【作者】 王虹；

【导师】 李发美；

【作者基本信息】 沈阳药科大学 ， 药物分析， 2007， 硕士

【摘要】 司他夫定是胸苷核苷类似物，通过细胞激酶磷酸化，形成司他夫定三磷酸盐而发挥强大的抗病毒活性。本文对司他夫定散和司他夫定缓释片做了全面的质量研究工作，建立了含量、有关物质、释放度等的测定方法；并应用所建立的方法进行稳定性研究，为司他夫定的制剂产品的质量控制提供科学依据；建立了测定犬血浆中司他夫定浓度的方法，并应用于犬灌胃司他夫定散后的药动学研究。1．司他夫定两种制剂含量、有关物质测定方法研究建立了高效液相色谱法进行了司他夫定散和司他夫定缓释片含量和有关物质检查的研究。色谱柱为Diamonsil^TM C₁₈柱；流动相为25mmol·L^-1乙酸铵水溶液-甲醇（80：20，v／v），流速为1.0mL·min^-1，检测波长为266nm，柱温为室温。方法平均回收率分别为99.7％和99.6％（n=9），RSD分别为0.53％和0.47％；含量测定的精密度分别为99.9％和100.1％（n=6），RSD分别为1.03％和0.88％；司他夫定浓度在5.0～50.0μg·mL^-1的范围内线性关系良好（γ=0.999 9）。对HPLC方法的耐用性进行了考察，在流动相配比、流速发生微小变化以及换用不同厂家色谱柱时，对含量测定结果无影响。专属性试验表明，在所采用的色谱条件下，司他夫定与其有关物质、降解产物及空白辅料能够得到良好的分离。司他夫定的检测限为0.02μg·m^-1。2．司他夫定缓释片释放度测定方法研究采用紫外分光光度法对司他夫定缓释片的释放度进行测定，在司他夫定最大吸收波长266nm处，空白辅料对释放度测定无干扰。根据《中华人民共和国药典（2005年版）》的要求，采用释放度测定第一法，分别考察其在水、0.1mol·L^-1盐酸溶液和pH6.8的磷酸盐缓冲液中的释放情况。在三种释放介质中的平均回收率分别为99.4％（n=12）、99.7％（n=6）和99.9％（n=6），RSD分别为0.32％、0.45％和0.47％；司他夫定浓度在1.0～12.0μg·mL^-1的范围内线性关系良好（γ=0.999 9）；从释放曲线测定结果可知，本品在三种释放介质中均表现出良好的缓释性能，均一性和重复性符合新药研究要求。3．司他夫定散及缓释片的稳定性研究参照新药稳定性研究的指导原则，应用所建立的两种制剂的质量控制方法进行研究，进行了影响因素试验、加速试验及长期稳定性试验。影响因素试验结果表明高温和高湿条件对司他夫定两种剂型的稳定性均有影响，而光照条件对稳定性结果基本无影响；加速试验和长期试验结果表明司他夫定两种制剂在规定的包装和贮存条件下基本稳定。4．犬血浆中司他夫定的药物动力学研究建立了高效液相色谱-紫外检测法测定犬血浆中的司他夫定含量。用乙酸乙酯提取司他夫定和内标脱水胸苷，以水-甲醇（20：80，v／v）为流动相，色谱柱为Diamonsil^TMC₁₈柱，流速为1.0mL·min^-1，检测波长为266nm，柱温为室温。司他夫定和内标的保留时间分别为9.1和12.3min，最低定量限为0.02μg·mL^-1，线性范围为0.02～5.0μg·mL^-1。日间和日内精密度RSD值分别为3.7％～12.5％和3.4％～7.6％。该方法应用于犬灌胃司他夫定散后药动学的研究，测得主要药动学参数C_max为2.65±0.31μg·mL^-1，T_max为0.58±0.13h，T_1/2为1.55±0.16h，AUC_0-t为6.19±0.35μg·h·mL^-1。更多还原

