Application Of HPCE In Quality Evaluation Of Guan-Xin-Ning Injection

Objective:The purpose of this study was to establish the high performance capillary electrophoresis(HPCE)fingerprint of Guan-Xin-Ning injection.And a micellar electrokinetic chromatography(MEKC)method was developed based on the fingerprint analysis to separate seven bioactive constituents(Tetramethylpyrazine(TMPZ),Isoferulic acid(IFA),Ferulic acid(FA),Danshensu(DSS),Caffeic acid(CA),Salvianolic acid A(SAA)and Salvianolic acid B(SAB))in Guan-Xin-Ning injection.Simultaneously,a capillary electrophoresis quantitative analysis of multi-components by single-marker(QAMS)method was used to determine the contents of seven components in Guan-Xin-Ning injection,and the quality of Guan-Xin-Ning injection was comprehensively evaluated.Methods:(1)Ten batches of Guan-Xin-Ning injections were analyzed by capillary electrophoresis and the fingerprint electropherogram was established.The Similarity Evaluation System for the Chromatographic Fingerprint of Traditional Chinese Pharmacopoeia Committee(Version 2012)was used as the software package to evaluate the similarity of samples.Separation parameters such as buffer concentration and type,p H,additives and separation voltage were investigated.The experiment was carried out on an uncoated fused silica capillary(38.8 cm×75 ?m,effective length 28.7 cm).Finally,30 m M borate buffer(p H 9.3),10 k V of voltage across the capillary,temperature of 25? and the detection wavelength of 212 nm were selected as the optimum condition in fingerprint analysis.(2)In order to further evaluate the quality of Guan-Xin-Ning injection,a micellar electrokinetic chromatography(MEKC)method was developed for simultaneous separation seven bioactive constituents in Guan-Xin-Ning injection by examining the sodium dodecyl sulfate(SDS)concentration,buffer concentration,p H,applied voltage and temperature.Ultimately,45 m M borate buffer containing 35 m M SDS at p H 9.3 with an voltage of 10 k V,temperature of 25 ? and the detection wavelength of 212 nm were chosen as the satisfactory separation condition.(3)In this paper,the Danshensu(DSS)was used as internal reference substance and the relative correction factors of the other six components were set and determined.And the content of seven components in Guan-Xin-Ning injection was determined by a capillary electrophoresis QAMS method,and compared with the content that determined by the method of MEKC.Meanwhile,the impact factors on the reproducibility of relative correction factor were investigated,such as capillary electrophoresis devices,the batches of capillary columns,temperatures and different laboratory technicians.Results:(1)Ten batches of samples were used to establish the fingerprint electropherogram and 34 common peaks were obtained within 20 min.Simulative mean electropherogram was generated by introducing ten batches of electropherograms into the similarity evaluation system,which was considered as reference fingerprint.And all of the similarity values were greater than 0.987.The RSD of relative migration times(RMT)and relative peak areas(RPA)were less than 5%.(2)Seven components reached baseline separation within 35 min under the optimal condition.A good linearity was obtained with correlation coefficients from 0.9906 to 0.9997.The LOD and LOQ ranged from 0.12 to 1.50 ?g/m L and from 0.40 to 4.90 ?g/m L,respectively.The recoveries ranged between 99.0% and104.4%.(3)The content of seven components in eight batches Guan-Xin-Ning injection were calculated by the capillary electrophoresis QAMS method.The content results were compared with the method of MEKC and the consistency was evaluated.All results indicated that,there was no significant differences between the two methods,and the relative error was less than 4.8%.Additionally,the results showed that the relative correction factor appeared good reproducibility on different batches of capillary columns and different capillary electrophoresis instruments.Conclusion:In this study,a fingerprint evaluation system for Guan-Xin-Ning injection was developed and a MEKC method was established to separate seven bioactive constituents in Guan-Xin-Ning injection,which was simple,economical and rapid.Meanwhile,the study showed that,the capillary electrophoresis QAMS method could effectively control the quality of Traditional Chinese Medicine injections when the reference substance was expensive or lacking.The study provided an effective method for the quality evaluation of Guan-Xin-Ning injection and valuable reference for further research and development of other Traditional Chinese Medicine injections.