UPLC-MS/QAMS测定小鼠血浆中石斛碱及其代谢产物M-250、M-280的浓度
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摘要
目的:首次建立测定小鼠血浆中石斛碱及其代谢产物M-250、M-280浓度的方法。方法:小鼠灌胃石斛碱60 mg/kg,1 h后收集血浆处理后,以盐酸伪麻黄碱为内标,以石斛碱对照品为对照,采用超高效液相色谱-质谱/一测多评法测定石斛碱及其代谢产物M-250、M-280的血药浓度。色谱柱为Hypersil Gold C18,流动相为0.1%甲酸水溶液-乙腈(梯度洗脱),流速为0.3 m L/min,柱温为40℃,进样量为5μL;采用可加热电喷雾离子源,全扫描/正离子模式,雾化温度为300℃,离子传输管温度为350℃,鞘气流速为35 arb,辅助气流速为15 arb,喷雾电压为3.5 k V,碰撞电压为30、40、50 e V,质荷比检测范围为100~1 500。结果:小鼠血浆中内源性物质对石斛碱及其代谢产物M-250、M-280浓度测定无干扰;石斛碱检测质量浓度线性范围为9.13~912.94 ng/m L(r=0.999 6);定量下限为3.04 ng/m L,日内、日间精密度的RSD≤7.5%(n=5或n=3);准确度为96.8%~107.5%(n=5);基质效应为97.1%~106.0%(RSD=1.8%~4.7%,n=5);质控血浆样品溶液15℃保存24 h、-70℃保存并反复冻融3次以及-70℃保存15 d后浓度的RSD≤12.8%(n=3)。小鼠血浆样品中石斛碱质量浓度为(41.3±5.7)ng/m L(n=12),以相对校正因子(RCF)按1.0计,代谢产物M-250、M-280的质量浓度分别为(493.0±73.1)、(41.4±3.0)ng/m L(n=12)。结论:该方法灵敏、准确,可用于小鼠血浆中石斛碱及其代谢产物M-250、M-280的浓度测定。
OBJECTIVE:To establish a method for the determination of dendrobine and its metabolites M-250 and M-280 in mice plasma for the first time. METHODS:Mice were given dendrobine 60 mg/kg by intragastric administration,1 h later plasma were collected and treated. Using pseudoephedrine hydrochloride as internal standard and dendrobine reference substance as control,the plasma concentrations of dendrobine and its metabolites M-250 and M-280 were determined by UPLC-MS combined with quantitative analysis of multi-components by single marker. The separation was performed on Hypersil Gold C18 column with 0.1%formic acid-acetonitrile(gradient elution)at the flow rate of 0.3 m L/min. The column temperature was set at 40℃,and sample size was 5 μ L. Heatable electrospray ionization(HESI) source,scan/ESI+were applied and operated in positive ion mode with atomization temperature of 300 ℃,ion transmission tube temperature of 350 ℃,the sheath gas velocity of 35 arb,the auxiliary air velocity of 15 arb,the spray voltage of 3.5 k V,the collision voltage of 30,40,50 e V. The mass-to-charge ratio of detection range were 100-1 500. RESULTS:The endogenous substances of mice plasma had no interference with the content determination of dendrobine and its metabolites M-250 and M-280. The linear range of dendrobine were 9.13-912.94 ng/m L(r=0.999 6). The limit of quantitation was 3.04 ng/m L. RSDs of intra-day and inter-day were all less than 7.5%(n=5 or n=3). The accuracy were96.8%-107.5%(n=5). Matrix effects were 97.1%-106.0%(RSD=1.8%-4.7%,n=5). RSDs of the content of sample at 15℃ for24 h,at-70 ℃ after three times freeze-thaw,at-70 ℃ for15 d were lower than 12.8%(n=3). The content of dendrobine in plasma sample of mice was(41.3±5.7)ng/m L(n=12). The contents of its metabolites M-250 and M-280 were(493.0 ± 73.1) and(41.4 ± 3.0) ng/m L(n=12) with Relative correction factor of 1.0. CONCLUSIONS: The method is sensitive and accurate,and can be used for content determination of dendrobine and its metabolites M-250 and M-280 in mice plasma.
