青霉素V钾及其有关物质分析方法的比较

Comparison of RP-HPLC methods for analysis of phenoxymethylpenicillin potassium and its related substances

目的:中国药典2005年版(chP 2005)中青霉素 V 钾有关物质分析方法与其他药典及文献报道方法在流动相、检测波长、杂质限度等方面存在较大差异,本文比较了几种典型的青霉素 V 钾有关物质分析方法,为完善中国药典中青霉素 V 钾有关物质控制方法提供科学依据。方法:采用 SinoChrom ODS-BP 柱(150 mm×4.6 mm,5μm),柱温35℃,以青霉素 V 钾及其杂质的分离情况为指标,比较了 ChP 2005方法和文献报道方法[流动相组成同欧洲药典5.1版方法,但采用等度洗脱]的专属性;由于在 pH 3.5时,醋酸盐缓冲系统的缓冲能力优于磷酸盐缓冲系统,故考察了将文献报道方法中 pH 3.5磷酸盐缓冲液换为 pH 3.5醋酸盐缓冲液后系统的专属性变化;比较了青霉素 V 钾及其有关物质的 UV 图谱,为检测波长的合理选择提供依据。结果:文献报道方法的专属性优于 ChP 2005方法,能将青霉素 V 钾及其有关物质较好地分离;将 pH 3.5磷酸盐缓冲液换为 pH 3.5醋酸盐缓冲液后,方法的专属性无明显改变;除青霉素外,青霉素 V 及其杂质的 UV 吸收值均为:A(254 nm)<A(268 nm)<A(220 nm),但考虑到220 nm 接近 UV 末端检测,容易受到试剂等因素的影响,故建议青霉素 V 钾有关物质控制采用268 nm 检测。结论:基于本文结果,为完善 ChP 2005中青霉素 V 钾质量标准,建议:(1)将青霉素 V 钾原料及制剂中有关物质检查方法中的流动相改为专属性更好的文献方法流动相或者改进后的流动相,检测波长仍采用原方法的268 nm 检测;(2)为促进青霉素 V 钾原料及制剂产品质量的提高,建议有关物质检查项中增加单一杂质的限度,采用外标法或者校正因子法控制单一杂质。

Objective： A comparison of testing methods for analysis of phenoxymethylpenicillin potassium and its related substances was performed to determine the most appropriate method for quality control. Methods ： The primary RP- HPLC methods reviewed were those adopted by Chinese Pharmacopoeia 2005 （Method 1 ） and a published method （ Method 2） , with similar mobile phase to that of European Pharmacopoeia 5.1 but without gradient elution. A modified Method 2 （ Method 3 ） was also investigated with pH 3.5 acetate buffer replacing the original pH 3.5 phosphate buffer, because of its better buffer capacity at pH 3.5. Results： Method 2 and Method 3 showed similar specificity and allowed for baseline separation of phenoxymethylpenicillin from its known related substances, including its precursor,possible degradations and by - products, demonstrating more specific than Method 1. And in general, phenoxymethylpenicillin and its related substances have similar UV spectra and their A （220 nm） 〉 A （268 nm） 〉 A （254 rim）, except benzylpenicillin, whose A （268 nm） being smaller than A （254 nm）. In considering the possible interferences from reagents ,268 nm was recommended as detection wavelength, according with that of ChP 2005. Conclusion：Based on the results of the paper, advices were proposed to improve the ChP 2005 methods for the quality control of phenoxymethylpenicillin potassium, e. g replacing the mobile phase with that of Method 2 or Method 3, and the limits for individual impurity should be added with impurity references or relative response factors ,etc.