四季青中原儿茶酸在beagle犬体内的药动学研究

目的 建立LC-MS/MS法测定血浆中原儿茶酸的浓度，并对beagle犬灌胃四季青水煎液后原儿茶酸的药动学进行研究。方法 Beagle犬血浆样品以酮洛芬为内标，用乙腈沉淀蛋白，采用LC-MS/MS法进行分离和测定。色谱柱为C18(4.6mm×100mm, 2.7μm)；流动相为80%乙腈-20%水(含0.1%甲酸)，流速为0.2mL/min，柱温为30℃。质谱条件：电喷雾离子源(ESI)，负离子模式，多反应离子监测(MRM)，检测离子为原儿茶酸m/z 153.0→109.0，内标酮洛芬m/z 253.1→209.1。6只beagle犬单次灌胃四季青水煎液50mL(0.2g/mL)后，于给药前及给药后0.083、0.167、0.333、0.5、0.667、0.833、1、1.5、2、3、4和6h采血，以LC-MS/MS法测定原儿茶酸在beagle犬体内的血浆浓度，并用非房室模型计算药动学参数。结果 原儿茶酸浓度在0.05~2.00μg/mL范围内线性关系良好，定量下限为0.05μg/mL，批内和批间精密度RSD均小于8.6%，准确度为92.3%~102.0%。Beagle犬分别单次灌胃1g/kg(生药量)四季青水煎液后，其主要药动学参数Cmax为(1.53±0.33)μg/mL，Tmax为(0.42±0.09)h，t1/2为(0.80±0.16)h，MRT0~t 为(1.04±0.14)h，MRT0~∞ 为(1.32±0.26)h，AUC0~t为(1.90±0.35)(μg/mL)·h，AUC0-∞为(2.02±0.33)(μg/mL)·h。 结论 本实验中建立的LC-MS/MS法专属性强、快速灵敏，可以用于犬血浆中原儿茶酸的测定。Beagle犬单次灌胃四季青水煎液后，血浆中原儿茶酸能快速吸收，达峰速度较快，在体内能滞留一定时间，有利于四季青药效作用的发挥。

Pharmacokinetic study of protocatechuic acid in beagle dogs after the oral administration of Ilex chinensis folium

Objective To develop an LC-MS/MS method for the determination of protocatechuic acid concentration in dog plasma, and study the pharmacokenitics of protocatechuic acid after a single intragastric administration of Ilex chinensis folium water decoction to beagle dogs. Methods The samples of plasma were deproteinated with acetonitrile. The protocatechuic acid was determined by an LC-MS/MS system using ketoprofen as the internal standard. The C18 column (4.6mm×100mm, 2.7μm) was used in the system, the mobile phase was acetonitrile-water(80:20,V/V) containing 0.1% formic acid, the flow rate was 0.2mL/min, and the column temperature was 30℃. The parameters for MS were set as follows: Electrospray ionization source (ESI) was operated in the negative ion mode, the quantification was performed using the multiple reaction monitoring (MRM) mode, andthe precursor-to-product ion transitions were m/z 153.0→109.0 for protocatechuic acid and m/z 253.1→209.1 for the internal standard ketoprofen. Six beagle dogs were administratedintragastrically withasingle dose of ilex chinensis sims folium water decoction 50mL (0.2g/mL). The plasma were collected respectively for the analysis before and at 0.083, 0.167, 0.333, 0.5, 0.667, 0.833, 1, 1.5, 2, 3, 4 and 6h, and the parametersof pharmacokinetic were calculated by a non-compartment model. Results The concentration of protocatechuic acid in dog plasma was linear in the range of 0.05~2.00μg/mL, and the lower limit of quantification (LLOQ) was 0.05μg/mL. Both the intra-and inter-batch precisions of RSD were less than 8.6%, and the accuracy were in the range of 92.3%~102.0%. After a single intragastric administration of ilex chinensis sims folium water decoction 1g/kg to the beagle dogs, the main pharmacokinetic parameters of protocatechuic acid were as follows: Cmax=(1.53±0.33)μg/mL, Tmax=(0.42±0.09)h, t1/2=(0.80±0.16)h, MRT0~t=(1.04±0.14)h, MRT0~∞=(1.32±0.26)h; AUC0-t=(1.90±0.35)(μg/mL)·h, AUC0-∞=(2.02±0.33)(μg/mL)·h. Conclusion The method of LC-MS/MS is specific, rapid and sensitive. It can be used to determine the concentration of protocatechuic acid in dog plasma. The absorption of protocatechuic acid was very quick after a single intragastric administration of Ilex chinensis folium water decoction. The Tmax can reach rapidly, and can maintain a certain period of time. These characteristics of Ilex chinensis folium showed better pharmacological