血清中罗红霉素浓度反相高效液相色谱测定法的改进

Determination of Roxithromycin in Serum by High-performance Liquid Chromatography with Ultraviolet Detection

目的 对测定血清中罗红霉素浓度的高效液相色谱法进行改进。方法 其改进要点是 :采用Penom enex L una C1 8分析柱 (5 μm,15 0 m m× 4.6 m m) ,以乙腈 -甲醇 - 0 .0 5 m ol/ L 磷酸 (39∶ 19∶ 42 ,用氨水调 p H至7.2 )为流动相 ,克拉霉素作内标。血清样品碱化后用二氯甲烷提取浓集 ,再经进一步纯化后进样 ,在 2 10 nm波长处检测 ,按内标法定量。结果 本法与原方法的标准曲线线性范围分别为 0 .2 5～ 32 mg/ L 和 0 .2 5～ 16 m g/ L,最低检出量分别为 5 ng和 2 0 ng,罗红霉素保留时间分别为 5 .0分钟和 13.2 7分钟 ,日内 RSD小于 2 .5 %和 7.5 % ,日间RSD小于 3.0 %和 5 .5 % ,萃取回收率和方法回收率一致。结论 本法与原法相比具有快速简便。

Objective To make better the RP HPLC method for the determination of roxithromycin(RM) in human serum. Methods RM and clarithromycin (internal standard) were extracted from alkalinized serum sample (500μl) with methylene chloride. After evaporation of the organic layer, the residue was dissolved in 100μl of acetonitrile ammonium phosphate (1∶1, pH6.0) and washed with n hexane, then 20μl was injected onto a column (5μm, 15cm×4.6mm) of Penomenex luna C 18 . The mobile phase was acetonitrile 0.05mol/L phosphoric acid (39∶19∶42, adjusted to pH7.2 with ammonia water) pumped at 1.2ml/min through the column. The variable wavelength UV detector operated at 0.01 aufs and the wavelength was set at 210nm.Results The retention times for RM and clarithromycin were 4.4min and 5.0min, respectively. Standard curve was linear in the concentration range of 0.25 to 32mg/L. The detection limit in serum was 0.06mg/L; the average method recovery 97.4%; the inter day RSD less than 3.0%. Conclusion This method was found to be simple, rapid, sensitive and accurate for determination of RM in human serum.