目的建立HPLC同时测定大鼠血浆中山楂叶总黄酮(HLF)的两个主要有效成分4″-O-牡荆素葡萄糖苷(GLV)和2″-O-牡荆素鼠李糖苷(RHV)的浓度,并用此方法对HLF在大鼠体内的药动学进行研究。方法大鼠尾静脉注射HLF,于不同时间点断尾取血,甲醇直接沉淀蛋白,HPLC测定GLV和RHV的血药浓度。色谱柱为Diamonsil^TM C18(4.6mm×200mm,5μm),流动相为四氢呋喃-甲醇-乙腈-0.5%冰醋酸(19.4∶1∶1∶78.6),检测波长为330nm。结果样品中GLV和RHV被成功分离,线性范围分别为0.23~138.42mg.L-1(r=0.9990)和0.36~218.49mg·L^-1(r=0.9995)。在测定浓度范围内,GLV和RHV的日内精密度(n=5)分别为0.69%~3.91%和0.51%~3.89%;日间精密度(n=5)分别为1.09%~5.03%和1.45%~2.16%;平均方法回收率(n=5)分别为(96.70±5.29)%~(102.20±1.15)%和(96.39±1.44)%~(103.64±2.23)%;平均提取回收率(n=5)分别为(92.68±2.19)%~(95.74±1.19)%和(93.45±1.85)%~(99.26±2.85)%。结论该分析方法简便快速,专属灵敏,精密度和回收率高,可满足药动学研究需要。静注HLF10~40mg·kg^-1,GLV和RHV在大鼠体内药动学特征相似,均符合三室开放药动学模型。
OBJECTIVE To establish a HPLC method for simultaneous determining rat plasma concentrations of 4″-O-glucosylvitexin (GLV) and 2″-O-rhamnosylvitexin (RHV) from Hawthorn Leaves Flavonoids (HLF) and study their pharmacokinetics in rats. METHODS Blood samples were collected via tail vein at time intervals after HLF injection. The plasma concentrations of the ingredients were analyzed by HPLC after the plasma protein was precipitated directly with methanol. The Diamonsil^TM C18 column (4.6 mm×200 mm;5μm) was used with a mobile phase consisting of tetrahydrofuran-methanol-acetonitrile-0. 5% acetic acid (19. 4: 1: 1: 78.6). The detection wavelength was 330 nm. RESULTS GLV and RHV were successfully separated and the linear range of them were 0. 23 ~ 138.42 mg · L^-1 ( r = 0. 999 0 ) and 0. 36 - 218. 49 mg · L^-1 ( r = 0. 999 5 ), respectively. The intraday precision of GLV and RHV (n = 5 ) within the concentration ranges were 0.69% -3.91% and 0. 51% - 3.89% , respectively;the interday precision ( n = 5 ) were 1.09% - 5.03% and 1.45% ~ 2. 16% , respectively; the mean method recoveries ( n = 5 ) were (96. 70 ± 5.29) % ( 102. 20 ± 1.15) % and (96. 39 ± 1.44) % ~ ( 103.64 ± 2.23 ) %, respectively ; the mean extraction recoveries ( n = 5 ) were ( 92. 68±2. 19)% (95.74 ±1.19)% and (93.45±1.85)% ~ (99.26 ±2.85)% ,respectively. CONCLUSION This HPLC method is simple and efficient with excellent specificity,sensitivity,precision, recovery and can be applied to pharmacokinetic investigation. The pharmacokinetic characteristics of GLV and RHV are similar after iv administration of HLF to rats at the doses of 10 -40 mg · kg^-1 and they were both best fitted to the three-compartment open pharmacokinetic model.