光动力药物血卟啉单甲醚HPLC-荧光检测法的建立及其药代动力学研究

Determination of Hematoporphyrin Monomethyl Ether in Dogs Plasma by HPLC Using Fluorescence Detection

目的建立测定血浆中血卟啉单甲醚(HMME)的HPLC-荧光检测法,并用以进行HMME在犬体内的药代动力学研究.方法血浆中的HMME采用乙酸乙酯液-液萃取,荧光素为内标.选用C18柱(4.6 mm×150 mm,5 μm),0.02 mol/L 醋酸钠(用冰醋酸调pH 6.0)-四氢呋喃(60∶40)组成为流动相,荧光检测器激发波长为 395 nm,发射波长为 613 nm.结果 HMME血药浓度的范围为0.025～5.000 μg/mL,采用两条标准曲线定量的方法,两条标准曲线的线性范围分别为:0.025～0.500 μg/ml(r=0.9973)和0.25～5.00 μg/ml(r=0.9999),提取回收率92.1%～99.2%,日内、日间RSD分别为3.3%～8.4%和3.8%～8.5%. 静脉给药后,HMME在犬体内的药代动力学符合开放二房室模型.其主要药代动力学参数:T1/2α(分布半衰期) =(0.18±0.16)h,T1/2β(消除半衰期) =(14.24±3.41)h, Vd(中央室表观分布容积)=(6.37±3.70)L/g,CL(清除率) =(0.31±0.17)L·kg-1·L-1,AUC(0-tn)(血药浓度-时间曲线下面积)=(27.40±11.72)mg·h-1L·-1.结论 HPLC-荧光检测法准确、灵敏,适用于HMME药代动力学研究.HMME在犬体内的药代动力学过程符合开放二室模型,其在犬体内可以迅速消除,可很好地消除光动力学疗法的主要副作用--正常组织的持久光毒反应.

Objective A HPLC with fluorescence detection method was developed and validated for the quantitative determination of hematoporphyrin monomethyl ether（HMME） in dog plasma. The validated method was applied to study the pharmacokinetics of HMME in beagle dogs.Methods The HMME and the internal standard were extracted from plasma samples by liquid-liquid extraction. Fluorescein sodium was applied as the internal standard. The HMME as separated on Diamonsil C18（4.6 mm × 150 mm,5μm） column and detected with fluorescence detector set at λex 395 nm, λem 613 nm. The mobile phase consisted of tetrahydrofuran/sodium acetate buffer solution （60：40） adjusted to pH 6.0 with acetic acid. Results The two standard curves for HMME were linear in the concentration range as follows ： 0.025-0. 500 μg/ml （ r = 0.9973 ） and 0.25-5.00 μg/ ml（r = 0. 9999）. The mean recovery of HMME from plasma was 92. 1%-99. 2%. The intra- and inter-day RSDs ranged from 3.3% -8.4% and 3.8% -8.5% , respectively. The method was applied to the pharmacokinetic study of HMME in beagle dogs and the pharmacokinetic parameters were determined after intravenous injections of HMME （5.0 mg/kg）. The main pharmaeoki- netic parameters were estimated to be as follows： T1/2α = （0. 18 ± 0. 16） h, T1/2β = （ 14. 24 ± 3.41 ） h , Vd = （6. 37 ± 3.70） L · kg^- 1, CL= （0. 31 ±0. 17）L · kg^- 1· h^-1 ,AUC（0-tn） = （27.40 ± 11.72）mg · h^- 1· L^-1.Conclusions The method is selective, sensitive and precise, and suitable to be applied in studying the pharmacokineties of HMME. It is indicated that the pharmacokinetics of HMME in beagle dogs after intravenous injection is fit for a two-compartment open model.