| Traditional Chinese medicine(TCM) is the natural therapeutic agent used under the guidance of the theory of traditional Chinese medical science LHQW capsule, approved by the China State Food and Drug Administration(CFDA) in 2004, has been recommended as one of the drugs to treat influenza A(H1N1) by the China health authority in 2009. Clinical studies over more than a decade have convincingly shown that LHQW capsule has anti-inflammatory and immune regulatory functions and may inhibit the airway inflammation that occurs during an acute exacerbation of chronic obstructive pulmonary disease(COPD). A recent meta-analysis on the efficacy of LHQW capsule compared with oseltamivir in treating influenza A virus infection conclude that LHQW capsule was superior to oseltamivir in improving the symptoms of influenza A virus infection. Much progress has been done in the clinical, Pharmacy and pharmacological of LHQW capsule over the years. But the index components study of LHQW capsule in vivo remains unclear.UPLC-MS/Q-TOF –MS is rapid and efficient technique for analysis of complex sample. The present study was implemented on the basis of multicomponent analysis, pharmacokinetic and excretion study, so as to take a view of the main active components and metabolites and to determine the metabolite profile and provide the basis for its pharmacological research. An UPLC-MS-MS/UPLC-Q-TOF-MS method of prescriptions of TCM and metabolites was established, and analysis methods in vivo and in vitro of LHQW multicomponents were improved. The plasma concentration of LHQW component was determined and the relevant pharmacokinetic parameters were calculated. The pharmacokinetic properties in rats were studied to provide the basis for its efficacy material base research. The metabolic profile of phillyrin in rat bile and urine was studied obtained by UPLC-Q-TOF-MS, and a modelof phillyrin metabolism in vitro was established. Metabolic differences in vivo and in vitro and metabolic differences of different species of animals and human were compared so as to clarify the mechanism of phillyrin in vivo and provide a theoretical basis. An ultra-performance liquid chromatography-mass spectrometry method was established to determine eight main active components in rat urine after an oral administration of LHQW capsule and excretion profiles of the main active components in rats were investigated which provide a basis for LHQW development and the safety of clinical drug application.Part one Simultaneous determination of LHQW multicomponent by UPLC-MS-MS methodObjective: Development of an UPLC-MS-MS method to determine the content of 8 active compounds of LHQW capsule.Methods: The dry extract power of LHQW capsule was ground to fine power, and 0.5 g of the power was weighed accurately and set in a conical flask with plug. 100 m L methanol was added and weighted the conical flask with plug and ultrasonically extracted for 30 min. The extracted solution was cooled and methanol was added to make up the weight loss and shake well.Then the aliquot of the supernatant was filtered with a 0.22-μm microporous membrane and sample was determined. The mass spectrometer was operated using an electrospray ionization source and multiple reaction monitoring(MRM). The main ionization settings were optimized as follows: capillary voltage was set at 3.5 k V, desolvation temperature was kept at 550 °C. Parent and daughter ion, cone voltage(CV) and collision energy(CE) for each investigated compounds were all optimized. Quasi-molecular ion [M+H]+ and daughter ion was set with the following transitions: m/z 253.0 ® 225.0 for chrysophanol, m/z 269.0 ® 225.0 for emodin, m/z 283.0 ® 239.0 for rhein,m/z 269.0 ® 240.0 for aloe-emodine, m/z 417.0 ® 255.0 for liquiritin, m/z287.0 ® 153.0 for luteolin, m/z 578.8 ® 370.8 for phillyrin, m/z 299.0 ®119.0 for salidroside, respectively, with a dwell time of 0.163 secs. The analysis was performed on a Waters ACQUITY UPLC® HSS T3 C18(2.1 × 50 mm, 1.8 μm) column, with a mobile phase of acetonitrile(solvent A) and water containing 0.1% formic acid and ammonium formate(v/v, solvent B) at a flow rate of 0.4 m L/min. The column temperature was set at 40 °C. A gradient program was optimized and set as 15% A at 0 min, 15%-85% A at0-2.5 min, 85%-15% A at 2.5-4.5 min, after holding the