Studies On The Methods For Quality Control Of Cortex Fraxini

Cortex Fraxini is originated from the dried bark of Fraxinusrhynchophylla Hance, Fraxinus chinensis Roxb., Fraxinus szaboana Lingelsh.or Fraxinus stylosa Lingelsh.. As recorded in Chinese Pharmacopoeia, it is animportant traditional Chinese herbal medicine commonly used for thetreatment of gout, arthritis, diarrhea and bacillary dysentery. More importantly,coumarins, the major bioactive constituents in Cortex Fraxini, are usually usedto treat primary hyperuricemia with renal dysfunction. Up to now, qualitativeand quantitative analysis of main active constituents in Cortex Fraxini waslimited in several analytes due to the complexity and low-content ofconstituents, it is difficult to conduct a comprehensive quality control. In thepresent study, HPLC-MS/MS for the quantitative and qualitative analysis ofconstituents in Cortex Fraxini were developed, and the HPLC fingerprint ofCortex Fraxini was established, which provided a reference for a morescientific and effective quality control of Cortex Fraxini.Part one Multi-responses extraction optimization based on responsesurface methodology combined with polarity switchingHPLC-MS/MS for the simultaneous quantitation of11compounds in Cortex Fraxini：Application to four species ofCortex Fraxini and its3confusable speciesObjective: To develop a novel polarity switching high performanceliquid chromatography-electrospray ionization tandem mass spectrometry(HPLC-ESI-MS/MS) approach combining optimization of extractioncondition by response surface methodology (RSM) for the simultaneousquantitative analysis of11compounds (aesculin, aesculetin, fraxin, fraxetin,scopoletin, isoscopoletin, oleuropein, salidroside, syringin, 6-hydroxy-7,8-dimethoxy coumarin and8-hydroxy-6,7-dimethoxy coumarin)in Cortex Fraxini.Methods: The ultrasonic extraction conditions of the11analytesincluding sample quantity, methanol concentration and extraction time weresimultaneously optimized with a Box-Behnken design (BBD). A second-orderpolynomial regression model was used to predict the experimental results, andDerringer’s desirability function was applied to optimize the11responsessimultaneously. Multiple-reaction monitoring (MRM) scanning was employedfor quantification with switching electrospray ion source polarity betweenpositive and negative modes in a single run of16min. The method wasapplied to differentiate55batches of Cortex Fraxini samples and itsconfusable species from different sources (21identified Cortex Fraxinisamples,3confusable species samples,31commercial Cortex Fraxinisamples).Results：The optimum conditions were as follows: sample quantity was11mg, methanol concentration was73%, and extraction time was65min. Thecontents of11compounds were obtained under the optimum conditions, andthere were no significant differences between the results of the optimizationexperiments and those predicted by the models. RSM could give measurablerelationship between responses and variables so that the optimal conditionsand predicted values were easy to be obtained. Moreover, interactive effects ofvariables on responses were considered and analyzed with RSM, which couldnot be implemented with OVAT experiment. The other advantage of RSM wasthat multiple responses could be optimized simultaneously. Meanwhile it wasdifficult to be performed with OVAT experiment for visual analysis of linegraph was not suitable for taking into account different responsessimultaneously. Quantitative parameters of the proposed method with respectto limit of detection (LOD), limit of quantification (LOQ), linearity, precision,accuracy and stability were evaluated under optimum conditions, and theresults indicated that the method was sensitive, specific and reliable. It was found that the Cortex Fraxini could be easily distinguished from theconfusable species according to the contents of the constituents.Conclusion：A novel sensitive and selective HPLC-ESI-MS/MS methodoperating negative and positive switching mode in a single analysis processwas developed and validated to simultaneously determine11constituents indifferent origins of Cortex Fraxini for the first time. The reported method herewas capable of providing higher sensitivity and selectivity. When applied tothe analysis the four species of Cortex Fraxini, the method was found to givesatisfactory applicability. The Cortex Fraxini could be easily distinguishedfrom the confusable species according to the contents of the constituents.Furthermore, this developed method exhibited powerful potential for theanalysis of coumarins in other medical herbs.Part two Study on fingerprint of Cortex Fraxini with HPLCObjective: To establish HPLC fingerprint of Cortex Fraxini and providereference for quality evaluation of Cortex Fraxini.Method:(1) Optimization of the chromatographic conditions:chromatographic conditions such as mobile phase systems, wavelength andcolumn temperature were tested for optimization of the chromatographicconditions.