| Drug ingested by organism must reach sufficient concentration in the site of function to produce its characteristic pharmacological effects. While the drug concentration in the function site not only influnced by the administration dose, but also effected by the physiological disposition — absorption,distribution, metabolism and excretion. Thus, the physiological disposition is vital for drug study.Peucedani radix, namely Qianhu in China, is the dried root of Peucedanum praeruptorum Dunn. It is a well-known traditional Chinese medicinal herb and has been widely used for treating common cold, cough,asthma and dyspnea. Courmarins were the major components in Peucedani radix and are divided into simple courmarin, furan courmarin and pyran courmarin. In the current study for Peucedani radix, most focused on the study in vitro, such as the identification and quantitation of the chemical components.In the physiological disposition study for Peucedani radix, most studies focused on pyran courmarin, such as dl-Praeruptorin A(PA),(+)-praeruptorin A(d PA)-and its chiral enantiomer(-)-praeruptorin A(l PA), but there was little knowledge about simple and furan coumarin studies in Peucedani radix.In our study, liquid chromatography–tandem mass spectrometry(HPLC–MS) was adopted to identify the metabolites and investigate the absorption and excretion process in rats after oral administration of Peucedani radix water extract and clarify the physiological disposition and pharmacological actions.Part Ⅰ Simultaneous determination of 5 courmarins in rat plasma and pharmacokinetic study after oral administration of Peucedani Radix aqueous extract in ratsObjective: To establish a HPLC-MS method for the simultaneous determination of praeruptorin A, praeruptorin B, praeruptorin E, scopoletin,xanthotoxin in rat plasma and the pharmacokinetic study of the 5 components after oral administration of Peucedani Radix aqueous extract in rats.Methods: 1 Plasma preparation: Liquid-liquid extraction method and protein precipitation method were compared for the sample preparation.Owing to that the polarity of the components differs largely, it is hard to find a proper extraction solvent. Thus, protein precipitation with acetonitrile was selected for plasma preparation. 2 Liquid Chromatography conditions:Welchrom C18 analytical column(150 mm × 4.6 mm, 5 μm, USA) column;Mobile phase: 1 mmol/L ammonium acetate buffer(A) and methanol(B);Total run time: 13.5 min; Flow rate: 1.0 m L/min; Injection volume: 10 μL.Mass spectrometry conditions: Ion source: electrospray ionization source;Monitoring mode: positive ion monitoring mode; MRM mode was used for quantitation.; The precursor-to-product ion pairs of 5 analytes were praeruptorin A m/z 404.2'327.1, praeruptorin B for m/z 444.2'327.1,praeruptorin E for m/z 446.3'327.1, scopoletin for 193.0'133.0,xanthotoxin for m/z 217.0'202.0, nodakenin(internal standard) for m/z409.1'247.1. 3 Method validation: Linerity, range, LLOQ, precision,accuracy, stability, extract recovery, matrix effect, carry-over were all performed for the method validation. 4 Pharmacokinetic study:(1)Peucedani Radix water extract preparation: Two hundreds grams of Peucedani Radix were cut into pieces and then extracted three times by decocting with boiling water(1:10) for 1h per time. The extraction solutions were combined and concentrated to 100 m L. The final concentration of Peucedani Radix extract was 2g/m L to Peucedani Radix.(2) Plasma collection and analysis: The Peucedani Radix water extract were orally administrated to the rats at a dose of 10g/kg. Blood samples were obtained at the rat’s medial angle of eye and then put in 1.5 m L heparinized tube. The blood samples were collected at0.083, 0.167, 0.333, 0.667, 1, 2, 4, 6, 8, 12, 24 h and then centrifuged 4500 rpm for 5 min at room temperature to obtain layer plasma. The plasma samples were injected into HPLC-MS for analysis and illustrate the mean plasma concentration-time profiles of the components.