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采用UPLC-MS/MS法研究辣薄荷基厚朴酚在不同种属肝微粒体中的代谢特征
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| 篇名： | 采用UPLC-MS/MS法研究辣薄荷基厚朴酚在不同种属肝微粒体中的代谢特征 |
| TITLE： | |
| 摘要： | 目的：建立测定肝微粒体孵育体系中辣薄荷基厚朴酚浓度的方法，并探讨其在不同种属肝微粒体中的代谢特征。方法：分别将辣薄荷基厚朴酚溶解于烟酰胺腺嘌呤二核苷酸磷酸（NADPH）启动的人、大鼠、小鼠、猴、犬肝微粒体孵育体系中，置于37 ℃水浴中进行孵育，分别于孵育的0、2、5、10、15、20、30、45、60 min时用甲醇终止反应，以厚朴酚为内标，采用超高效液相色谱-串联质谱法（UPLC-MS/MS）测定各孵育体系中辣薄荷基厚朴酚的质量浓度。色谱柱为Acquity UPLCTM CSH C18，流动相为0.1％甲酸溶液-甲醇（梯度洗脱），流速为0.3 mL/min，柱温为30 ℃，进样量为2 μL；离子源为电喷雾离子源，以多反应监测模式进行正离子扫描，用于定量分析的离子对分别为m/z 401.2→331.1（辣薄荷基厚朴酚）、m/z 265.1→247.0（内标）。以孵育0 min时辣薄荷基厚朴酚的质量浓度为参照，计算其在不同孵育体系中的药物剩余百分比、体外代谢半衰期（t1/2）和固有清除率（CLint）。采用化学抑制剂法探讨辣薄荷基厚朴酚的代谢途径；在上述色谱条件下，采用一级全扫描以正离子方式检测，初步分析其体外代谢产物。结果：辣薄荷基厚朴酚质量浓度检测的线性范围为3.91～500.00 ng/mL，定量下限为3.91 ng/mL；日内、日间RSD均小于10％，准确度为87.40％～103.75％，基质效应不影响待测物的测定。辣薄荷基厚朴酚在人、大鼠、小鼠、犬肝微粒体中代谢明显，而在猴肝微粒体中代谢不明显；孵育30 min后，其在各种属肝微粒体的药物剩余百分比趋于稳定。辣薄荷基厚朴酚在人、大鼠、小鼠、猴、犬肝微粒体中的t1/2分别为12.07、17.68、17.59、216.56、61.88 min，CLint分别为0.115、0.078、0.079、0.006、0.022 mL/（min·mg）。细胞色素P450（CYP）2A6、CYP2D6、CYP2C19、CYP3A4、CYP2C9、CYP2E1、CYP1A2酶对该化合物代谢的抑制率分别为55.76％、93.94％、96.01％、93.69％、71.81％、23.25％、28.04％。辣薄荷基厚朴酚在人肝微粒体中两个主要代谢产物的准分子离子峰分别为m/z 441.2（[M+Na]+）、m/z 337.2（[M+H]+）。结论：本研究建立的UPLC-MS/MS法简便、快速、专属性强，可用于肝微粒体孵育体系中辣薄荷基厚朴酚浓度的测定及药动学的研究。该化合物在人、大鼠、小鼠、猴、犬等5种肝微粒体中的代谢特征有差异，且其代谢过程可能与CYP2D6、CYP2C19、CYP3A4、CYP2C9等酶有关。 |
| 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. |
| 期刊： | 2019年第30卷第2期 |
| 作者： | 邓星，罗莉娅，苟立平，温倩雯，汤明海，万丽 |
| AUTHORS： | DENG Xing，LUO Liya，GOU Liping，WEN Qianwen，TANG Minghai，WAN Li |
| 关键字： | 辣薄荷基厚朴酚；不同种属；肝微粒体；体外代谢稳定性；代谢酶；代谢产物 |
| KEYWORDS： | Piperitylmagnolol； Different species； Liver microsomes； Metabolic stability in vitro； Metabolic enzyme； Metabolites |
| 阅读数： | 491 次 |
| 本月下载数： | 8 次 |
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