RESUMO
OBJECTIVE： To establish a method for the determination of piperitylmagnolol in the incubation system of liver microsomes， and to investigate the metabolic characteristics of it in different species of liver microsomes. METHODS： The piperitylmagnolol were respectively dissolved in NADPH activated liver microsome incubation systems of human， rat， mouse， monkey and dog， and then incubated in water at 37 ℃. The reaction was terminated with methanol at 0， 2， 5， 10， 15， 20， 30， 45 and 60 minutes of incubation， respectively. Using magnolol as internal standard， UPLC-MS/MS method was used to determine the concentration of piperitylmagnolol in the incubation system. The determination was performed on Acquity UPLCTM CSH C18 column with mobile phase consisted of 0.1％ formic acid-methanol （gradient elution） at the flow rate of 0.3 mL/min. The column temperature was set at 30 ℃， and the sample size was 2 μL. The ion source was electrospray ion source， and the positive ion scanning was carried out in the multiple reaction monitoring mode. The ion pairs used for quantitative analysis were m/z 401.2→331.1 （piperitylmagnolol） and m/z 265.1→247.0 （internal standard）， respectively. Using the concentration of piperitylmagnolol at 0 min of incubation as a reference， the residual percentage， metabolism half-life in vitro （t1/2） and intrinsic clearance （CLint） were calculated for different incubation systems. The metabolic pathway of piperitylmagnolol was studied by chemical inhibitor method. Under the above chromatographic conditions， the metabolites in vitro were preliminarily analyzed by first-order full scanning and positive ion detection. RESULTS： The linear range of piperitylmagnolol was 3.91-500.00 ng/mL. The limit of quantitation was 3.91 ng/mL. RSDs of intra-day and inter-day were less than 10％. The accuracy ranged 87.40％-103.75％. Matrix effect didn’t affect the determination of the substance to be measured. The piperitylmagnolol was metabolized significantly in human， rat， mouse and dog liver microsomes， but not in monkey liver microsomes. After incubating for 30 min， residual percentage of piperitylmagnolol kept stable in different species of liver microsomes. The t1/2 of piperitylmagnolol were 12.07， 17.68， 17.59， 216.56 and 61.88 min in human， rat， mouse， monkey and dog liver microsomes； CLint were 0.115， 0.078， 0.079， 0.006， 0.022 mL/（min·mg）， respectively. Inhibitory rates of CYP2A6， CYP2D6， CYP2C19， CYP3A4， CYP2C9， CYP2E1 and CYP1A2 to compound metabolism were 55.76％， 93.94％， 96.01％， 93.69％， 71.81％， 23.25％， 28.04％， respectively. Quasi-molecular ion peaks of the two main metabolites of piperitylmagnolol in human liver microsomes were m/z 441.2（[M+Na]+） and m/z 337.2（[M+H]+）， respectively. CONCLUSIONS： Established UPLC-MS/MS method is simple， rapid and specific， and can be used for the determination of piperitylmagnolol concentration in the incubation system of liver microsomes and pharmacokinetic study. The metabolic characteristics of the compound are different among liver microsomes of human， rat， mouse， monkey and dog. Its metabolism process may be associated with CYP2D6， CYP2C19， CYP3A4， CYP2C9， etc.