composition of 15%solvent A for 3 min and the mobile phase composition was returned to its starting conditions for column balance. And the injection volume was 8 μL.Results: Eight compounds showed good linearity among the determination concentration, r2>0.99. And the precisions(RSD) were less than1.73% which showed that the precision of the method is good. The average recovery was 92.8%~101.8% with RSD range from 1.09% to 3.79%. The test solution has good stability in room temperature within 12 h and RSD was less than 2.83%. An analysis cycle was completed in 7.5 min by UPLC-MS-MS method scanning with positive/negative ion-switching.Conclusions: The method was simple, sensitive and specific and can be used for quality control of LHQW capsule.Part two Study on the pharmacokinetic of LHQW capsule in rat by UPLC-MS/MS methodObjective: Development of an UPLC-MS-MS method to determine rhein, chrysophanol, phillyrin and salidroside of LHQW. The plasma concentration was determined and mean plasma concentration-time profiles of4 analytes was established after the intragastric administration of LHQW capsule to rats. The relevant pharmacokinetic parameters were calculated and pharmacokinetic properties in rats were clarified.Methods: Blood samples were collected from the orbital vein into heparinized centrifuge tubes at 15, 30, 60, 90, 120, 150, 180, 210, 240, 270,300, 360, 420, 480 and 600 min after the intragastric administration of LHQW capsule to male SD rats. Blood samples were immediately centrifuged at 3500 rpm for 10 min and then the supernatant were stored at-80 °C until analysis. The samples were pretreated and extracted by liquid-liquid extraction prior to UPLC-MS/MS. A 400 μL aliquot of rat plasma samples spiked with 5 μL of IS solutions(5 μg/ml chloramphenicol methanol solution) were taken. After vortex-mixing, 10μL HCl(1mo L/L) was added and all samples were extracted with 1.5m L ethyl acetate vortex-mixed thoroughly for 2.0 min and centrifuged at 16,000 rpm for 10 min to remove the protein. The supernatants were concentrated to dryness under N2. Dried samples were dissolved with 100μL methanol vortex-mixed 1.0 min and then was centrifuged at 16000 rpm for 10 min. Follow that, the solution were filtered by a 0.22-μm filter membrane before UPLC/MS/MS analysis. The analysis was performed on a Waters ACQUITY UPLC® HSS T3 C18(2.1 × 50 mm, 1.8 μm) column, with a mobile phase of acetonitrile(solvent A) and water containing 0.1% formic acid and ammonium formate(v/v, solvent B). A gradient program was optimized and set as 15% A at 0 min, 15%-85% A at 0-2.5 min, 85%-15% A at2.5-4.5 min. The flow rate was 0.4 m L/min. The column temperature was set at 40 °C. The mass spectrometer was operated using an electrospray ionization source and negative ion(NI) detection mode was used for detection. The capillary voltage was set at 3.5 k V, desolvation temperature was kept at550 °C. The response ratios(peak areas of rhein, chloramphenicol, phillyrin and salidroside to IS) were determined and the concentrations were calculated by calibration curves. The plasma concentration-time curves were constructed by plotting the concentration of each analyte vs time. The pharmacokinetic parameters were analyzed by DAS3.0 software.Results: The main pharmacokinetic parameters of rhein, chrysophanol,phillyrin and salidroside after intragastric administration of LHQW capsule were as follows: Cmax 1.813μg/m L, 0.336μg/m L, 0.622μg/m L and0.579μg/m L;Tmax 1.6 h, 2.3 h, 1.0 h and 2.4h;t1/2 2.213 h, 3.165 h, 1.512 h and4.13h; AUC0-t 4.36μg/m L·h2, 1.54μg/m L·h2, 1.106μg/m L·h2 and3.229μg/m L·h2;AUC0-∞ 4.843μg/m L·h2, 1.74μg/m L·h2, 1.202μg/m L·h2 and4.313μg/m L·h2;MRT 3.152 h, 3.37 h, 1.574 h and 4.233 h.Conclusions: The proposed method was successfully applied to pharmacokinetic evaluation of rhein, chrysophanol, phillyrin and salidroside in rat after oral administration of LHQW capsule. The exploration of action material foundation in vivo will build a foundation for further study of the pharmacological mechanisms of LHQW.Part three Metabolic profile of phillyrin in rats obtained by UPLC-Q-TOF-MSObjective: Development of an UPLC-Q-TOF-MS method for identification the structure of phillyrin and its metabolites and to determine the metabolite profile of phillyrin.Methods: After orally administration, all