LC-MS/MS法测定大鼠血浆中芦荟苦素浓度及其药动学研究

Determination of Aloesin in Rat Plasma by LC-MS/MS and Its Pharmacokinetic Study

目的:建立测定大鼠血浆中芦荟苦素浓度的方法,并考察芦荟苦素的药动学特征。方法:血浆样品经甲醇沉淀蛋白后,以芦荟新苷D为内标,采用液相色谱-串联质谱法测定大鼠血浆中芦荟苦素的血药浓度。以Synergi Hydro-RP为色谱柱,以0.1‰甲酸溶液-甲醇为流动相进行梯度洗脱,流速为0.50 mL/min,柱温为30℃,进样量为5μL;采用电喷雾离子源,以多反应监测模式进行负离子检测,用于定量分析的离子对分别为m/z 393.1→272.9(芦荟苦素)、m/z 555.3→144.9(内标)。采用上述方法测定大鼠尾静脉注射(3.35 mg/kg)和灌胃(16.75 mg/kg)芦荟苦素后0.083、0.167、0.333、0.667、1、1.5、2.5、4、6、8、10 h静脉血中芦荟苦素的质量浓度,采用DAS 3.0软件计算药动学参数。结果:芦荟苦素检测质量浓度的线性范围为1~600 ng/mL(r=0.9945),定量下限为1 ng/mL,批内、批间RSD均小于15%,批内、批间准确度在±15%内,基质因子为(92.74±4.33)%~(94.84±2.57)%,提取回收率为(69.04±2.13)%~(75.03±2.84)%,稳定性试验的实测结果与理论值的偏差在±15%内。大鼠静脉注射和灌胃芦荟苦素后,主要药动学参数c_(max)分别为(10693.3±2745.3)、(223.3±36.2)ng/mL,t_(1/2)分别为(2.45±1.45)、(3.33±1.91)h,AUC_(0-24 h)分别为(4190.6±883.6)、(1210.1±93.9)ng·h/mL(n=3),口服绝对生物利用度为11.13%。结论:本研究成功建立了一种快速、灵敏的芦荟苦素血药浓度测定方法,适用于其药动学研究。

OBJECTIVE:To establish a method for the determination of aloesin in plasma of rats,and to investigate pharmacokinetic characteristics of aloesin.METHODS:The plasma samples were precipitated with methanol.Using aloeresin D as internal standard,the plasma concentration of aloesin was determined by LC-MS/MS.The determination was performed on Synergi Hydro-RP column with mobile phase consisted of 0.1‰formic acid-methanol(gradient elution)at the flow rate of 0.50 mL/min.The column temperature was 30℃,and sample size was 5μL.The electrospray ionization source was applied to carry out negative ion detection with multiple reaction monitoring mode.The ion transitions for quantitative analysis were m/z 393.1→272.9(aloesin)and m/z 555.3→144.9(internal standard),respectively.The concentration of aloesin in venous blood was determined by above method at 0.083,0.167,0.333,0.667,1,1.5,2.5,4,6,8,10 h after intravenous injection(3.35 mg/kg)and intragastric administration(16.75 mg/kg)of aloesin.DAS 3.0 software was used to calculate pharmacokinetic parameters.RESULTS:The linear range of aloesin were 1-600 ng/mL(r=0.9945).The lower limit of quantification was 1 ng/mL,and RSDs of within and between batches were less than 15%;accuracies within and between batches were within±15%.The matrix factors were(92.74±4.33)%-(94.84±2.57)%,and extraction recoveries were(69.04±2.13)%-(75.03±2.84)%;the deviation between the measured results of the stability test and the theoretical values were within±15%.After intravenous injection and intragastric administration of aloesin,main pharmacokinetic parameters were as follows:c_(max) were(10693.3±2745.3)and(223.3±36.2)ng/mL;t_(1/2) were(2.45±1.45)and(3.33±1.91)h;AUC_(0-24 h) were(4190.6±883.6)and(1210.1±93.9)ng·h/mL(n=3).Absolute bioavailability was 11.13%.CONCLUSIONS:The established method is rapid and sensitive for plasma determination of aloesin,and suitable for its pharmacokinetic study.