| Objective:Liquid phase microextraction is a new sample pretreatment technology developed on conventional liquid-liquid extraction,which integrates sampling,purification,and enrichment.It can adapt to the analysis of complex matrix,trace components and special components.In order to further improve the enrichment efficiency of target compounds,reduce the use of organic solvents,and increase the sensitivity of detection,we propose to ameliorate the conventional microextraction model and establish a more simple,efficient,and environmentally friendly method,which combined with HPLC to realize the purification and efficient determination of the target compounds in traditional Chinese medicine samples.Methods:A hydrophobic deep eutectic solvent(tetrabutylammonium chloride-hexanoic acid)was used as both extraction phase and acceptor phase to impregnate the wall-pores and lumen of hollow fiber.Two fibers full of deep eutectic solvent were bent to U-shape,and submerged in the traditional Chinese medicine sample solution.Four cinnamic acid derivatives were extracted to the deep eutectic solvent phase at a certain temperature,and analyzed by HPLC-UVD.Because the fiber's wall pores can intercept and separate biological macromolecules,the proposed method can be also used to determine the active compound-protein binding rate in plasma samples.The influencing factors of microextraction were optimized,and methodological evaluation were conducted.Furthermore,both the extraction mechanism of target active compounds and the determination mechanism of active compound-plasma protein binding rate were also explored and illustrated.In the self-made extraction device,a hydrophobic deep eutectic solvent synthesized from tetrabutylammonium chloride and hexanoic acid was added to the sample solution as an extractant.The dispersing power was provided by hand,so that deep eutectic solvent was dispersed into tiny droplets to reach the maximum contact area with sample solution,and complete the extraction of target compounds.Finally,the two phases(deep eutectic solvent phase and aqueous phase)were separated by centrifugation.The extraction conditions were optimized and methodology was investigated under the optimal conditions.The developed method combined with HPLC-UVD was applied to the separation,concentration,and determination of four cinnamic acid derivatives in Chuanxiong Rhizoma and Angelicae Sinensis Radix.Meanwhile,the proposed procedure was also compared with other extraction methods of active compounds in traditional Chinese medicines.Results:The optimum extraction conditions for deep eutectic solvent-based hollow fiber liquid-phase microextraction were as follows: the extractant was tetrabutylammonium chloride-hexanoic acid(1:3,n:n);sample phase p H was 2;salt concentration was 20%;extraction temperature was 55?;stirring speed was 800 rpm;the extraction time was 40 min.Under these conditions,the enrichment factors of four target compounds were from 82 to 127.Their linear ranges were 0.002-5.6 ?g/m L for caffeic acid,0.001-3.2 ?g/m L for p-hydroxycinnamic acid,0.0016-4.8 ?g/m L for ferulic acid,and 0.0012-4 ?g/m L for cinnamic acid,with r2 ? 0.9985.Detection limits and quantitative limits were 0.1-0.3 ng/m L and 0.4-1.0 ng/m L,respectively.The relative standard deviations of intra-day and inter-day precision were 1.0%-6.1% and 0.7%-6.0%.The average recoveries were 89.9%-105.2%.The average contents of caffeic acid and ferulic acid in Chuanxiong Rhizoma were 64.1±2.4 and 928.9±27.1 ?g/g.The average contents of caffeic acid,p-hydroxycinnamic acid,ferulic acid and cinnamic acid in Mai-luo-ning injection were 629.3±10.1?61.1±1.2?23.8±0.8 ? 576.4±16.4 ?g/m L.In addition,the incubation time between the active compounds and plasma protein was 90 min,and the binding rates of the four cinnamic acid derivatives were 39.3±1.4%,42.0±1.3%,52.9±2.6% and 19.8±1.1%.The optimum extraction conditions for deep eutectic solvent-based dispersive liquid phase microextraction were as follows: the extractant was 150 ?L of tetrabutylammonium chloride-hexanoic acid(1:2,n:n);sample phase p H was 3;salt concentration was 15%;the extraction time was 15 s;the centrifugation time was 2 min.Under the optimized experimental conditions,the enrichment factors of 135,171,146,220 for caffeic acid,p-hydroxycinnamic acid,ferulic acid and cinnamic acid.The calibration graphs were linear(with r2 ? 0.9954)in the range of 0.02-8 ?g/m L,0.01-6.4 ?g/m L,0.016-7.2 ?g/m L,and 0.012-7.2 ?g/m L,respectively.The limits of detection and limits of quantitation were 0.2-0.4 ng/m L and 0.6-1.4 ng/m L.The relative standard deviations of intra-day and inter-day precision were 1.6%-7.2% and 1.3%-8.5%.The average recoveries were 90.0%-104.6%.The average contents of caffeic acid and ferulic acid in Chuanxiong Rhizoma were 70.2±4.0 and 1004.1±40.9 ?g/g.The average contents of ferulic acid in Angelicae Sinensis Radix were 1029.4±17.7 ?g/g.Conclusions:In this paper,two novel liquid-phase microextraction methods of deep eutectic solvent-based hollow fiber liquid-phase microextraction and deep eutectic solvent-based dispersive liquid phase microextraction were established.Deep eutectic solvent-based hollow fiber liquid-phase microextraction not only improved the enrichment factors of the traditional hollow fiber liquid-phase microextraction on these compounds,but also completed the determination of the active compound-plasma protein binding rate.The extraction time and operating procedure of deep eutectic solvent-based dispersive liquid phase microextraction have been improved to a great extent.It has the characteristics of high enrichment and friendly environment.This study made an active and effective exploration to establish a more environmentally friendly,simple,efficient,and economical liquid-phase microextraction method.The three methods combined with HPLC-UVD have been successfully applied to the extraction,concentration and determination of the quality markers of cinnamic acid derivatives in complex samples(traditional Chinese medicine and human plasma). |
PDF Full Text Request