(3) Non-compartmental model was used for the calculation of pharmacokinetic parameters: Cmax, Tmax,t1/2, Ke, AUC0-t, AUC0-∞.Results: 1 Method validation: The correlation coefficients(r) of calibration curves were all higher than 0.9915, which exhibited good linearity.The LLOQs of the coumarins were lower than 2.763 ng/m L, which presented the method was sensitive. The RSDs of intra-day and inter-day precisions were lower than 6.0%, while the accuracy were in the range of-4.2%-3.7%.The extract recoveries of the coumarins were in the range of 72.5%-98.0%,and the matrix effect were in the range of 78.1%-98.6 %. The REs value of stability were lower than 5.3%. The peak area of the analytes obtained from the blank sample injected after the upper limit of quantitation(ULOQ) sample were lower than that of 20% of the average of LLOQ samples, which showed that the carry-over would not influence the detection. 2 Pharmacokinetic study:Tmax of 5 analytes were 0.333 h, which showed that 5 analytes absorbed quickly.While t1/2 were 7.65 h, 8.36 h, 16.70 h, 12.53 h and 10.03 h,which exhibited the elimination was slow. The slow elimination phenomenon of praeruptorin A was consistent with the conclusion in previous literatures. A double-peak phenomenon was presented in concentration-time profiles of praeruptorin B and xanthotoxin, and the second peak appeared at 4 h and 2h separately. The double-peak phenomenon for praeruptorin B was caused by reabsorption. The double-peak phenomenon for xanthotoxin was much higher than the first one was the courmarins were metabolized to xanthotoxin to produce the second large peak.Conclusion: A sensitive, specific and reproducible HPLC-MS method was developed for the simultaneous determination of 5 courmarins in rat plasma and pharmacokinetic study after oral administration of Peucedani Radix water-extract in rats. The results showed that 5 components in rats plasma absorbed quickly and eliminated slowly. The pharmacokinetic studies for scopoletin and xanthotoxin were investigated for the first time in rats after oral administration of Peucedani Radix water extract.Part Ⅱ Simultaneous determination of courmarins in rat bile and urine and excretion study after oral administration of Peucedani Radix aqueous extract in ratsObjective: To establish a HPLC-MS method for the simultaneous determination of praeruptorin A, praeruptorin B, praeruptorin E, xanthotoxin,bergapten in rat bile and praeruptorin A, umbelliferon, scopoletin, xanthotoxin,bergapten, isopsoralen, isoimperatorin in rat urine and the excretion study after oral administration of Peucedani Radix aqueous extract in rats.Methods: 1 Bile and urine sample pretreatment: Protein pretatation with acetonitrile method was used for sample preparation. 2 Liquid chromatography conditions: Welchrom C18 analytical column(150 mm × 4.6mm, 5 μm, USA) column; Mobile phase: 1 mmol/L ammonium acetate buffer(A) and methanol(B); Total run time: 13.5 min; Flow rate: 1.0 m L/min;Injection volume: 10 μL. Mass spectrometry conditions: Ion source:electrospray ionization source; Monitoring mode: positive ion monitoring mode; MRM mode was used for quantitation. The precursor-to-product ion pairs of the analytes were praeruptorin A m/z 404.2'327.1, praeruptorin B for m/z 444.2'327.1, praeruptorin E for m/z 446.3'327.1, umbelliferon for m/z163.0'107.1, scopoletin for 193.0'133.0, xanthotoxin for m/z 217.0'202.0,bergapten for m/z 217.0'202.0, isopsoralen for m/z 217.0'202.0, and isoimperatorin for m/z 271.0'203.0, and nodakenin(internal standard) for m/z 409.1'247.1. 3 Method validation: Linerity, range, LLOQ, precision,accuracy, stability, extract recovery, matrix effect and carry-over were all performed for the method validation. 4 Excretion study:(1) Bile collection:six rats were anesthetized and a cannula was implanted into the bile duct to collect bile. The Peucedani Radix water extract at a single dose of 10 g/kg was oral administered. Bile samples were collected at 0-2, 2-4, 4-6, 6-8, 8-12,12-24, 24-36, 36-48, 48-60 and 60-72 h post-dosing and stored at-80℃ after the volume of each collection was recorded. Urine collection: The Peucedani Radix water extract at a single dose of 10 g/kg was oral administered to rats. The rats were housed in stainless-steel metabolic cages with free access to water and food was returned approximately 4 h post-dosing. Urine were collected at 0-4, 4-8, 8-12, 12-24, 24-36, 36-48, 48-60 and 60-72 h post-dosing and stored at-80℃ after the volume of each collection was recorded. The concentrations of coumarins in urine and bile were determined by the established HPLC-MS method.