Application Of Liquid Phase Microextraction Based On New Green Solvents In The Analysis Of Traditional Chinese Medicine

Objective:Liquid phase microextraction（LPME）is a sample pretreatment method which integrates extraction,concentration and purification.Compared with the traditional sample pretreatment method,LPME provides unique advantages,such as high enrichment factor（EF）,low cost,simple,sample extraction and concentration are accomplished in one step,and can be combined with various analytical instruments.However,conventional LPME methods have the following disadvantages:（1）most of the organic solvents used in conventional LPME methods are potentially toxic;（2）some operations are complex and time-consuming.Therefore,due to the need of green chemistry,some new green solvents are proposed to be used in LPME to reduce the harm to the experimenter and the environment,at the same time,the extraction and enrichment of target compounds in the complex matrix of traditional Chinese medicine samples were realized with high efficiency and simple operation.Methods:In the first part of this paper,the switchable solvent（SS）was used in liquid-phase microextraction,and the surfactant was used as sensitizing agent,we established a method of sodium dodecyl sulfate sensitized switchable solvent liquid-phase microextraction（SDS-SSSLPME）.Hydrophobic triethylamine（TEA）,as a switchable extraction solvent,was first converted to protonated triethylamine carbonate（TEA-C-P⁺）in the presence of CO₂,and then the TEA-C-P⁺combined with the alkaline target analytes.When sodium hydroxide was added,the TEA-C-P⁺was deprotonated and switched to TEA,so as to realize phase separation and target analyte enrichment.The proposed SDS-SSSLPME combined with high performance liquid chromatography-diode array detection（HPLC-DAD）was applied to simultaneous extraction,enrichment,and determination of active protoberberine alkaloids in Rhizoma coptidis.Several parameters affecting the performance and methodology research were optimized and investigated,and the possible microextraction mechanism was analyzed and discussed.Also,the proposed method was compared with other liquid-phase microextraction methods for the determination of alkaloids in Rhizoma coptidis.In the second part of this paper,the deep eutectic solvents（DES）was used in hollow-fiber liquid-phase microextraction,and we introduced a method of three-phase hollow-fiber liquid-phase microextraction based on DES as acceptor phase（DES/3p-HFLPME）.The DES of methyltrioctylammonium chloride-glycerol was used as acceptor phase,and the complex alcohols was used as extraction phase.The target analytes in the sample solution were first extracted through the extraction phase,and further back-extracted to the acceptor phase during the stirring process.DES/3p-HFLPME combined with HPLC was successfully applied to the simultaneous extraction,concentration,and determination of the main active compounds in a formula of Zi-Cao-Cheng-Qi decoction（ZCCQD）.The important extraction parameters and methodology research were optimized and investigated,and the extraction mechanism was clarified.In the third part of this paper,hydrophobic DES was used in dispersive liquid phase microextraction,a novel method of vortex-assisted dispersive liquid phase microextraction based on deep eutectic solvent（DES/VA-DLPME）was established,and DES/VA-DLPME coupled with HPLC was applied for the enrichment and determination of the six main active compounds in a formula of ZCCQD.In this method,hydrophobic DES was prepared by mixing tetrabutylammonium chloride（TBAC）and hexanoic acid,DES as extraction solvent was added to sample solution containing the analytes,then the analytes were adequately contacted with DES fine droplets by vortex-assisted,the obtained cloudy solution was separated by centrifugation.The extraction conditions of the proposed method were optimized and the methodology was investigated,and the proposed method was compared with other extraction methods of active compounds in ZCCQD.Results:（1）in the first part of this study,under the optimised extraction conditions,the enrichment factors of epiberberine,coptisine,palmatine,and berberine by SDS-SSSLPME were separately 102,103,125,and 152.The linear ranges were0.032-4.23μg/mL for epiberberine,0.031-4.33μg/mL for coptisine,0.0026-10.04μg/mL for palmatine,and 0.0013-4.13μg/mL for berberine,respectively.The correlation coefficients（r²）was ranged from 0.9906 to 0.9935.The limits of detection（LOD）were ranged from 0.2 to 0.3 ng/mL,the limits of quantitation（LOQ）were ranged from 0.3 to 0.6 ng/mL.The relative standard deviations（RSD）of intra-day precision were 0.1%-7.6%,and the RSD of inter-day precision were 1.9%-10.9%.The average recoveries were 100.2%-105.0%.（2）in the second part of this study,under the optimal experimental conditions of DES/3p-HF-LPME,the enrichment factors of the main active compounds（hesperidin,honokiol,shikonin,magnolol,emodin,andβ,β′-dimethylacrylshikonin）in Zi-Cao-Cheng-Qi decoction were from 6 to 114.The calibration graphs were linear（with r²≥0.9901）in the range of 0.002-10,0.001-5,0.0026-13,0.014-7,0.005-1,and 0.02-10μg/mL for hesperidin,honokiol,shikonin,magnolol,emodin,andβ,β′-dimethylacrylshikonin,respectively.Detection limits and quantitative limits were 0.3-0.8 ng/mL and 0.6-2 ng/mL,respectively.The RSD of intra-day and inter-day precision were 0.3-7.1%and 0.3-8.4%,respectively,and average recoveries were 92.5%-102.4%.（3）in the third part of this study,under the optimized experimental conditions of DES/VA-DLPME,the enrichment factors of the main active compounds（hesperidin,honokiol,shikonin,magnolol,emodin,andβ,β′-dimethylacrylshikonin）in Zi-Cao-Cheng-Qi decoction were in the range of3–330.The calibration graphs were linear（with r²≥0.9859）in the range of 0.0015-3,0.003-6,0.05-10,0.0075-3,0.001-10,and 0.01-4μg/mL for honokiol,shikonin,hesperidin,magnolol,emodin,andβ,β′-dimethylacrylshikonin.Detection limits and quantitative limits were 0.3-0.9 ng/mL and 1-3 ng/mL,respectively.The RSD of intra-day and inter-day precision were 0.5-8.9%and 0.8-8.3%,respectively,and average recoveries were 91.1%-102.2%.Conclusion:In this paper,three novel liquid-phase microextraction methods based on the new green solvents were established,namely SDS-SSSLPME,DES/3p-HFLPME,DES/VA-DLPME.Compared with other traditional liquid-phase microextraction methods,the proposed methods have the advantages of greener operation,high enrichment factor,wide application range.The three methods combined with HPLC has been successfully used for simultaneous extraction,enrichment,and effective determination of various trace components in Chinese herbal medicine and traditional Chinese medicines formula.