芪雪浸膏剂中大黄素、大黄酸和毛蕊异黄酮在大鼠体内的药动学研究

Study on Pharmacokinetics of Emodin,Rhein and Calycosin from Qixue Extract in Rats in vivo

目的:建立大鼠血浆中大黄素、大黄酸和毛蕊异黄酮含量的测定方法,并研究大鼠体内芪雪浸膏剂的药动学。方法:采用液相色谱-质谱联用(LC-MS/MS)法。色谱柱为Thermo C_(18),流动相为乙腈-0.1%甲酸溶液(梯度洗脱),流速为0.2 mL/min,柱温为30℃,进样量为20μL,定量离子为大黄素质荷比(m/z)269.7→226.1,大黄酸m/z 283.7→239.9,毛蕊异黄酮m/z 283.5→268.5,地西泮(内标物)m/z 285.0→154.1。6只大鼠灌胃芪雪浸膏剂8.91 g/kg(按临床用量换算而得)后0.08、0.17、0.25、0.5、0.75、1、2、4、6、8、12 h眼眶取血测定血药浓度。采用DAS 1.0药动学软件计算各药动学参数。结果:大黄素、大黄酸和毛蕊异黄酮质量浓度线性范围分别为1.988～198.8 ng/mL(r=0.998 8)、2～200 ng/mL(r=0.998 8)、2.092～209.2 ng/mL(r=0.998 5),精密度、准确度试验的RSD均小于7.78%(n=5),基质效应分别为92.13%～102.23%、90.15%～93.67%、90.33%～104.63%(n=5),稳定性试验的RSD均小于6.54%(n=3);3种成分在大鼠体内的t1/2分别为(1.95±0.53)、(3.55±1.25)、(4.21±1.14)h,AUC0-12 h分别为(192.67±40.58)、(93.67±20.26)、(608.41±108.36)μg·h/L,cmax分别为(89.56±12.87)、(31.84±10.58)、(300.56±50.57)μg/L,tmax分别为(0.50±0.00)、(0.75±0.23)、(1.00±0.00)h,CL分别为(5.45±1.12)、(2.09±0.47)、(10.01±1.25)L(/h·kg)(n=6)。结论:建立的方法灵敏度高、准确可靠,大黄素、大黄酸、毛蕊异黄酮在大鼠体内的消除过程为一级消除,且符合二室开放模型。

OBJECTIVE:To establish a method for the determination of emodin,rhein and calycosin in rat plasma,and to study pharmacokinetics of Qixue extract in rats.METHODS:LC-MS/MS method was adopted.The determination was performed on Thermo C18 column with mobile phase consisted of acetonitrile-0.1%formic acid solution(gradient elution),and flow rate of 0.2 mL/min.The column temperature was set at 30℃,and sample size was 20μL.The quantitative ions were emodin m/z 269.7→226.1,rhein m/z 283.7→239.9,calycosin m/z 283.5→268.5 and diazepam m/z 285.0→154.1.The blood concentration of samples from orbital cavity of 6 rats were determined 0.08,0.17,0.25,0.5,0.75,1,2,4,6,8,12 h after intragastric administration of Qixue extract 8.91 g/kg(by clinical dosage).DAS 1.0 pharmacokinetic software was employed to calculate pharmacokinetic parameters.RESULTS:The linear ranges of emodin,rhein and calycosin were 1.988-198.8 ng/mL(r=0.998 8),2-200 ng/mL(r=0.998 8)and 2.092-209.2 ng/mL(r=0.998 5),respectively.RSDs of precision and accuracy tests were all less than 7.78%(n=5).The recoveries were 92.13%-102.23%,90.15%-93.67%and 90.33%-104.63%(n=5),respectively.RSDs of stability tests were all less than 6.54%(n=3).The pharmacokinetic parameters of emodin,rhein and calycosin were as follows as t1/2 were(1.95±0.53),(3.55±1.25),(4.21±1.14)h;AUC0-12 h were(192.67±40.58),(93.67±20.26),(608.41±108.36)μg·h/L;cmax were(89.56±12.87),(31.84±10.58),(300.56±50.57)μg/L;tmax were(0.50±0.00),(0.75±0.23),(1.00±0.00)h;CL were(5.45±1.12),(2.09±0.47),(10.01±1.25)L/(h·kg)(n=6),respectively.CONCLUSIONS:Established method is highly sensitive,accurate and reliable.The elimination process of emodin,rhein and calycosin are first-order elimination,which is in line with two-compartment model.