(2) System suitability test: under the above chromatographicconditions, the number of theoretical plate of aesculin peak and resolutionbeween adcajent peaks were calculated.(3) Method validation: precision,reproducibility and stability were determined.(4) Identification of main peaks:main peaks were identified by HPLC and HPLC-MS/MS.(5) Sample analysis:each sample solution of Cortex Fraxini was prepared and analyzed to get theirfingerprints. Then the similarity was calculated and evaluated.Results：(1) Optimization of the chromatographic conditions:chromatographic experiments were performed on an Agilent Zorbax SB-C18column (250mm×4.6mm i.d.5μm) with solvent A (methanol) and solventB (water containing0.5‰acetic acid) as the mobile phases in gradient elution.The column temperature was maintained at25C. The detection wave length was230nm, the flow rate was1.0mL/min,10μL of samples were injected inevery case.(2) System suitability test: under the above chromatographicconditions, each peak was well separated, and the number of theoretical plateof aesculin peak was about16000.(3) Method validation: designating themajor peaks for reference peak, the RSD valus of relative retention time andpeak areas of were less than0.38%and3.55%for precision, less than0.67%and3.98%for reproducibility and less than0.86%and4.98%for stability.(4)Identification of main peaks: nine peaks were identified.(5) Sample analysis:The similarities of the chromatograms of the52Cortex Fraxini samples werecompared with the reference chromatogram, and ten common peaks were got.According to the similarity results,52samples were divided into three types,their similarities were0.8-1.0,0.6-0.8and0.4-0.6, respectively. It indicatedthat the method was a supplement to the quality control of Cortex Fraxini.Fingerprints of four kinds of Cortex Fraxini were analysed. The similarities ofinter-species for each kind were high, which indicated that samples in onespecies were similar. The number and content of common peaks were notexactly the same among the four kinds of Cortex Fraxini, which suggested thevariance among the species. Three chromatograms of Cortex Fraxiniconfusable species were compared with Cortex Fraxini standard sample, allthe similarities were lower than0.47, and only two common peaks were foundthere. It indicated that significant diversity could be found between CortexFraxini and confusable species, and the Cortex Fraxini could be easilydistinguished from the confusable species by the established method.Conclusion： HPLC fingerprint of Cortex Fraxini were established in thepresent study. Difference among four kinds of Cortex Fraxini samples andtheir confusable species were studied by similarity analysis, then the quality ofCortex Fraxini were generally evaluated. It is simple and quick to distinguishCortex Fraxini with confusable species by the method, which can be used forthe quality control of Cortex Fraxini. Part three The characterization of constituents in Cortex Fraxini byHPLC-Q-TOF-MS/MSObjective: To develop a practical method using high performance liquidchromatography with quadruple time-of-flight mass spectrometry(HPLC-Q-TOF-MS/MS) technology for the rapid separation and identificationof the complicated constituents in Cortex Fraxini.Method: First, we studied the mass fragmentation patterns of11standards of Cortex Fraxini in the positive and negative ion mode by full scan,product ion scan and precursor ion scan, and summarized the fragmentationrules. Then, on the basis of the summarized rules, Cortex Fraxini sample wasanalyzed by HPLC-Q-TOF-MS/MS in the scanning mode ofTOF-MS-IDA-8MS/MS to acquire exact mass and fragment ions of theunknown compounds. Then, the combination use of Peak View toolsincluding “XIC Manager” and “show XIC”(extracted ion chromatography)were chosen to identify the constituents of Cortex Fraxini and deduce itsfragmentation rule. Chromatographic experiments were performed on anAgilent Zorbax SB-C18column (250mm×4.6mm i.d.5μm) with solvent A(methanol) and solvent B (water containing0.5‰acetic acid) as the mobilephases in gradient elution, and the flow rate was1.0mL/min.Results：Seven fragmentation rules of11standards (8coumarins,1phenylpropanol and2phenolic compounds) were summarized by studying themass fragmentation patterns of11standards in the positive and negative ionmode by full scan, product ion scan and precursor ion scan. A total of35compounds (8coumarins,1phenylpropanol,5escucide,1phenolic acid and2phenolic compound) in crude extract of Cortex Fraxini were characterized byHPLC-Q-TOFMS/MS according to the summarized fragmentation rules,accurate molecular weights and characteristic fragment ions. Nine of whichwere identified by comparing with the standards, and the other24compoundswere identified by analyzing the information provided by XIC manager andthe XIC patterns, the error tolerance is±5ppm. Conclusion: HPLC-Q-TOF-MS/MS was high sensitive, accurate andselective for the TCM identification.