Qualitative Analysis Of Isomeric Furanocoumarins In The Complex Matrix Of TCM With HPLC-MS Technology And Pharmacokinetic Studies Of Flavonoids From Scutellaria Barbata

The high performance liquid chromatography-mass spectrometry (HPLC-MS) technology which possesses the functions of good chromatographic separation and exact structure characterization has been accepted to be the powerful tool for the analysis of the compounds in traditional Chinese medicines (TCMs). Furanocoumarins are active biological components in many TCMs. They have attracted great interest in the world due to their various biological and pharmacological effects, such as the anti-virus, anti-cancer and anti-bacterial, especially the anti-HIV effect. Nevertheless, furanocoumarins have multiple isomers with the same molecular weight, which leads to a serious challenge for HPLC-MS method since it mainly depends on the different molecular weight to label the coupounds. In the present study, the mass fragmentation patterns and rules of the isomeric furanocoumarins were systematically studied firstly. Then isomeric furanocoumarins in5herbal medicines including Radix Glehniae, Angelica dahurica, Radix Angelicae Pubescentis, Radix Peucedani and Fructus Psoraleae were characterized and a feasible HPLC-MS methodology for analyzing components in complex TCM system was developed.Scutellaria Barbata is derived from the dried whole plant of Scutellaria barbata (Labiatae). It is one of the most commonly used TCMs and is used to treat urinary, ophthalmic, respiratory and digestion system disorders in China. Flavonoids are the main and effective components which are the most contributing to the pharmacological efficacy of Scutellaria Barbata. In the present study, a new HPLC-MS method was developed and validated for the determination of five flavonoids (the free and the conjugated) including scutellarin, naringenin, apigenin, luteoline and wogonin in rat plasma, and then was applied to reseach their pharmacokinetic characteristics.Part one Study on the mass fragmentation rules of isomeric furanocoumarinsObjective: To study the fragmentation pathways and differences of isomeric furanocoumarins by using the soft ionization technologies (ESI and APCI), and to develop a stable and fast MS method for identification of the isomeric furanocoumarins.Methods:The reference compounds including psoralen, isopsoralen, xanthotoxin, bergapten, impinellin, isoimpinellin, imperatorin and isoimperatorin were dissolved in methanol and then diluted with methanol-water (1:1, v/v) to a concentration of1μg/mL, respectively. The solutions were introduced via sringe pump at a flow rate of10μL/min. Combined using of multiple ions scan, precusor scan and netural loss scan in the ESI and APCI were performed. The main fragmentation ions were collected in the different collsion energies and the acquisition time were2mm.Results:1.7MS fragmentation pathways for differentiation of the isomeric furanocoumarins were summarized. The ratio of the relative abundance method was introduced. The ratios of relative abundance of typical ion m/z203to m/z185and [M+NH4]+to [M+Na]+could be used to characterize the position of the subsitituted group in the skeleton structures.2. A new rule named the ratio of the relative abundance to differentiate the isomeric furanocoumarins was developed. The formula is R=M/N, M and N present the value of each furanocoumarin's ratio of the relative abundance, respectively. For the mono-furanocoumarins, if R was more than1, the oxysubsituent group was at C-5, on the contrary, was at C-8. For the double-furanocoumarins, if the value of R was more than1, the more active oxysubsituent group was at C-8, on the contrary, was at C-5.Conclusion:It was the first time to use the method of the ratio of the relative abundance to differentiate the isomeric furanocoumarins. The7MS fragmentation pathways were stable and reliable and were applied to characterize the isomeric furanocoumatins quickly.Part two Development of the HPLC-EPI method and its application to quantitative identification of TCM componetsObjective: To develop a stable, reproduceable and specific mass spectrimetric information acquired method for the detection and characterization of chemical compounds in the complex system of traditional Chinese medicine.Methods:The reference compounds including psoralen, isopsoralen, xanthotoxin, bergapten, impinellin, isoimpinellin, imperatorin and isoimperatorin were dissolved in methanol and then diluted with methanol-water (1:1, v/v) to a concentration of1ug/mL, respectively.50%methanol extractions of the5traditional Chinese medicines were acquired respectively by ultrasonic extracting and then were filtered. The filtrate was evaporated to dryness and the residue was dissolved in water-methanol (1:1, v/v). The supernatant was injected into the HPLC-MS instrument after high speed centrifugaion. The chromatographic separation was performed on a C18column with1mmol/L ammonium acetate and methanol as the mobile phase. The detection was performed with ESI source in positive mode and enhanced product ion (EPI) scan mode were used. The fixed line ion fill time was30ms and Qo was in the condition of trapping. Different collision energies including25,30and40eV were used. The information of the relative abundance of the main fragmentation ions for each analyte was used to study the precision and stability of HPLC-EPI method. These informations obtained from HPLC-EPI, HPLC-MRM-IDA-EPI and HPLC-PREC-IDA-EPI scan modes were also compared. The STDEV analysis with SPSS16.0were used for data processing.Results:In the HPLC-EPI scan mode, the RSDs of the precision and stability of each fragmentation ion in the analytes were all less than10%which indicated that the mass information was stable. The STDEV for each fragmentation ion in the HPLC-MRM-IDA-EPI and HPLC-PREC-IDA-EPI scan modes were all between0.0-13.7%, while in HPLC-EPI scan mode was between0.0-2.3%. It