(2) The accumulation amount of coumarins excreted in rat urine and bile were depicted. The accumulation excretion profile was constructed by plotting the accumulation excretion amount(y) of each analyte versus time(x). The accumulation excretion percent was the ratio of accumulation excretion amount and administration dose.Results: 1 Method validation: The correlation coefficients(r) of calibration curves were all higher than 0.9907, which exhibited good linearity.The LLOQs of the coumarins were lower than 2.763 ng/ml, which presented the method was sensitive. The RSDs of intra-day and inter-day precisions were lower than 13.7%, while the accuracy were in the range of-11.3%-14.0%. The extract recoveries of the coumarins were in the range of72.5%-98.0%, and the matrix effect were in the range of 82.1%-101.7 %. The REs value of stability were lower than 15.5%. The peak area of the analytes obtained from the blank sample injected after the upper limit of quantitation(ULOQ) samples were lower than that of 20% of the average of LLOQ samples, which showed that the carry-over would not influence the detection.2 Excretion study: The accumulation excretion percent of the components in rat bile: praeruptorin A for 0.004%, praeruptorin B for 0.011%, praeruptorin E for 0.0003%, xanthotoxin for 0.87%, bergapten for 0.169%. The accumulation excretion percent of the components in rat urine: praeruptorin A for 0.0001%,umbelliferon for 117.4%, scopoletin for 122.3%, xanthotoxin for 0.362%,bergapten for 0.193%, isopsoralen for 0.033%, isoimperatorin for 0.045%.The results showed that the accumulation excretion percent of scopoletin and umbelliferon exceeded 100%, which meant that some courmarins might be metabolized to them. Few ingredients in prototype form were excreted in urine and bile, which indicated that they were metabolized or excreted in other pathway.Conclusion: A sensitive, specific and reproducible HPLC-MS method was developed for the simultaneous determination 5 courmarins in rat bile, 7courmarins in rat urine and excretion study after oral administration of Peucedani Radix water extract in rats. Few ingredients in prototype form were excreted in urine and bile, which indicated that they were metabolized or excreted in other pathway.Part Ⅲ Metabolite identification for Peucedani radix in ratsObjective: To establish a high performance liquid chromatography with quadruple time-of-flight mass spectrometry(HPLC-Q-TOF-MS) method for the metabolites identification in rat plasma, bile and urine after oral administration Peucedani radix extract, and to investigate the metabolic pathway.Methods: 1 The fragmentation rules of 3′, 4′-diacyloxykhellactone pyran coumarin, simple courmarins and furan courmarins were summed up according to literatures and previous study results in our laboratory. 2Biological samples were analyzed by HPLC-Q-TOF-MS in the scanning mode of TOF-MS-IDA-8MS/MS. Then, the combination use of data post-processing softwares Metabolitepilot 1.5 and Peak View2.0 including “Fragment and neutral loss defect filter” function were chosen to find metabolites.Furthermore, “Master View”function in Peak View2.0 was used to obtain the accurate molecular weight, formula, spectra to identify the metabolites and speculate metabolic pathway. The compounds which have standards were speculated by comparing the retention times and fragment ions with standards.The analytes which have no standards were speculated by analyzing the fragment ions. The metabolites were compared with Chemspider database to investigate they were reported or not.Results: A total of 16 compounds(10 prototypes, 6 metabolites) in plasma, 19 compounds(12 prototypes, 7 metabolites) in bile, 30 compounds(15 prototypes, 15 metabolites) in urine were detected by HPLC-Q-TOF-MS according to the fragmentation rules and the metabolic pathway of the metabolites were analyzed.Conclusion: 1 A total of 30 compounds were detected in which 6 new metabolites were not reported. 2 Hydrolysis, dehydration after hydrolysis and oxidation were metabolic pathway for 3′, 4′-diacyloxykhellactone pyran coumarin in which hydrolysis was the major metabolic pathway. But oxidation and hydrogenation were the main metabolic pathway for simple courmarin and furan courmarins. Ⅱ phase metabolites were not found for all the courmarins. 3 HPLC-Q-TOF-MS method was a sensitive, specific, accurate,simple and practicable method for the metabolite identification. |
PDF Full Text Request