Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt.
Pharmaceutical Service Center, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt.
J Pharm Biomed Anal. 2019 Mar 20;166:421-434. doi: 10.1016/j.jpba.2019.01.001. Epub 2019 Jan 3.
A highly sensitive, selective and precise HPTLC method coupled with fluorescence detection was developed and validated for the determination of α-aminocephalosporin antibiotics; namely cefalexin, cefadroxil and cefradine in their standard solutions. The applicability of the developed methodology was demonstrated via analysis of cefalexin in goat milk samples. Full optimization of the fluorescence derivatization reaction was carried out with regard to the standard solutions of the studied compounds or after extraction of milk samples. The separation of the studied compounds was performed on HPTLC precoated silica gel plates 60 F using acetonitrile: water in a ratio 85:15 (v/v) as a mobile phase. The retention behavior of the formed derivatives was discussed in detail. It was found that hydrophilic interaction mode is the main interaction mechanism governing the retention of the formed derivatives. In addition, an experimental design approach was conducted for optimization of the chromatographic conditions. Modified QuEChERS was applied as an efficient extraction technique of cefalexin from both spiked and real goat milk samples. Optimization of QuEChERS extraction technique to achieve the highest extraction recovery was performed and the results indicate that this method provides a good extraction recovery (83-116%) for cefalexin from goat milk samples. Limit of detection (LOD) of the developed method was found to be 0.023, 0.005, and 0.023 ng band for cefalexin, cefadroxil and cefradine, respectively in their standard solutions and 0.165 ng band for cefalexin in goat milk samples. According to the achieved LOD values, the method sensitivity was quasi-equivalent to other methods based on expensive techniques such as HPLC-UV and HPLC-MS and it is sufficient to determine cefalexin below its MRL in milk samples. Moreover, the method was successfully applied to a pharmacokinetic study of cefalexin in goat milk after single intramuscular injection of 10 mg of cefalexin kg per body weight.
开发并验证了一种高灵敏度、选择性和精确的 HPTLC 方法，结合荧光检测，用于测定α-氨基头孢菌素抗生素；即头孢氨苄、头孢羟氨苄和头孢拉定在其标准溶液中的含量。通过对山羊奶样品中头孢氨苄的分析，证明了所建立方法的适用性。对研究化合物的标准溶液或提取牛奶样品后的荧光衍生反应进行了充分优化。采用 HPTLC 预涂硅胶板 60 F，以乙腈：水 85:15（v/v）为流动相，对研究化合物进行分离。详细讨论了形成的衍生物的保留行为。结果表明，亲水相互作用模式是控制形成的衍生物保留的主要相互作用机制。此外，还进行了实验设计方法来优化色谱条件。改良的 QuEChERS 被用作从添加和真实山羊奶样品中提取头孢氨苄的有效提取技术。对 QuEChERS 提取技术进行了优化，以实现最高的提取回收率，结果表明该方法可提供良好的提取回收率（83-116%）用于从山羊奶样品中提取头孢氨苄。在其标准溶液中，头孢氨苄、头孢羟氨苄和头孢拉定的检出限（LOD）分别为 0.023、0.005 和 0.023 ng 带，而在山羊奶样品中，头孢氨苄的检出限为 0.165 ng 带。根据所获得的 LOD 值，该方法的灵敏度与基于 HPLC-UV 和 HPLC-MS 等昂贵技术的其他方法相当，足以在牛奶样品中低于其 MRL 来测定头孢氨苄。此外，该方法成功应用于山羊奶中单次肌肉注射 10 mg/kg 头孢氨苄后的头孢氨苄药代动力学研究。
