and then combined with emodin (EMO) to form a 1: 2 ionic group Compound. Iron (II) -1,10-phenanthroline solution: Dissolve 0.278 0 g FeSO 4 · 7H 2 O in 70 m L water and add 0.595 0 g phenanthroline (1,10-phen) Ascorbic acid was diluted to 100 mL, and a stock solution of 1 × 10-2 mol·L-1 was obtained. The working solution was diluted to 2.0 × 10-3 mol·L-1. At room temperature, 10 mL L of BR buffer solution of 2 μL, appropriate amount of EMO standard solution, and 2.0 × 10-3 mol·L-1Fe (phen) 2 + 3 solution of 10 mL . After each addition of a reagent, mix well and dilute to the mark with twice-distilled water. Shake well and allow to stand for 10 min. Scanning synchronously with λex = λem on a fluorescence spectrophotometer. Resonance Rayleigh scattering (RRS) Spectra were obtained by scanning with λem = 2λex and λem = 1 / 2λex, respectively measuring the scattering intensities ISOS and IFDS at different incident wavelengths (λex), and then plotting the corresponding wavelengths of ISOS and IFDS respectively to obtain the second-order scattering (SOS) Spectral and frequency doubling scattering (FDS) spectra. Scattering intensities IRRS, ISOS, IFDS and IORRRS, IOSOS, IODF, and ΔI = I-I0 were measured for the blank and reagent blank at their respective maximum scattering wavelengths. Results: The RRS, SOS and FDS of the system were significantly enhanced and new scattering peaks appeared. The corresponding maximum scattering peaks were at 349, 684 and 351 nm, respectively. When the concentration of EMO was 0.8 ~ 10.4μg · m L-1, there was a good linear relationship between the scattering intensity of RRS, SOS and FDS. The detection limits (3σ) were 10.1, 32.8 and 28.6 ng · m L -1. The recoveries of EMO measured in blood and urine samples (3 batches each) ranged from 94.9% to 102.4% and 99.4% to 102.7% with RSDs of 1.5% to 3.1% and 1.1% to 3.0%, respectively. The method and UV-visible spectrophotometry, the results are satisfactory. CONCLUSION: The method of Resonance Rayleigh scattering of the system can be used for the determination of emodin in urine and serum after verification by method.