Determination of the chiral and achiral related substances of methotrexate by cyclodextrin-modified micellar electrokinetic chromatography.
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.
Gotti
Roberto
R
Dipartimento di Scienze Farmaceutiche, Università di Bologna, Bologna, Italy. gottir@alma.unibo.it
El-Hady
Deia Abd
DA
Andrisano
Vincenza
V
Bertucci
Carlo
C
El-Maali
Nagwa Abo
NA
Cavrini
Vanni
V
D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods* / Q000706:statistics & numerical data; D004340:Drug Contamination; D004573:Electrolytes; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification; D015394:Molecular Structure; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012967:Sodium Dodecyl Sulfate; D012997:Solvents; D013237:Stereoisomerism; D013501:Surface-Active Agents; D047392:beta-Cyclodextrins
D004573:Electrolytes; D012997:Solvents; D013501:Surface-Active Agents; D047392:beta-Cyclodextrins; D012967:Sodium Dodecyl Sulfate; C031215:betadex; D008727:Methotrexate