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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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 UPLC~(TM)CSH C_(18),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2μL;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(t_(1/2))和固有清除率(CL_(int))。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t_(1/2)分别为12.07、17.68、17.59、216.56、61.88 min,CL_(int)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/z441.2([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。
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 UPLC~(TM)CSH C_(18) 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(t_(1/2))and intrinsic clearance(CLin)t werecalculated 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 t_(1/2) of piperitylmagnolol were 12.07,17.68,17.59,216.56 and 61.88 min in human,rat,mouse,monkey and dog liver microsomes;CL_(int)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.
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 UPLC~(TM)CSH C_(18) 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(t_(1/2))and intrinsic clearance(CLin)t werecalculated 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 t_(1/2) of piperitylmagnolol were 12.07,17.68,17.59,216.56 and 61.88 min in human,rat,mouse,monkey and dog liver microsomes;CL_(int)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.
引文
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[13]李春正,林庆辉,庄笑梅,等.重组人源CYP同工酶介导的罗通定O-去甲基代谢[J].药学学报,2010,45(3):307-313.
[14]焦士勇,艾常虹,李艾芳,等.补骨脂酚的体外肝微粒体代谢及代谢减毒作用的种属比较[J].中国药理学通报,2011,27(2):216-220.
[15]樊慧蓉,董世奇,李全胜,等.甘草素在体外不同种属肝微粒体中的代谢差异研究[J].中草药,2017,48(2):320-326.
[16]国家食品药品监督管理局.化学药物非临床药代动力学研究技术指导原则[S].2005-03-18.
[17]刘晓东,柳晓泉.药物代谢动力学教程[M].南京:江苏凤凰科学技术出版社,2015:296-316.
[2]张国良,李娜,林黎琳,等.木脂素类化合物生物活性研究进展[J].中国中药杂志,2007,32(20):2089-2094.
[3] YAHARA S,NISHIYORI T,KOHDA A,et al. Isolation and characterization of phenolic compounds from Magnoliae cortex produced in China[J]. Chem Pharm Bull,1991,39(8):2024-2036.
[4] KUO WL,CHUNG CY,HWANG TL,et al. Biphenyl-type neolignans from Magnolia officinalis and their anti-inflammatory activities[J]. Phytochemistry,2013. DOI:10.1016/j.phytochem.2012.08.014.
[5] YOUN UJ,LEE IS,CHEN QC,et al. A cytotoxic monoterpene-neolignan from the stem bark of Magnolia officinalis[J]. Nat Prod Sci,2011,17(2):95-99.
[6] SYU WJ,SHEN CC,LU JJ,et al. Antimicrobial and cytotoxic activities of neolignans from Magnolia officinalis[J].Chem Biodivers,2004,1(3):530-537.
[7]吴慧,彭英,孙建国,等.体外代谢在新药早期评价中的应用与发展[J].药学学报,2013,48(7):1071-1079.
[8] COHEN LH,REMLEY MJ,RAUNING D,et al. In vitro drug interactions of cytochrome P450:an evaluation of fluorogenic to conventional substrates[J]. Drug Metab Dispos,2003,31(8):1005-1015.
[9]国家药典委员会.中华人民共和国药典:四部[S].2015年版.北京:中国医药科技出版社,2015:363-368.
[10] MOHUTSKY MA,CHIEN JY,RING BJ,et al. Predictions of the in vivo clearance of drugs from rate of loss using human liver microsomes for phaseⅠand phaseⅡbiotransformations[J]. Pharm Res,2006,23(4):654-662.
[11]刘治军,傅得兴,汤光. FDA药物相互作用研究指南(草案)2006版解读[J].国际药学研究杂志,2008,35(1):50-58.
[12] CARLILE DJ,ZOMORODI K,HOUSTON JB. Scaling factors to relate drug metabolic clearance in hepatic microsomes,isolated hepatocytes,and the intact liver:studies with induced livers involving diazepam[J]. Drug Metab Dispos,1997,25(8):903-911.
[13]李春正,林庆辉,庄笑梅,等.重组人源CYP同工酶介导的罗通定O-去甲基代谢[J].药学学报,2010,45(3):307-313.
[14]焦士勇,艾常虹,李艾芳,等.补骨脂酚的体外肝微粒体代谢及代谢减毒作用的种属比较[J].中国药理学通报,2011,27(2):216-220.
[15]樊慧蓉,董世奇,李全胜,等.甘草素在体外不同种属肝微粒体中的代谢差异研究[J].中草药,2017,48(2):320-326.
[16]国家食品药品监督管理局.化学药物非临床药代动力学研究技术指导原则[S].2005-03-18.
[17]刘晓东,柳晓泉.药物代谢动力学教程[M].南京:江苏凤凰科学技术出版社,2015:296-316.