基于LC-MS研究交泰丸调控睡眠剥夺小鼠胆汁酸代谢变化

Study on bile acid metabolism of sleep-deprived mice regulated by Jiaotai Pills based on LC-MS

为了探讨睡眠剥夺状态下小鼠体内胆汁酸水平变化,考察交泰丸对睡眠剥夺小鼠胆汁酸代谢的调控作用,采用UPLC-MS/MS建立同时测定小鼠体内23种胆汁酸的分析方法。将24只ICR小鼠随机分成正常组、模型组、交泰丸组,交泰丸组每天灌胃交泰丸3.3 g·kg^(-1),连续给药8 d,正常组、模型组灌胃同等体积的纯净水;采用睡眠剥夺仪剥夺模型组、交泰丸组小鼠睡眠20 h·d^(-1),连续剥夺8 d,实验后对小鼠血清、肝脏、回肠中胆汁酸水平进行检测。选用Waters ACQUITY UPLC BEH C18色谱柱(2.1 mm×100 mm,1.7μm),以0.1%甲酸水-甲醇作为流动相进行梯度洗脱,采用电喷雾离子源负离子模式,多反应监测(MRM)扫描方式建立23种胆汁酸的检测方法并进行方法学研究,同时考察小鼠血清、肝脏、回肠中胆汁酸的组成、含量。结果显示,23种胆汁酸可在15 min内快速测定,线性相关系数均大于0.99,准确度高,精密度良好,专属性、稳定性、提取回收率和基质效应等均符合生物样品分析要求;睡眠剥夺可显著升高小鼠血清、肝脏、回肠中的胆汁酸水平,给药交泰丸后,小鼠体内胆汁酸水平显著回调。以上结果表明,该分析方法可用于生物样品中23种胆汁酸的定量分析;给药交泰丸可显著降低睡眠剥夺小鼠升高的胆汁酸水平。

To investigate the changes of bile acid(BA)levels in mice with sleep deprivation and the regulatory effect of Jiaotai Pills(JTP)on bile acid metabolism,this study established an ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)method for simultaneous determination of 23 BAs in mice.A total of 24 ICR mice were randomized into normal group,model group,and JTP group.Mice in the model group and JTP group were deprived of sleep at 20 h·d^(-1)by sleep deprivation apparatus for 8 consecutive days.Mice in the JTP group were given(ig,qd)JTP 3.3 g·kg^(-1)and those in the normal group and model group received(ig)the same volume of purified water.UPLC conditions are as follows:Waters ACQUITY UPLC BEH C18 column(2.1 mm×100 mm,1.7μm),gradient elution with the mobile phase of 0.1%formic acid in water-methanol.MS conditions are as below:negative-ion electrospray ionization,multiple reaction monitoring(MRM).Thereby,the content of 23 BAs in serum,liver,and ileum was determined and methodological investigation of the method was performed.The results showed that 23 BAs could be ac-curately determined within 15 min and the correlation coefficients were all higher than 0.99.The precision,accuracy,specificity,reproducibility,matrix effect,and recovery of BAs all met the requirement.The levels of BAs were significantly increased in the serum,liver,and ileum of sleep-deprived mice,but JTP can significantly reduce the levels.The UPLC-MS/MS method is simple,rapid,and accurate,which can be used for the determination of 23 BAs in biological samples,and JTP can adjust the elevated BA levels of sleepdeprived mice.