RESUMO
OBJECTIVE:To establish the detection method for anti-inflammatory compound HB0314 in plasma of rats,and study on its pharmacokinetic characteristics in rats in vivo. METHODS:UPLC-MS/MS was performed on the column of Waters Ac-quity UPLCTM BEH C18 with mobile phase of 0.1% formic acid aqueous solution(A)-methanol(B)by gradient elution(0-2 min, 70%-90% B) at flow rate of 0.25 mL/min,column temperature was 30 ℃,and injection volume was 5 μL. Electrospray ion source was used,capillary voltage was 3 kV,ion source temperature was 150 ℃,desolvation gas temperature was 450 ℃,desol-vent air flow volume was 600 L/h,cone air flow volume was 45 L/h,and the inner standard was tetrahydropalmatine. 12 rats were randomly divided into iv group and ig group,6 in each group. Rats were intravenously injected and intragastrically administrated HB0314 solution 5,10 mg/kg. Sample blood 0.4 mL were taken from the jugular vein blood before administration and after 5,15, 30,60,120,240,360,480,600,720,1440 min of administration to determine the HB0314 plasma concentration. DAS 2.0 software was used to calculate the pharmacokinetic parameters and absolute bioavailability. RESULTS:The linear range of HB0314 was 1-1000 ng/mL(r=0.9955),and the lower limit of quantification was 1 ng/mL. RSDs of extra-day and daytime precision,sta-bility were not higher than 8.45%(n=5);recovery were 68.21%-90.29%(RSD≤11.20%,n=5),and matrix effects were 82.63%-106.90%(RSD≤6.75%,n=5). After intravenous injection and intragastric administration,AUC0-24 h were (270.267 ± 21.164), (252.755 ± 26.169)μg·h/L (n=6);t1/2z were (8.722 ± 2.266),(11.877 ± 4.517) h (n=6);and absolute bioavailability was 56.79%. CONCLUSIONS:The method is rapid,simple,and can be used for the determination of HB0314 content in plasma of rats. HB0314 shows high oral absolute bioavailability in rats in vivo,indicating that post-dosage form design may be considered as oral anti-inflammatory drugs.
RESUMO
OBJECTIVE:To establish the detection method for anti-inflammatory compound HB0314 in plasma of rats,and study on its pharmacokinetic characteristics in rats in vivo. METHODS:UPLC-MS/MS was performed on the column of Waters Ac-quity UPLCTM BEH C18 with mobile phase of 0.1% formic acid aqueous solution(A)-methanol(B)by gradient elution(0-2 min, 70%-90% B) at flow rate of 0.25 mL/min,column temperature was 30 ℃,and injection volume was 5 μL. Electrospray ion source was used,capillary voltage was 3 kV,ion source temperature was 150 ℃,desolvation gas temperature was 450 ℃,desol-vent air flow volume was 600 L/h,cone air flow volume was 45 L/h,and the inner standard was tetrahydropalmatine. 12 rats were randomly divided into iv group and ig group,6 in each group. Rats were intravenously injected and intragastrically administrated HB0314 solution 5,10 mg/kg. Sample blood 0.4 mL were taken from the jugular vein blood before administration and after 5,15, 30,60,120,240,360,480,600,720,1440 min of administration to determine the HB0314 plasma concentration. DAS 2.0 software was used to calculate the pharmacokinetic parameters and absolute bioavailability. RESULTS:The linear range of HB0314 was 1-1000 ng/mL(r=0.9955),and the lower limit of quantification was 1 ng/mL. RSDs of extra-day and daytime precision,sta-bility were not higher than 8.45%(n=5);recovery were 68.21%-90.29%(RSD≤11.20%,n=5),and matrix effects were 82.63%-106.90%(RSD≤6.75%,n=5). After intravenous injection and intragastric administration,AUC0-24 h were (270.267 ± 21.164), (252.755 ± 26.169)μg·h/L (n=6);t1/2z were (8.722 ± 2.266),(11.877 ± 4.517) h (n=6);and absolute bioavailability was 56.79%. CONCLUSIONS:The method is rapid,simple,and can be used for the determination of HB0314 content in plasma of rats. HB0314 shows high oral absolute bioavailability in rats in vivo,indicating that post-dosage form design may be considered as oral anti-inflammatory drugs.
RESUMO
In order to affirm the cardioactive components in Fuzi, we identified a group of aminoalcohol- diterpenoid alkaloids in Fuzi using ultra high-performance liquid chromatography coupled with electrospray ionization mass spectrometer (UPLC-ESI-MS) method. Among a total of forty-one isolated ingredients, thirteen major aminoalcohol-diterpenoid alkaloids were identified by comparing their retention times and MS spectra with those of the reference substances. Moreover, Fuzi samples from different places of origin and with different processing methods were examined and their components displayed a pattern of high similarity, though the relative abundance varies probably due to their different processing methods. Furthermore, the cardiac effect of each identified alkaloid was individually evaluated using the isolated bullfrog heart perfusion experiment. Among the thirteen aminoalcohol diterpenoid alkaloids tested, six of them significantly enhanced the amplitude rates. Taken together, we affirm that the cardioactive components in Fuzi are aminoalcohol-diterpenoid alkaloids, shedding light on future studies of the mechanisms and development of these cardioactive compounds.