Studies On Chemical Composition Analysis And Pharmacokinetics Of Radix Ophiopogonis Based On LC-MS/MS Technology

Ophiopogon japonicus(Chinese name: Maidong) is one of the most frequently used Chinese herbs, whose tubers are considered effective in treating a wide range of disorders, mainly thrombosis,myocardial ischemia, arrhythmias, respiratory disease and hyperglycemia. Modern pharmacological investigations revealed that the physiological and pharmacological activity of Ophiopogon japonicus in hypoxia tolerance, enhancing myocardial contractility, resisting arrhythmia, anticancer, antitumor and hypoglycemic was obvious significant. Previous phytochemical investigations revealed that steroidal saponins and homoisoflavonoids are rich in O. japonicus. Because these two types of compounds have special structures and low content, the study of such components are very limited. Liquid chromatography-mass spectrometry technology(LC-MS) is exclusive, rapid and sensitive, which have a tremendous advantage in qualitative and quantitative analysis on active components of complex TCM system.In this paper, we developed a simple, sensitive and reliable LC-MS/MS method for qualitative and quantitative analysis of Radix Ophiopogonis and Radix Liriopes; validated a UPLC-MS/MS method for studing pharmacokinetic and excretion in rat after oral administration of ophiopogonis extract; and investigated ophiopogonones and their metabolites in rat urine after oral administration of ophiopogonis extract. Part one Identification of components in Radix Ophiopogonis by UPLC-Q-Tof-MS/MSObjective: To develop a novel ultra high performance liquid chromatography coupled with tandem quadrupole time-of-flight mass spectrometry(UPLC-Q-Tof-MS/MS) approach for component analysis of Radix Ophiopogonis.Methods: We investigated the mass fragmentation patterns of 9 standards of Radix Ophiopogonis in the positive and negative ion mode by full scan, product ion scan and precursor ion scan, and then summarized the fragmentation rules. The ethanol extract of Ophiopogon japonicus was analyzed by UPLC-Q-Tof-MS/MS in the scanning mode of Tof-MS-IDA-8MS/MS to acquire the full-scan chromatogram. The data acquisirion and processing analysis were conducted using the "Master View", "Formular Finder" analysis function of Peak View 2.0 data processing software. The chemical constituents of Ophiopogon japonicus were identifid by the comparison of compound retention time, precise molecular mass, elemental composition, isotopic abundance and mass spectrogram. Chromatographic conditions: Kinetex XB-C18 column(100 mm * 2.1 mm i.d. 2.6 m); the column temperature: 25℃; mobile phase acetonitrile(A)-1‰ acetic acid water(B), gradient elution, flowing rate: 0.4 m L/min. Injection volumn: 5 μL.Results : Eleven fragmentation rules were concluded by the mass fragmentation patterns of 9 standards(4 ophiopogonins and 5 ophiopogonones) in the positive and negative ion mode by full scan, product ion scan and precursor ion scan. A total of 30 compounds(12 ophiopogonins and 18 ophiopogonones) in ethanol extract of Radix Ophiopogonis were identified by UPLC-Q-Tof-MS/MS by accurate molecular weights and characteristic fragment ions. Nine of these compounds were character ized by comparing with the standards, and the other 21 compounds were identified by analyzing the information with elemental composition, isotopic abundance and mass spectrogram, the error tolerance is ±5 ppm.Conclusion:The method is simple, rapid, and high sensitivity, which can be used for qualitative analysis of the bioactive components of Radix Ophiopogonis. The method provided a new way to study the pharmacodynamic material basis of Traditional Chinese Medicine. Part two Comparative studies on Ophiopogonis and Liriopes based on the determination of 11 bioactive componentsObjective: To establish a LC-MS/MS method for simultaneous determination of 6 saponins and 5 flavonoids in Ophiopogonis and Liriopes, which was applied for determination the 11 active components from different habitats of Ophiopogonis and Liriopes. Based on these results, Ophiopogonis and Liriopes were compared by the column percentage content of bar chart and cluster analysis.Method: Chromatographic