【Abstract】 Stavudine, a nucleoside analogue of thymidine, is phosphorylated by cellular kinases to the active metabolite, stavudine triphosphate, which has shown powerful activity against human immunodeficiency virus（HIV）. The quality of stavudine powder and stavudine sustained-release tablet has been comprehensively investigated. Firstly, an HPLC method was developed for the determination of stavudine content and related substance, and a UV spectrophotometry method for the determination of sustained-release tablet’s drug release. Secondly, a stability test was carried out for both formulations using the developed methods. Thirdly, a reliable reversed-phase isocratic HPLC method with UV detection for the determination of stavudine in dog plasma was developed and applied to the pharmacokinetic study of stavudine in dog after administration on stavudine powder.1. The content and related substance determination of stavudine preparationsAn HPLC method was developed for the content and related substance determination of stavudine powder and stavudine sustained-release tablet. The separation was performed on a Diamonsil C₁₈ column. The mobile phase was composed of 25mmol·L^-1 ammonium acetate solution-methanol（80:20, v/v） with flow rate of 1.0mL·min^-1. The detection was set at 266nm and column temperature at ambient temperature. The method was accurate and the average recoveries of stavudine powder and stavudine sustained-release tablet were 99.7% and 99.6% with RSDs of 0.53% and 0.47%（n=9）. The precisions of content determination of stavudine perparations were 99.9% and 100.1% with RSDs of 1.03% and 0.88%（n=6）. The linear range for stavudine was 5.0～50.0μg·mL^-1（Y=0. 999 9）. The robustness of the method was demonstrated by little variations in the mobile phase composition, the columns and the flow rate. The specificity test demonstrated that the related substances and degradation products of stavudine and excipients were completely separated from stavudine and had no influence on the determination. The lower limit of detection of stavudine was 0.02μg·mL^-1. 2. The determination of drug release of stavudine sustained-release tabletA UV spectrophotometry was used for determining the drug release of stavudine sustained-release tablet. At the maximum absorption wave length（266nm） of stavudine, the excipients had no influence on the determination. The sustained-release properties of stavudine sustained-release tablet were investigated in water, 0.1mol·L^-1 hydrochloride solution and phosphate buffer solution（pH6.8） with the methodⅠof drug release ditermination. In the three dissolution mediums, the average recoveries were 99.4%（n=12）, 99.7%（n=6） and 99.9%（n=6） with RSDs of 0.32%, 0.45% and 0.47%, respectively. The linear range for stavudine was 1.0～12.0μg·mL^-1（γ=0. 999 9）. The release curves demonstrated that stavudine sustained-release tablet had good sustained-release properties and its uniformity confirmed the requirement in the Guideline for new drug development.3. The stability study on stavudine preparationsThe stress test, accelerating test and long-term stability test were carried out by using the developed methods according to Guideline of study on stability of new drug. The results of stress test demonstrated that humidity and temperature had effects on the stability of stavudine preparations. While light had no effect on the stability of stavudine preparations. Both accelerated test and long-term test demonstrated that stavudine powders and stavudine sustained-release tablets were stable under the packaging and storage conditions.4. Determination of stavudine in dog plasmaAn HPLC method with UV detection（266nm） was developed and validated for the determination of stavudine in dog plasma. Stavudine and the internal standard（dehydrate-thymidine） were extracted into ethyl acetate and separated using an isocratic mobile phase of methanol-water（20:80, v/v） on a Diamonsil C₁₈ column. The retention times for stavudine and internal standard were 9.1 and 12.3 min, respectively. No endogenous interferences were observed. This HPLC method was fully validated. The lower limit of quantification was 0.02μg·mL^-1. A linear range of 0.02～5.0μg·mL^-1（γ=0.998 9） was established. The interday and intraday RSDs were within 12.5～3.7% and 7.6～3.4%, respectively. This method developed was easily applied to the pharmacokinetic study of stavudine in dog plasma after i.g. administration stavudine powder. The pharmacodinetic parameters: C_max of 2.65±0.31μg·mL^-1, T_max of 0.58±0.13h, T_1/2 of 1.55±0.16h and AUC_0-t of 6.19±0.35μg·h·mL^-1 were observed.更多还原