samples were collected over0-4 h, 4-8 h, 8-12 h, 12-24 h periods respectively. The bile, urine and feces samples were extracted with ethyl acetate. The chromatography separations were performed on a C18 reversed phase LC column(Phenomenex Kinetex C18100×2.1mm, 2.6μm), and the column temperature was maintained at 50 °C.The mobile phase consisted of water-formic acid(100:0.1, v/v)(A) and methanol(B) at the flow rate of 400 μL/min. A gradient program was adopted,specific as 5% B at 0-1 min, 5%-50% B at 1-10 min, 50%-70% B at 10-11 min,70%-100% B at 11-15 min, 100%-5% at 15-15.5 min, and 5% B at 15.5-18 min. A Triple TOF? 5600 system with a Duo Spray TM source operating in both positive ion(PI) and negative ion(NI) detection mode was used for detection. The following parameter settings were used: ion spray voltage floating,(+) 5.5 k V,(-)4.5 k V; ion source heater, 550 °C; curtain gas, 25 psi;ion source gas 1, 55 psi; and ion source gas 2, 55 psi. The full MS experiments were run with a scan range from m/z 100 to m/z 1200, and the MS/MS experiments were run with a scan range from m/z 50 to m/z 1200, and a250-ms accumulation time for the full MS and a 100-ms accumulation time for MS/MS experiments. The CE was(+) 45 e V;(-) 45 e V; the CES was 15 e V; and DP was(+) 60 e V;(-) 60 e V in the MS/MS experiment. The IDA was used to trigger the acquisition of MS/MS spectra for ions matching the IDA criteria. A real-time MMDF and DBS were used to fulfill the IDA criteria and metabolites were identified by Metabolite Pilot TM metabolite software.Results : In this study, an UPLC-Q-TOF-MS method was applied to identify the structure of phillyrin and its metabolites and to determine the metabolite profile of phillyrin. A total of 34 metabolites, including 30 phase I and 4 phase II metabolites, were detected and tentatively identified in rat bile,urine and feces after orally administration of phillyrin, and 28 new metabolites were reported for the first time. UPLC-Q-TOF-MS analysis of the phillyrin samples showed that phillyrin was converted to a major metabolite, M26,which underwent deglucosidation, further dehydration and desaturation.Conclusions: This study is the first to explore the metabolic profile of phillyrin in rats by method. The main biotransformation route of phillyrin was identified as hydrolysis, oxidation, and sulfation. The glucosyl group, the methoxy group and tetrahydrofuran ring were the metabolic soft spots. These findings enhance our understanding of the metabolism and effective forms of phillyrin.Part four Metabolism of phillyrin in liver microsomes and study of interspecies differencesObjective: Phillyrin metabolism in liver microsomes obtained from different species(rat and human) was studied in vitro and compared using UPLC-Q-TOF-MS. Species-specific metabolic profiles of phillyrin were obtained by analyzing the reaction mixtures and the metabolism in vivo and in vitro were also compared.Methods: Phillyrin was incubated with rat and human liver microsomes along with NADPH(containing β-NADP, G-6-P, G-6-PD and Mg Cl2),K2HPO4 buffer solution(0.1 mo L/L, p H 7.4), liver microsome proteins(1mg/m L) to a final volume of 1 m L to evaluate the interspecies differences in phillyrin metabolism. Phillyrin was incubated with rat and human liver microsomes. The substrate concentration was 25 μg/m L and the final solution contained 0.5% methanol. Bake in a preheated 37℃ for 5 min, NADPH redox system was added to the hepatocyte incubation solutions to initiate the response at the adsorptiongal condition of 37℃(150 r/min, 1h). The reaction system was taken out and ended immediately in-20 ℃ refrigerator and parallel determination was done 3 times. Liquid-liquid extraction was used as sample preparation technique. Samples were extracted with ethyl acetate(twice amount of the samples v/v for 2 times). The supernatants were combined and concentrated to dryness under reduced pressure at 40 °C using a Heidolph Laborota 4001 rotatory evaporator(Heidolph Instruments Gmb H &Co., Schwabach, Germany). Dried samples were dissolved with 200 μL methanol in an ultrasonic bath for 5 min and then was centrifuged at 14000 rpm for 10 min and filtered through a 0.22-μm GHP filter(Mfd. For Waters Corporation, USA). Afterward, the supernatant was transferred to a clean polypropylene