Application Of Cyclodextrin-modified Micellar Electrokinetic Capillary Chromatography For Separation And Analysis Of Active Components In Uncaria

Objective:In this article,by optimizing the extraction method of the alkaloids in Uncaria and its preparations,an effective method for extracting hirsutine and hirsuteine in Uncaria rhynchophylla and its preparations is established.Cyclodextrin-modified micellar electrokinetic capillary chromatography (CD-MEKC) was used to separate and analyze hirsutine and hirsuteine in Uncaria rhynchophylla and its preparations.A simple,convenient and effective capillary electrophoresis (CE)method was developed for the separation and determination of two alkaloids in Uncaria rhynchophylla and its preparations.Besides,the proposed method provides scientific reference for the quality control of Uncaria rhynchophylla and its preparations.Method:(1)The optimal extraction method of alkaloids in Uncaria rhynchophylla and its preparations was obtained by investigating the three extraction solvent and two extraction methods.(2)Mono-(6-ethylenediamine-6-deoxy)-β-cyclodextrin(ED-β-CD),sodium cholate(SC)and 2,6-dimethyl-β-cyclodextrin(DM-β-CD)were used as additives for the separation and determination of two alkaloids in Uncaria rhynchophylla and its preparations by CD-MEKC.The optimal method was developed by investigating influences of significant factors on the separation including concentration and pH of buffer,concentration of additives(DM-β-CD,ED-β-CD and SC),separation voltage and system temperature.The optimal separation condition including 150 mM DM-β-CD,3.0mM ED-β-CD and 30 mM SC in 40 m M phosphate buffer(pH 7.0)at the separation voltage of 15 kV,temperature of 25℃,inject pressure of 0.5psi,inject time of 5s and the detection wavelength of 224nm.Results:(1)Chloroform was used as the extraction solvent,and the method of heating and refluxing was used to effectively extract the hirsutine and hirsuteine in Uncaria rhynchophylla and its preparations.Under this extraction condition,the hirsutine and hirsuteine can be obtained to the maximum extent.(2)Under the condition,hirsutine and hirsuteine were successfully separated within 13min at the separation voltage of 15 kV,temperature of 25℃ and the detection wavelength of 224 nm.For the analytes,linear calibration curves were performed within the range of 5.0-160.0 μg/mL respectively,the correlation coefficient was from 0.9934 to 0.9969.The RSDs of intraday<1.3% and the RSDs of interday<0.9%.The limits of detection(LOD,S/N=3)and the limits of quantitation(LOQ,S/N=10)were 0.41,1.42μg/mL for hirsutine and 0.60,2.17μg/mL for hirsuteine.The recovery rate of three samples within the range of 97.9-102.3%.Conclusion:The optimal extraction method of alkaloids in Uncaria rhynchophylla and its preparations was obtained by investigating the three extraction solvent and two extraction methods.The CD-MEKC method was successfully applied to the determination of hirsutine and hirsuteine in uncaria rhynchophylla(UR)and its formulations.Meanwhile,it provides an effective reference of the quality control of the UR and its formulations.Additionally,the CD-MEKC method could provide the potential of analysing and separating other Hydrophobic compounds.