conditions: phenomenex Luna C18(2) column(150 × 2.00 mm, 5μm); the column temperature: 25℃; mobile phase acetonitrile(A) – 0.03% acetic acid water(B), gradient elution,flowing rate: 0.8 m L/min. Injection volumn: 10 μL. The electrospray ionization source was operated between positive and negative modes in a single run by multiple-reaction monitoring(MRM) modes using the following conditions: ion spray voltage, 5500 and-4500 V; the turbo spray temperature, 600 °C; curtain gas(CUR), 1.72×105 Pa and interface heater was turned on; collision gas, medium; nebulizer gas(gas 1) and heater gas(gas 2), 4.14×105 and 4.48×105 Pa. The precursor-to-product ion pairs of 11 analytes were m/z 719.5/393.3(ophiopogon Ra), 851.6/393.3(ophiopojaponin C), 855.8/287.3(ophiopogonin D), m/z 855.8/253.4(ophiopogonin D’), m/z 872.3/413.5(liriope muscari baily saponins C), m/z 723.6/269.3(liriopesides B), 361.2/237.8(ophiopogonanone E), m/z 341.1/178.0(methylophiopogonone A), 327.1/178.0(methylophiopogonone B), m/z 355.0/193.2(ophiopogonanone C), m/z 339.0/217.8(methylophiopogonanone A), respectively.Results: The developed method was performed for analyzing 53 batch of Ophiopogonis and Liriopes from different sources(including the 16 batches of O. japonicus, 19 batches of L.muscari, 9 batches of L. spicata and 9 batches of the mixture of O. japonicus and L. muscari)Conclusion: All calibration curves showed good linearity(r>0.9915) within the test ranges. The intra-day and inter-day precision for 11 analytes were less than 3.7% and 4.2%, respectively. The recoveries were between 95.3%~106.7%. The stability, LOD and LOQ of the method were good for the 11 components. Hierarchical clustering analysis(HCA) and content analysis were performed to differentiate and classify the samples.The results demonstrated that steroidal saponins were themajor components in both Liriopes species,and a low content of flavonoids could become an important symbol of distinction between Ophiopogonis and Liriopes.The developed method could successfully differentiate between Ophiopogonis and Liriopes.Part three Pharmacokinetic investigations of five constituents in rat plasma after oral administration of Radix Ophiopogonis extractObjective: To establish a UPLC-MS/MS method for the simultaneous determination of one ophiopogonins and four ophiopogonones in rat plasma. The method was applied to evaluate pharmacokinetics after oral administration of Radix Ophiopogonis extract in rats.Method: Blood samples were collected from the from the vein of the eye ground at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24 and 36 h after oral administration of Radix Ophiopogonis extract(10m L/kg). Then, the plasma samples was analyzed by protein precipitation with acetonitrile, psoralen as internal standard. Separation condition: Kenetix C18 column(50 mm × 3 mm, 2.6 μm); column temperature : 25 °C; mobile phase acetonitrile(A)-0.03% acetic acid water(B), gradient elution,flowing rate: 0.4 m L/min. Injection volumn: 10 μL. The electrospray ionization source was operated between the positive and negative by MRM modes.Results:The selectivity, sensitivity, linearity, accuracy, precision, and stability were within the acceptable ranges. The method was linear for all analytes over investigated range with all correlation coefficients greater than 0.9931. The method described was successfully applied to reveal the pharmacokinetic properties of ophiopogonin D, ophiopogonanone E, methylophiopogonanone A, methylophiopogonone A, and methylophiopogonone B.Conclusion: A UPLC-MS method was established for determination ophiopogonin D, ophiopogonanone E, methylophiopogonanone A, methylophiopogonone A, and methylophiopogonone B in rat plasma after oral administration of Radix ophiopogonis extract, which was used for the pharmacokinetic study of these components. The validation results of this method were simple, rapid and reliable. Part four Excretion investigations of ophiopogonins and ophiopogonones in rats after oral administration of Radix Ophiopogonis extract.Objective: The aim of this study was to develop a UPLC-MS/MS method for the simultaneous determination of 8 components in urine and 4 ingredients in bile after oral administration of Radix ophiopogonis