tube. Sample analysis were performed by UPLC-Q-TOF-MS.Combining with metabolites identified in vivo in our previous studies,metabolites were elucidated by comparing retention time and MS/MS fragment ions.Results: After comparing with the blank sample,16 phase Ⅰmetabolites were detected in rat and human liver microsomes. Among of them 6metabolites were consistent with the metabolites detected in rat bile. The others were new metabolites in vivo. The main biotransformation route of phillyrin in vitro was identified as hydrolysis, oxidation, and demethylation.Similarity index(SI=0.952) indicated that phillyrin metabolism in rat was highly analogous to that in humans. These results suggest that UPLC-Q-TOF-MS-based multivariate analytical approaches are useful for the evaluation of interspecies differences in the metabolism of phillyrin.Conclusions: The metabolism study in vitro indicted that the phase Ⅰmetabolites of phillyrin was similar in the liver microsomes for the two species. The results indicate that no species difference exists among rats and humans. This study provided the most comprehensive picture for phillyrin in vivo and in vitro metabolism till now, and may predict the metabolism in humans.Part five Excretion study of the main active components in rats after an oral administration of Lianhuaqingwen capsule by ultra-performance liquid chromatography mass spectrometryObjective: A rapid and sensitive ultra-high performance liquid chromatography-mass spectrometry(UPLC-MS/MS) method was developed for quantification of chrysophanol, emodin, rhein, aloe-emodine, liquiritin,luteolin, phillyrin, and salidroside in rat bile and urine after oral administration of LHQW capsule. The excretion profiles of the main active components in rats were investigated.Methods: SD rats were anesthetized with 2% pentobarbital sodium salt to achieve biliary cannulation with standard silicone tubing for the collection of bile samples, and administration was carried out after five animals fully recovered from the surgery. Bile samples were collected during 0-2, 2-4, 4-6,6-8, 8-10, 10-24, 24-36 h periods. After oral administration, urine samples were obtained during 0-2, 2-4, 4-6, 6-8, 8-10, 10-12, 12-36 h periods from five animals. The samples were pretreated and extracted by a single protein precipitation prior to UPLC-MS/MS. Chromatographic separation was achieved on a Waters ACQUITY UPLC® HSS T3 C18(2.1 × 50 mm, 1.8 μm)column using a gradient elution program with 0.1% formic acid aqueous solution and acetonitrile at a flow rate of 0.4 m L/min. The column temperature was set at 40 °C. The mass spectrometer was operated using an electrospray ionization source and multiple reaction monitoring(MRM) scanning with positive/negative ion-switching.Results: Detection and quantitation were accomplished by a hybrid quadrupole mass spectrometer using electrospray ionization source and MRM scanning with positive/negative ion-switching. The mass transition ion-pairs(m/z) for quantitation were all optimized and the total run time was 7.5 min.Full validation of the assay was implemented and this method demonstrated good linearity and specificity. The validation results indicated that this method was simple, rapid, specific and reliable. Eight compounds were detected in rat urine and five compounds were detected in rat bile after the intragastric administration of LHQW capsule. Eight compounds attained the maximum in12-h cumulative urinary excretion and tended to be constant after that. The urine excretion rate of rhein, chrysophanol, aloe-emodine were 78.0%, 43.9%and 37.0%, respectively. The results confirmed that urine is the main elimination pathway. The urine excretion rate of phillyrin, liquiritin, emodin,luteolin and salidroside were very low, which revealed that there may exit other elimination way or extensive metabolism. The bile excretion rate of rhein, chrysophanol, emodin, phillyrin and salidroside were lower than4.25%。Conclusions: The proposed method was successfully applied to investigate the bile and urinary excretion study of eight compounds in rat after oral administration of LHQW capsule.The exploration of action material foundation in vivo will build a foundation for further study of the pharmacological mechanisms of LHQW. |
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