Chemical Components Analysis Of Radix Peucedani By HPLC-MS And Library Searching Technologies

Radix Peucedani, the dry roots of Peucedanum Praeruptorum Dunn., isone of the most commonly used traditional Chinese medicines (TCM). It hasbeen recorded in Chinese Pharmacopoeia and is used to treat cough with thicksputum and dyspnea, upper respiratory infections, and nonproductive coughchiefly in China. Radix Glehniae contains a large number of coumarins whichare active constituents. Up to now, qualitative and quantitative analysis ofmain active constituents in Radix Peucedani was limited in several analyte dueto complex matrix and low contents. HPLC-MS technology has demonstratedits value in analyzing complex mixtures and has become a powerful tool in thequalitative and quantitative analysis of various natural compounds owing to itsshort running time, high accuracy, sensitivity and specificity. In the presentstudy, HPLC-MS was performed to analyze chemical components in RadixPeucedani. First,3′,4′-diacyloxykhellactone coumarins were characterized incrude extract of Radix Glehniae with the summarized fragmentation rules.Second, a novel specific approach based on library searching for simultaneousidentification and quantitation of coumarins by HPLC-ESI-MS/MS wasdeveloped and validated to simultaneously determinate and identifyconstituents in commercial Radix Peucedani samples and adulterants.Part one The characterization of main coumarins in Radix Peucedani byhigh performance liquid chromatography-mass spectrometryObjective: To summarize fragmentation rules and develop a highsensitive and efficient liquid chromatography-mass spectrometry (LC-MS)method for detection and characterization of the main active coumarins inRadix Peucedani.Methods: First, we studied the mass fragmentation patterns of33′,4′-diacyloxykhellactone coumarin standards in the positive ion mode by full scan, product ion scan and precursor ion scan. On the basis of the summarizedrules, coumarins in a crude extract of Radix Peucedani were characterized bythe combined use of the PREC-IDA-EPI and MIM-IDA-EPI modes on ahybrid triple quadrupole-linear ion trap mass spectrometer for the first time.Based on the simultaneous appearance of [M+NH4]+and [M+Na]+in thespectrum of Prec, the molecular weight could be identified unambiguously.The chromatographic separation was achieved on a C18column with gradientelution of methanol and1mmol/L ammonium acetate.Results: Five fragmentation rules of3′,4′-diacyloxykhellactonecoumarins were summarized:①The distinctive fragmentation pattern of3′,4′-diacyloxykhellactone coumarins was α decomposition of alkoxy bonds oftwo acyloxys on4′-C and3′-C orderly;②The distinctive fragment ion at m/z227could be used to identify the skeleton of khellactone coumarins;③Thecommon substituted groups in this kind of coumarins were acetoxy, isobutylacyloxy, angelica acyloxy, tiglic acyloxy,2-methylbutyroxy, isoamyl acyloxyand senecio acyloxy, the alkoxy bonds on4′-C broke easily, so fragment ionsat m/z287、m/z315、m/z327and m/z329could be used to deduce thesealkoxy groups on3′-C;④Molecular weights of unknown compounds culd beconfirmed by [M+NH4]+and [M+Na]+;⑤The distinctive fragmentationpattern of3′,4′-diacyloxykhellactone coumarins was not the successive loss ofCO which was different from simple coumarins and linear-type furocoumarins.A total of24coumarins in crude extract of Radix Peucedani werecharacterized by combination use of the two scan modes according to thesummarized fragmentation rules, accurate molecular weights andcharacteristic fragment ions.Conclusion: In this study, a high sensitive and effective LC-MS methodfor qualitative analysis of the main active coumarins in Radix Peucedani hasbeen developed. It is demonstrated that the combination use of thePREC-IDA-EPI and MIM-IDA-EPI scan mode could provide more credibleand abundant structure information and has played an important role inTraditional Chinese Medicine quality control. Part two Qualitative and quantitative analysis of14coumarins in RadixPeucedani and adulterants by HPLC-MS coupled with librarysearching technologyObjective: To establish an EPI spectra library of14coumarins and todevelop a novel specific HPLC-ESI-MS method combining with librarysearching technology for qualitative and quantitative analysis of14coumarins in Radix Peucedani and adulterants.Methods: MRM-IDA-EPI scan was employed for spectra collection. Theobtained spectra were used for identification of chromatographic peaks withlibrary searching technology. MRM mode was employed for quantification of14coumarins. The developed method was applied to the evaluation of RadixPeucedani and commercial samples. Liquid Chromatography conditions:Agilent Zorbax Extend-C18(250×4.6mm,5μm) column; Columntemperature:25℃; Mobile phase: acetonitrile (A) and0.1‰formic acid(v/v)-0.2mmol/L ammonium acetate buffer (B); Total run time:36min; Flowrate:0.8mL/min; Injection volume:10μL. Mass spectrometry conditions: Ionsource: electrospray ionization source; Monitoring mode: positive ionmonitoring mode; Ion spray voltage:5500V; Curtain gas:20psi; Ion sourcegas1:60psi; Ion source gas2:65psi; Turbo spray temperature:650℃. MRMmode was used for quantitation.; The precursor-to-product ion pairs of14analytes were404.1/327.1(praeruptorin A),444.1/327.1(praeruptorin B),446.1/327.1(praeruptorin E),409.1/247.1(nodakenin),271.0/203.0(imperatorin),271.0/203.0(isoimperatorin),163.0/107.0(umbelliferon),217.0/202.0(bergapten),217.0/202.0(xanthotoxin),187.0/131.0(psoralen),187.0/131.0(isopsoralen),193.0/133.0(scopoletin),247.0/231.0(pimpinellin),247.0/217.0(isopimpinellin) was employed for quantification; Mass spectrumacquisition mode: MRM-IDA-EPI; EPI scan range:50~500amu; Fixed filltime:30ms; Scan rate:4,000amu/s; CES:35±15eV.Results: The mass spectra library was successfully applied forchromatographic peak identification. The linear relationships, linearity, limitof detection, limit of quantification, precision, accuracy, repeatability and stability were good for14coumarins. The results of sample analysis showedthat only three batches of samples were Radix Peucedani in commercial RadixPeucedani samples and the content of praeruptorin A and praeruptorin B wereconsistent with the requirements in Chinese Pharmacopiea, which conformedto the results of pharmacognosy identification of medicinal materials.Praeruptorin A, praeruptorin B and praeruptorin E were the principalcomponents in Peucedanum praeruptorum and nodakenin was thecharacteristic component in Peucedanum decursivum. The types and contentsof coumarins in other samples were significantly different from those inPeucedanum praeruptorum.Conclusion: The method combining LC-MS and library searchingimproved the specificity of quantitation and accuracy of results. It played animportant role in the quanlity control of Radix Peucedani. The method wasemployed to evaluate35batches of Radix Peucedani and adulterants collectedfrom different resources. It was showed that the content of praeruptorin A,praeruptorin B and praeruptorin E were the key factors for distinguishingRadix Peucedani and adulterants. It was also indicated that the quality ofcommercial Radix Peucedani samples was uneven and the supervision of theTCM market and the quality control of Radix Peucedani should be reinforcedurgently.