Determination of the chiral and achiral related substances of methotrexate by cyclodextrin‐modified micellar electrokinetic chromatography

Abstract

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.

Chiral separations by capillary electrophoresis: Recent developments and applications

“…The addition of b-CD to the MEKC system based on SDS was essential for the enantioresolution of methotrexate and for the separation of achiral impurities. The application of this method for the analysis of tablets and injections showed that the major impurity was D-methotrexate [157].…”

“…Baseline separation was obtained using b-CD in a phosphate [157] buffer pH 4.0. The method was applied to measure cefoperazone enantiomers in pharmaceutical doses.…”

Capillary electrophoresis for the analysis of small‐molecule pharmaceuticals

“…A CD-MEKC method to simultaneously determine the chiral and achiral purity of methotrexate was published by Gotti [77]. The method was validated and proved to be sufficiently robust and sensitive for the analysis of commercially available tablets and injection solutions.…”

Impurity analysis of pharmaceuticals using capillary electromigration methods

“…Impurities of L-methotrexate, an antimetabolite of folic acid, and its chiral and achiral impurities were separated using MEKC with cyclodextrin as additive [107]. The method was validated concerning ICH guidelines and applied to quantify the impurities in tablets and injection formulations.…”