高效液相色谱-荧光检测法测定盐酸帕罗西汀在小鼠血浆和脑组织中的蛋白结合率

Determination of protein binding of paroxetine hydrochloride in mouse plasma and brain by HPLC-FLU

目的:建立生物样品中盐酸帕罗西汀浓度的高效液相色谱-荧光检测方法,测定盐酸帕罗西汀在小鼠血浆和脑组织中的蛋白结合率。方法:以盐酸普萘洛尔为内标,血浆和脑组织匀浆样品采用醋酸乙酯萃取,采用高效液相色谱-荧光检测方法,其中色谱柱为Eurospher 100-5 C18柱(250 mm×4 mm,5μm);流动相为乙腈-0.5%三乙胺水溶液=35∶65(磷酸调pH值至3.5);柱温:40℃;流速:1 mL·min^(-1);检测波长:激发波长295 nm,发射波长350 nm,进样量:20μL。蛋白结合率的测定采用平衡透析法。结果:血浆和脑匀浆液中盐酸帕罗西汀在0.05~10μg·mL^(-1)范围内线性关系良好,血浆与脑匀浆液基质提取回收率均>75%;日内、日间精密度均<10%。磷酸盐缓冲液盐酸帕罗西汀在0.005~0.5μg·mL^(-1)内线性良好,定量下限为0.005μg·mL^(-1),且日内、日间精密度均符合要求。当血浆和脑匀浆液中盐酸帕罗西汀质量浓度为1.0,3.0,6.0μg·mL^(-1)时,血浆蛋白结合率分别为(96.14±0.38)%,(96.07±0.88)%,(97.25±0.23)%;脑组织蛋白结合率分别为(99.84±0.031)%,(99.83±0.021)%,(99.80±0.041)%。结论:本研究建立的分析方法快速、简单、灵敏、准确,可用于生物样品中盐酸帕罗西汀含量的测定。小鼠血浆和脑组织中盐酸帕罗西汀蛋白结合率高,只有极少的药物以游离形式存在而发挥作用。

OBJECTIVE To develop an HPLC method for determination of paroxetine hydrochloride in biological samples and measure its protein binding in plasma and brain tissues of mice.METHODS Propranolol hydrochloride was used as internal standard,plasma and brain homogenates were extracted with ethyl acetate.Analysis was conducted by HPLC-fluorescence detector on a C18 column.Mobile phase consisted of acetonitrile-0.5% triethylamine（35∶65,pH 3.5 adjusted by phosphoric acid）.Flow rate was 1.0mL·min-1 and column temperature was 40 ℃.Excitation and emission wavelengths were 295 nm and350 nm,respectively.Equilibrium dialysis method was used to investigate reversible binding of paroxetine in mouse plasma and brain tissues.RESULTS Linear range of paroxetine hydrochloride in plasma and brain homogenates was 0.05-10μg·mL-1.Extraction recoveries in plasma and brain homogenates were more than75%.Intra-and inter-day precisions were both less than10%.Linear range of paroxetine hydrochloride in phosphate buffer was 0.005-0.5μg·mL-1 and the lower limit of quantitation was 0.005μg·mL-1.Intra-and inter-day precisions all met the requirements.Plasma protein binding rates of paroxetine were（96.14±0.38）%,（96.07±0.88）%,（97.25±0.23）% at three concentration levels（1,3,6μg·mL-1）of paroxetine,respectively,while those for mouse brain were（99.84±0.031）%,（99.83±0.021）%,（99.80±0.041）%,respectively.CONCLUSION This method is rapid,simple,sensitive and accurate,can be used to determine paroxetine hydrochloride in biological samples.Protein binding rates of paroxetine in mouse plasma and brain tissue are consistently high,only a tiny part of paroxetine in form of original drug can produce therapeutic effects.