Gotti Roberto, El-Hady Deia Abd, Andrisano Vincenza, Bertucci Carlo, El-Maali Nagwa Abo, Cavrini Vanni
Dipartimento di Scienze Farmaceutiche, Università di Bologna, Bologna, Italy.
Electrophoresis. 2004 Aug;25(16):2830-7. doi: 10.1002/elps.200405926.
A cyclodextrin-modified micellar electrokinetic chromatographic (CD-MEKC) method for the determination of the most important potential impurities of methotrexate (MTX): 2,4-diamino-6-(hydroxymethyl)pteridine, aminopterine hydrate, 4-[N-(2-amino-4-hydroxy-6-pteridinylmethyl)-N-methylamino] benzoic acid, 4-[N-(2,4-diamino-6-pteridinylmethyl)-N-methylamino] benzoic acid, and the distomer D-MTX is presented. The MEKC separation of these compounds was optimized by applying a step-by-step approach. The addition of beta-CD to a conventional MEKC system, based on sodium dodecyl sulfate (SDS) as surfactant, showed to be essential for the enantioresolution of racemic MTX as well as for the separation of the achiral impurities. To achieve high-resolution factor between the peaks adjacent to the main component (L-MTX), as required in the analysis of related impurities, the separation conditions were stressed; in particular, the addition of methanol to the CD-MEKC system resulted in a very effective choice. Under the optimized final conditions (100 mM SDS and 45 mM beta-CD in a mixture of 50 mM borate buffer, pH 9.30-methanol (75:25 v/v)), the method was validated showing a general adequate accuracy (93-106% recovery) in the determination of L-MTX related substances at the impurity level of 0.12% w/w with a relative standard deviation (RSD)% lower than 8% (n = 4). The method was successfully applied to the analysis of pharmaceuticals (tablets and injections) which showed to contain the distomer D-MTX as major impurity and aminopterine hydrate as a further related substance in the commercial tablets.
介绍了一种用于测定甲氨蝶呤（MTX）最重要潜在杂质的环糊精修饰胶束电动色谱（CD - MEKC）方法，这些杂质包括：2,4 - 二氨基 - 6 -（羟甲基）蝶啶、氨基蝶呤水合物、4 - [N -（2 - 氨基 - 4 - 羟基 - 6 - 蝶啶基甲基）- N - 甲基氨基]苯甲酸、4 - [N -（2,4 - 二氨基 - 6 - 蝶啶基甲基）- N - 甲基氨基]苯甲酸以及异构体D - MTX。通过逐步方法对这些化合物的MEKC分离进行了优化。在以十二烷基硫酸钠（SDS）作为表面活性剂的传统MEKC系统中添加β - CD，对于外消旋MTX的对映体拆分以及非手性杂质的分离至关重要。为了在相关杂质分析中达到与主要成分（L - MTX）相邻峰之间的高分辨率因子要求，对分离条件进行了强化；特别是向CD - MEKC系统中添加甲醇是一个非常有效的选择。在优化的最终条件下（50 mM硼酸盐缓冲液，pH 9.30 - 甲醇（75:25 v/v）的混合物中含有100 mM SDS和45 mMβ - CD），该方法经验证，在测定杂质水平为0.12% w/w的L - MTX相关物质时，总体准确度良好（回收率为93 - 106%），相对标准偏差（RSD）%低于8%（n = 4）。该方法成功应用于药物（片剂和注射剂）分析，结果表明市售片剂中含有异构体D - MTX作为主要杂质，以及氨基蝶呤水合物作为进一步的相关物质。
