Studies On The Application Of High-speed Counter Current Chromatography And Liquid-phase Microextraction In Separation And Determination Of Aconitum Alkaloids

Aconitum alkaloids are the main constituents of plants of the genus aconitum.They have the pharmaceutical effects of anti-inflammatory,analgesic,anti-tumor,cardiotonic and so on,meanwhile,they are toxic components.Such herbal medicines must be strictly processed and tested before use.Otherwise,it is easy to cause poisoning and even death.Accordingly,it is necessary to establish perfect analytical methods accurately monitor the contents of aconitum alkaloids to ensure the safety and therapeutic effect.At the same time,the establishment of the determination method also require standard substance with high purity.In this paper,we first investigated an efficient method for separation and purification of aconitum alkaloids.On the basis of this research,a series of novel methods were established for green determination of aconitum alkaloids in different matrix including Chinese medicinal herbs,Chinese patent medicine,human plasma and urine in order to provide new methods for quality control,toxicological research,forensic medicine and therapeutic drug monitoring.Firstly,we investigated an efficient method using HSCCC for separation of three monoester-diterpenoid aconitines from lateral roots of Aconitum carmichaeli Debx?Fuzi in Chinese?.The separation was performed by injecting the ethanolic extract of processed Fuzi into HSCCC system directly without treatment.Impacts of the solvent systems,the rotary speed as well as the flow rates of the mobile phases on the separation efficiency were investigated.An optimized HSCCC condition with two-phase solvent system composed of chloroform-methanol-n-butanol-Hydrochloric acid aqueous solution?4:1:1:5,v/v?was used with a flow rate of 4.0 ml/min and a rotary speed of 800 rpm.Under the optimized conditions,three target compounds including benzoylaconine?BAC?,benzoylmesaconine?BMA?,benzoylhypaconine?BHA?with purities above 98.0% were successfully separated in 200 min.The results showed that the present HSCCC method was a highly effective process with high recovery and loading capacity,low solvent consumption,simple operation,less time-consuming.Furthermore,this research provided standard substance for the follow-up study of determination methods.Aiming at the determination of aconitum alkaloids in Fuzi herbs,a green analytical method based on the use of ionic liquid aqueous two-phase system?IL-ATPS?coupled with HPLC was developed for determination of six aconitum alkaloids in Fuzi.Aconitum alkaloids in Fuzi were extracted with ionic liquid aqueous solution and then concentrated by adding inorganic salts to form IL-ATPS.Aconitum alkaloids were enriched into the upper IL-rich phase which can be injected into the HPLC for analysis.Various parameters that influence the extraction efficiency,including type and amount of ionic liquid,amount of salts,pH value as well as extraction time were investigated.Under optimal conditions,the recoveries of the analytes ranged from 80.37 to 107.18%,and the relative standard deviations?RSDs?ranged from 2.13 to 4.87%.The proposed method provided lower cost of extractant and sample,shorter extraction time and higher sensitivity in comparison with conventional extraction methods.In order to obtain the extract of aconitum alkaloids,we investigated organic solvent and non-organic solvent methods to back extract aconitum alkaloids from ionic liquids.Four organic solvents including chloroform,dichloromethane,ethyl acetate and n-hexane were investigated.The results showed that the back extraction efficiency was greater than 75% using chloroform,dichloromethane and ethyl acetate as extractant.The back extraction efficiency was very low when n-hexane was used.The non-organic solvents method was performed by conversion hydrophilic ionic liquids into hydrophobic ionic liquids by means of in situ metathesis reaction.Hydrochloric acid aqueous solution was used as back extraction agent to recover ionic liquids.Although the approach was complicated,it had the advantage of not using volatile organic solvents.In view of the low concentration of diester-diterpenoid aconitines and the complex matrix in xiaohuoluo pills which was a common Chinese patent medicine,two dispersive liquid-liquid microextraction?DLLME?approaches including Gas-assisted ionic liquid dispersive liquid-liquid microextraction?GA-IL-DLLME?and ionic liquid homogeneous liquid-liquid microextraction?IL-HLLME?were developed for enrichment of three diester-diterpenoid aconitines in xiaohuoluo pills prior to their determination by HPLC-DAD.Several experimental parameters affecting GA-IL-DLLME including type and amount of ionic liquid,pH value as well as gas bubbling time and IL-HLLME including type and amount of metathesis reagent,amount of ionic liquid as well as pH value were investigated and optimized.Under optimal conditions,The spiked recoveries of each analyte for GA-IL-DLLME and IL-HLLME were in the range of 85.83⁹4.44% and 81.67⁹3.06%,respectively.The results showed that GA-IL-DLLME was a much more effective method,providing high recoveries and consuming less ionic liquid.Therefore,GA-IL-DLLME coupled with HPLC-DAD was successfully applied to determination of the three diester-diterpenoid aconitines in xiaohuoluo pills.The results showed that the contents of the diester-diterpenoid aconitines in xiaohuoluo pills had certain difference from different manufacturers.Nevertheless,the total contents were comparatively low in all samples.On the basis of previous research,the determination methods of aconitum alkaloids in biological samples,including plasma and urine were also studied in this paper.In view of the plasma samples containing protein,fat and other interfering substances,a novel method for determination of six aconitum alkaloids in human plasma was developed using matrix solid-phase dispersion combined with vortex-assisted dispersive liquid-liquid microextraction and HPLC-DAD.Several experimental conditions,including type and amount of dispersing sorbent,type and volume of elution solvent,type and volume of extraction solvent,salt concentration as well as pH value,were investigated and optimized.Under optimal conditions,the recoveries of the aconitum alkaloids ranged from 73.81 to 101.82%,and RSDs ranged from 2.35 to 6.56%,indicating that sample matrices originating from the plasma had no significant interferences on the performance of MSPD-VADLLME.Therefore,The proposed method can be satisfactorily used to enrich and determine of aconitum alkaloids in plasma samples.In view of the low viscosity and low protein content in urine samples,a novel effervescence-assisted ionic liquid dispersive liquid-liquid microextraction technique with extractant removal by magnetic nanoparticles was proposed for the determination of six aconitum alkaloids in urine coupled with HPLC-DAD.The extraction technique makes use of a mixture of ionic liquid,Fe₃O₄ magnetic nanoparticles?MNPs?and acetic acid solidified under the low temperature.The mixture of ice chip was added into the sample,which was previously dissolved sodium carbonate,and CO2 bubbles were generated to disperse the extraction solvent.Then,the extraction solvent was easily retrieved through Fe₃O₄ magnetic nanoparticles and external magnet.The main factors affecting the recovery,including component of the effervescent agent,type and amount of ionic liquid,amount of Fe₃O₄ as well as the kind of eluent were investigated and optimized.Under the optimal conditions,the recoveries of six aconitum alkaloids in real urine samples ranged from 81.11% to 106.67%,and RSDs ranged from 2.70% to 6.49%,indicating the proposed method had good recoveries and could be satisfactorily used to enrich and determine of aconitum alkaloids in urine samples.In summary,this paper provide a rapid and efficient separation method for the preparation of standard substance of aconitum alkaloid and a series of sample pretreatment methods for green determination of aconitum alkaloids in different medicinal and biological samples.Moreover,this study also provides a new research idea for the analysis and quality control of Chinese herbal medicines.