extract in rats. The method was applied to evaluate uriniary and biliary excretion after oral administration of Radix Ophiopogonis extract in rats.Method: The chromatographic separation was performed using a Kenetix C18 column(50 mm × 3 mm, 2.6 μm); and gradient elution was used,flowing rate: 0.4 m L/min. Injection volumn: 5 μL. Ion source temperature, 600℃. Rats(n=12) were randomly divided into two groups. Both groups of rats were orally administrated at a single dose of 10 m L/kg Radix Ophiopogonis extract. One group was for the bile collection, and bile samples from 0-1h, 1-3h, 3-5h, 5-7h, 7-9h, 9-12 h and 12-24 h were obtained through bile duct cannulas. Another group was for the urine collection, and urine samples from 0-4h, 4-8h, 8-12 h, 12-24 h, 24-36 h, 36-48 h, 48-60 h, 60-72 h and 72-84 h post dosing were obtained. The established method was applied to determination for the accumulative excretion of ophiopogonins and ophiopogonones in rat urine and bile.Results:There were no endogenous interferences to be observed at the retention times of any analytes or the I.S in urine and bile samples. For urine, all calibration curves showed good linearity with correlation coefficients greater than 0.9947; the extraction recoveries of the analytes were greater than 63.2% and the matrix effect values obtained for analytes ranged from 87.3% to 105.7%. For bile, all calibration curves showed good linearity with correlation coefficients greater than 0.9956; the extraction recoveries of the analytes were greater than 68.9% and the matrix effect values obtained for analytes ranged from 84.5% to 103.6%. In bile, low recoveries of the analytes indicated that few unconverted forms were excreted by bile, which might be resulted from liver first pass effect and produced a large number of metabolites.Conclusion: This is the first study to determine 8 active components in urine and 4 active components in blie after oral administration of Radix ophiopogonis extract in rats. The method is enough simple and sensitive to satisfy the requirements of determination of ophiopogonins and ophiopogonones in biological sample, it is important to study further the material basis of Ophiopogon japonicas. Part five Identification of ophiopogonins, ophiopogonones and their metabolites in rats using UPLC-Q-Tof-MS/MS MethodObjective: A UPLC-Q-Tof-MS/MS Method was established to identified ophiopogonins, ophiopogonones and their metabolites in urine, bile, and plasma after oral administration of Radix ophiopogonis extract in rats which was used for exploring the metabolism mode of Radix Ophiopogonis in vivo.Method: Chromatographic conditions: Kinetex XB-C18 column(100 × 2.1 mm, i.d. 2.6μm); the column temperature: 25℃; mobile phase acetonitrile(A)-0.1% acetic acid water(B), gradient elution,flowing rate: 0.4m L/min. Injection volumn: 5 μL. Collect the urine,bile and plasma samples from 1-24 h after oral administration of Radix ophiopogonis extract. The urine samples and control samples were analyzed by UPLC-Q-Tof-MS/MS in the scanning mode of Tof-MS-IDA-8MS/MS to acquire the full-scan chromatogram. The data acquisirion and processing analysis were conducted using Peak View 2.0 and metabolitepilot data processing software. Ophiopogonones and their metabolites were identifid by the comparison of compound retention time, precise molecular mass, elemental composition, isotopic abundance and mass spectrogram.Results : The developed method is applicable to the analysis and identification of ophiopogonins, ophiopogonones and their metabolites in biological matrices after administration of Radix Ophiopogonis. Based on the investigation of the metabolism of ophiopogonins and ophiopogonones, 19 parent compounds with 20 metabolites in rat urine, bile and plasma were detected after oral administration of Radix ophiopogonis extract.Conclusion: In this study, the developed method is simple and efficient, which provides the novel pattern and illumination for the research of material basis of Radix Ophiopogon. The results showed that the main metabolite pathways for ophiopogonones included oxidation, hydrogenation, demethylation and ketone formation. There were not found for the metabolites of ophiopogonins in rat.