ABSTRACT
OBJECTIVE： To establish a determination method for the concentration of cajanonic acid A （CAA） in liver microsome incubation system， and to compare the metabolism characteristics of it in different species of liver microsomes. METHODS： CAA was dissolved in liver microsome incubation system of rat， Beagle dog and human initiated by reduced nicotinamide adenine dinucleotide phosphate （NADPH）， and was incubated in water at 37 ℃. The reaction was terminated with acetonitrile at 0， 5， 10， 15， 30， 45 and 60 min， respectively. Using genistein as internal standard， the concentration of CAA in different incubation systems was determined by UPLC-MS/MS. The determination was performed on Waters BEH C18 column with mobile phase consisted of water （containing 0.1％ formic acid）-acetonitrile （containing 0.1％ formic acid） （45 ∶ 55， V/V） at the flow rate of 0.25 mL/min. The column temperature was 30 ℃， and the sample size was 2 μL. The electrospray ionization source was used to the select reaction monitoring mode for negative ion scanning. The ion pairs for quantitative analysis were m/z 353.14→309.11 （CAA）， m/z 269.86→224.11 （internal standard） respectively. The residual percentage and enzymatic kinetic parameters of CAA in different incubation systems were calculated according to the mass concentration of CAA at 0 min. RESULTS： The linear range of CAA was 0.05-20 μg/mL； the limit of quanti- tation was 0.05 μg/mL， and the lowest detection limit was 0.01 μg/mL. RSDs of intra-day and inter-day were lower than 10％； relative errors ranged －4.83％-8.94％； extraction method and matrix effect did not affect the determination of the substance to be measured. At 60 min of incubation， residual percentages of CAA in rat， Beagle dog and human liver microsomes were（62.79±9.99）％，（64.07±11.59）％，（96.66±5.71）％， respectively. The half-life period （72.19， 68.61 min） of CAA in rat and Beagle dog liver microsomes were significantly shorter than human liver microsome （364.74 min）. The clearance rates [0.019 2， 0.020 2 mL/（min·mg）] were significantly higher than human liver microsome [0.003 8 mL/（min·mg）] （P＜0.05）. CONCLUSIONS： Established UPLC-MS/MS method is simple， rapid， specific and sensitive， and can be used for the determination of CAA concentration in liver microsome incubation system and the study of metabolism stability in vitro. The stability of CAA metabolism in rat and Beagle dog liver microsomes are poorer than human liver microsome.
ABSTRACT
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.
ABSTRACT
@#To construct nanostructured lipid carriers(NLCs)with different particle sizes but the same other physicochemical properties, central composite design was adopted. Coumarin-6(C-6)was selected as the model drug due to its high lipophilicity and high fluorescence intensity. Physicochemical properties of NLCs with 100 nm, 200 nm and 300 nm in particle size could remain stable during certain time in K-R solution and PBS. Release experiments in vitro showed that cumulative release of C-6 in NLCs was less than 7% after 24 h. The MTT assay indicated that both blank NLCs and C-6 loaded NLCs showed low toxicity. To confirm the integrity of NLCs in gastrointestinal tract, DiR-loaded NLCs were prepared and the distribution in vivo was monitored by fluorescence imaging. After 6 h oral administration, intact DiR-loaded NLCs could stiu be found, suggesting that NLCs could be used to characterize the uptake in gastrointestinal tract.