RP-HPLC法测定β-司他夫定的有关物质

目的: 建立了β-司他夫定原料有关物质的RP-HPLC测定方法和水解破坏制备系统适用性试验溶液方法. 方法: 采用Agilent 1100型高效液相色谱仪,使用SUPELCOSIL LC-18-DB(4.6 mm×250 mm,5 μm)色谱柱,以0.01 mol·L^-1醋酸铵溶液-乙腈(96.5:3.5)和0.01 mol·L^-1醋酸铵溶液-乙腈(75:25)为流动相,梯度洗脱,检测波长254 nm,柱温25 ℃. 结果: β-司他夫定和4种已知杂质及其他未知杂质均能达到有效分离;经水解破坏产生的α-司他夫定与β-司他夫定的分离度均达2.8;β-司他夫定、胸腺嘧啶、β-胸苷与5-O'-苯甲酰-司他夫定线性范围分别为0.51~26 μg·mL^-1(r=1.000)、0.13~27 μg·mL^-1(r=1.000)、0.50~25 μg·mL^-1(r=1.000)、1.7~6.3 μg·mL^-1(r=1.000),已知杂质胸腺嘧啶、β-胸苷与5-O'-苯甲酰-司他夫定的平均加样回收率(n=9)分别为102.8%(RSD=1.5%)、100.6%(RSD=0.9%)、101.9%(RSD=2.1%);β-司他夫定与3种已知杂质的最小检出量均在2.5 ng以下;经水解破坏制备的系统适用性溶液的重复性良好;供试品溶液在4 ℃下的30 h内基本稳定. 结论: 本方法灵敏、准确、可靠,专属性强,可用于β-司他夫定原料的有关物质测定.

Objective: To develop a method for RP-HPLC determination of related substances in β-stavudine and prepare the solution for system suitability test by sample hydrolysis. Methods: The chromatographic separation was achieved on a SUPELCOSIL LC-18-DB column(4.6 mm×250 mm,5 μm)with Agilent 1100 liquid chromatography system.The mobile phases consisted of 0.01 mmol·L^-1 ammonium acetate solution-acetonitrile(96.5:3.5)and 0.01 mmol·L^-1 ammonium acetate solution-acetonitrile(75:25)with gradient elution.The detection wavelength was 254 nm,and the column temperature was 25 ℃. Results: Good separation between β-stavudine and 4 identified impurities and other unknown impurities could be achieved,the resolution between β-stavudine and α-stavudine produced by hydrolysis was 2.8.Good linear relationship was observed in the concentration range of 0.51-26 μg·mL^-1 (r=1.000)for β-stavudine,0.13-27 μg·mL^-1 (r=1.000)for thymine,0.50-25 μg·mL^-1 (r=1.000)for β-thymidine and 1.7-6.3 μg·mL^-1 (r=1.000)for 5-O'-benzoyl-stavudine.The average recoveries of thymine,β-thymidine and 5-O'-benzoyl-stavudine were 102.8%(RSD=1.5%),100.6%(RSD=0.9%)and 101.9%(RSD=2.1%),respectively.The limits of detection of β-stavudine and identified impurities were all below 2.5 ng·mL^-1.The solution for system suitability test prepared by hydrolysis also showed good reproducibility.Sample solutions were stable at 4 ℃ in 30 hours. Conclusion: This method is sensitive,accurate,specific and reproducible,which can serve as a reliable analytical method for the determination of related substances in β-stavudine.