was demonstrated that the acquisition of the mass information in HPLC-EPI scan mode was more stable than in HPLC-MRM-IDA-EPI and HPLC-PREC-IDA-EPI scan modes.Conclusion:The developed method was stable, reproducible and specific and could be applied for the detection and characterization of the chemical compounds in complex systems of TCMs.Part three Characterization of isomeric furanocoumarins in five traditional Chinese medicines by HPLC-MS techniqueObjective: To recognize and identify the isomeric furanocoumarins in Radix Glehniae, Angelica dahurica, Radix Peucedani, Fructus Psoraleae and Radix Angelicae Pubescentis.Methods:50%methanol extractions of the5traditional Chinese medicines were acquired respectively by ultrasonic extracting and then filtered. The filtrate was evaporated to dryness and the residue was dissolved in water-methanol (1:1, v/v). The supernatant was injected into the HPLC-MS instrument after high speed centrifugation. The isomeric furanocoumarins in five herbals were then characterized and differentiated by the combined use of HPLC-EPI, HPLC-MRM-IDA-EPI and HPLC-PREC-IDA-EPI modes on a hybrid triple quardruple-linear ion trap mass spectrometer. Based on the simultaneous appearance of [M+H]+and [M+NH4]+in the spectra of PREC mode, the molecular weight of compound could be identified. Based on the generous mass fragmentation ions of MRM mode, the skeleton structures of the compounds could be characterized. According to the stable, reproducible relative abundance of the main ions of the HPLC-EPI mode, the isomeric furanocoumarins could be differentiated. The chromatographic separation was performed on a C18column with1mmol/L ammonium acetate and methanol as the mobile phase.Results:Total18pairs of isomeric furanocoumarins were identified in the5traditional Chinese medicines. There were9pairs,12pairs,8pairs,7pairs and5pairs in Radix Glehniae, Angelica dahurica, Radix Peucedani, Radix Angelicae Pubescentis and Fructus Psoraleae, respectively. Conclusion: It is the first time to combine the HPLC-EPI, HPLC-MRM-IDA-EPI and HPLC-PREC-IDA-EPI scan modes for the characterization and differentiation the isomeric furancoumarins in Radix Glehniae, Angelica dahurica, Radix Peucedani, Fructus Psoraleae and Radix Angelicae Pubescentis. It is an important meaning to differentiate the isomeric furanocoumarins in the complex systems of the TCMs. It also supplied an idea for the characterization of other isomeric compounds from the complex matrix.Part four Pharmacokinetic studies of five flavonoids from Scutellaria BarbataObjective: To develop a HPLC-MS method for the quantification of5flavonoids (scutellarin, naringenin, apigenin, luteoline and wogonin) in rat plasma and to study the pharmacokinetics of these flavonoids after oral administration of Scutellaria Barbata extract to rats.Methods:Blood samples of approximately0.3mL were collected from the vein of the eye ground at0.08,0.5,4,5,6,8,12,24,36,48h after a single oral administration of Scutellaria Barbata extract (8mL/kg). Plasma was obtained by centrifugation and duplicate50μL plasma samples were dispensed to heparinized tubes and stored at-20℃until use. Plasma samples were pretreated by liquid-liquid extraction procedure with ethyl acetate and acid hydrolysis method was used for converting conjugated flavonoids to their respective free forms. The chromatographic separation was performed on a C18column with a linear gradient elution using a mobile phase consisted of0.01%acetic acid and methanol (35:65,v/v). The total run time was12.5min between injections. The internal standard (IS) was sulfamethalazole. The detection was accomplished by MRM scan with ESI source operating in the negative ionization mode. The optimized mass transition ion-pairs (m/z) monitored for scutellarin, naringenin, apigenin, luteoline, wogonin and IS were461.1/285.1,271.0/119.0,269.0/117.0,285.0/132.9,283.0/268.0and252.0/155.9, respectivelyResults:The calibration curves were linear over the investigated concentration range with all correlation coefficients higher than0.9917. The lower limit of quantitation (LLOQ) of scutellarin was9.15ng/mL and other compounds were all less than2.0ng/mL. The intra-and inter-day RSD were no more than14.8%and the relative errors (RE) were within the range of-12.4to14.0%. The extraction recoveriesand matrix effects for all compounds were between53.8-84.2%and81.9-103.3%, respectively. The free five analytes could achieve the maximum plasma concentration between5.75-8.25h after oral administration. The t1/2was between4.95-9.56h. The values of the K ranged from0.001to0.0024, which indicated that the free five analytes had slow elimination rates. A double-peak phenomenon was showed in the profiles of scutellarin. And other free four flavonoids had parallel plasma concentration-time profiles and pharmacokinetic parameters in vivo. It was demonstrated that the flavonoids aglycone had the similar oral administration and absorption from the Scutellaria Barbata extract. It also revealed that the AUCoâ†'∞of the four flavonoids conjugated metabolites constituted between54.5-81.3%of the total AUCoâ†'∞. The Cmax of the conjugates metabolites were also approximately twice more than their respective free form. These parameters clearly demonstrated that the amounts of the4compounds (especially the apigenin) could be extensively converted to its conjugated forms and undergo rapid first-pass effect by the gut and liver∞after oral administration.Conclusion:A sensitive and specific HPLC-MS method was developed for the simultaneous analysis of five flavonoids in rat plasma for the first time. The analytical procedure was successfully applied to pharmacokinetic study of the free and conjugated forms of the analytes after oral administration of Scutellaria Barbata extract. The results would be helpful to study the action mechanism of major flavonoids in Scutellaria Barbata and provided a useful tool for pharmacokinetic investigation of TCM.