OBJECTIVE:To establish a method for the determination of dendrobine and its metabolites M-250 and M-280 in mice plasma for the first time. METHODS:Mice were given dendrobine 60 mg/kg by intragastric administration,1 h later plasma were collected and treated. Using pseudoephedrine hydrochloride as internal standard and dendrobine reference substance as control,the plasma concentrations of dendrobine and its metabolites M-250 and M-280 were determined by UPLC-MS combined with quantitative analysis of multi-components by single marker. The separation was performed on Hypersil Gold C18 column with 0.1%formic acid-acetonitrile(gradient elution)at the flow rate of 0.3 m L/min. The column temperature was set at 40℃,and sample size was 5 μ L. Heatable electrospray ionization(HESI) source,scan/ESI+were applied and operated in positive ion mode with atomization temperature of 300 ℃,ion transmission tube temperature of 350 ℃,the sheath gas velocity of 35 arb,the auxiliary air velocity of 15 arb,the spray voltage of 3.5 k V,the collision voltage of 30,40,50 e V. The mass-to-charge ratio of detection range were 100-1 500. RESULTS:The endogenous substances of mice plasma had no interference with the content determination of dendrobine and its metabolites M-250 and M-280. The linear range of dendrobine were 9.13-912.94 ng/m L(r=0.999 6). The limit of quantitation was 3.04 ng/m L. RSDs of intra-day and inter-day were all less than 7.5%(n=5 or n=3). The accuracy were96.8%-107.5%(n=5). Matrix effects were 97.1%-106.0%(RSD=1.8%-4.7%,n=5). RSDs of the content of sample at 15℃ for24 h,at-70 ℃ after three times freeze-thaw,at-70 ℃ for15 d were lower than 12.8%(n=3). The content of dendrobine in plasma sample of mice was(41.3±5.7)ng/m L(n=12). The contents of its metabolites M-250 and M-280 were(493.0 ± 73.1) and(41.4 ± 3.0) ng/m L(n=12) with Relative correction factor of 1.0. CONCLUSIONS: The method is sensitive and accurate,and can be used for content determination of dendrobine and its metabolites M-250 and M-280 in mice plasma.
引文
[1]国家药典委员会.中华人民共和国药典:一部[S].2015年版.北京:中国医药科技出版社,2015:92.
[2]马继兴.神农本草经辑注[S].北京:人民卫生出版社,1995:37.
[3]王亚芸,任建武.石斛碱的研究进展[J].山东农业大学学报(自然科学版),2015,46(1):152-158.
[4]陶凤,金徽,杨贵忠,等.金钗石斛水提物对糖尿病大鼠肾组织非酶糖基化及氧化的影响[J].山东大学学报(医学版),2012,50(10):11-15、22.
[5]白金丽,温淑香.金钗石斛提取物抗白内障的体外实验研究[J].云南中医中药杂志,2009,30(9):57-59.
[6]刘名昆,李国庆.石斛提取物抗肿瘤作用研究进展[J].现代医药卫生,2015,31(23):3571-3574.
[7]YANG S,GONG QH,WU Q,et al.Alkaloids enriched extract from Dendrobium nobile Lindl.attenuates tau protein hyperphosphorylation and apoptosis induced by lipopolysaccharide in rat brain[J].Phytomedicine,2014,21(5):712-716.
[8]宾捷,胡余明,尹进,等.金钗石斛对老龄小鼠抗氧化作用的实验研究[J].实用预防医学,2010,17(6):1063-1064.
[9]姚寅.金钗石斛生物碱对老年痴呆症的保护作用和机制的研究进展[J].医药前沿,2013,2(4):47-48.
[10]鲁艳柳,黄思,汪敏,等.UPLC/LTQ-Orbitrap-MS技术鉴定石斛碱在人肝微粒体中的代谢产物[J].中国实验方剂学杂志,2018,24(4):32-37.
[11]左岚,孟胜男.一测多评法在中药药物分析中的应用进展[J].中国药房,2016,27(18):2589-2592.
[12]王欣,覃瑶,王德江,等.一测多评法在中药质量控制中的应用进展[J].中成药,2016,38(2):395-402.
[2]马继兴.神农本草经辑注[S].北京:人民卫生出版社,1995:37.
[3]王亚芸,任建武.石斛碱的研究进展[J].山东农业大学学报(自然科学版),2015,46(1):152-158.
[4]陶凤,金徽,杨贵忠,等.金钗石斛水提物对糖尿病大鼠肾组织非酶糖基化及氧化的影响[J].山东大学学报(医学版),2012,50(10):11-15、22.
[5]白金丽,温淑香.金钗石斛提取物抗白内障的体外实验研究[J].云南中医中药杂志,2009,30(9):57-59.
[6]刘名昆,李国庆.石斛提取物抗肿瘤作用研究进展[J].现代医药卫生,2015,31(23):3571-3574.
[7]YANG S,GONG QH,WU Q,et al.Alkaloids enriched extract from Dendrobium nobile Lindl.attenuates tau protein hyperphosphorylation and apoptosis induced by lipopolysaccharide in rat brain[J].Phytomedicine,2014,21(5):712-716.
[8]宾捷,胡余明,尹进,等.金钗石斛对老龄小鼠抗氧化作用的实验研究[J].实用预防医学,2010,17(6):1063-1064.
[9]姚寅.金钗石斛生物碱对老年痴呆症的保护作用和机制的研究进展[J].医药前沿,2013,2(4):47-48.
[10]鲁艳柳,黄思,汪敏,等.UPLC/LTQ-Orbitrap-MS技术鉴定石斛碱在人肝微粒体中的代谢产物[J].中国实验方剂学杂志,2018,24(4):32-37.
[11]左岚,孟胜男.一测多评法在中药药物分析中的应用进展[J].中国药房,2016,27(18):2589-2592.
[12]王欣,覃瑶,王德江,等.一测多评法在中药质量控制中的应用进展[J].中成药,2016,38(2):395-402.